Expression Profiling In Acute and Chronic Cardiac Allogr

心脏移植术后第一年内，急性心脏移植物细胞排斥反应仍然是发病率和死亡率的重要来源。心脏移植物血管病（cardiac allograft vasculopathy，CAV）是心脏移植术后慢性血管排斥反应的主要原因。在移植后的第一年内，几乎三分之二的受者将经历至少一次排斥反应。在移植后五年，近50%的幸存者将患有CAV。在移植后的第一年，近63%的患者经历至少一次心脏排斥反应，其中约三分之一的患者将有多次发作。急性心脏排斥反应的临床症状相对非特异性（疲劳、呼吸困难、低热）。大多数CAV患者在出现严重问题（如心肌梗死、心力衰竭、室性心律失常或心源性猝死）之前一直无症状。目前尚无无创性方法可用于诊断急性或慢性心脏排斥反应。非侵入性方法，如心电图，超声心动图和核研究都已研究，但迄今为止，对任何一种情况都不成功。已经研究了几种方法，包括心电图、超声心动图、核成像和磷光谱，但没有成功。目前诊断急性细胞排斥反应的金标准仍然是右心室肌内膜活检。这是一种侵入性的诊断方法，易受发病率、随机抽样和解释错误的影响。同样，诊断CAV的金标准是心导管插入术和血管内超声，这是一种侵入性手术，也会导致发病。 我们正在应用功能基因组学研究心脏移植急性细胞排斥反应和CAV。我们的团队已经开发并测试了用于样品处理和必要时扩增的标准实验室程序。我们已经建立了实验室和生物信息学基础设施，以支持寡核苷酸微阵列研究。当地两个主要的移植项目（约翰霍普金斯大学和INOVA费尔法克斯）已经同意合作。我们有IRB批准的方案，目前正在积极招募患者（迄今为止招募了77名患者）。我们假设循环外周血单核细胞（PBMC，主要是T淋巴细胞）的大规模表达谱将识别可作为急性和慢性心脏细胞排斥反应的可靠生物标志物的基因。在该项目的初始实验室阶段，从同种异体移植物的免疫耐受（无急性排斥）和同种异体移植物的免疫不耐受（急性排斥）期间的心脏移植受者以及从具有和不具有CAV的心脏移植受者获得PBMC，以确定独特的基因表达模式是否与每种状态相关。可以采用的其他分析工具包括蛋白质组学、RT-PCR、Western印迹、原位杂交、免疫组织化学和组织病理学。在该项目的后期，我们希望将这些特征转化为可接受的急性和慢性心脏同种异体移植细胞排斥反应的床边试验。除了开发用于诊断心脏移植患者排斥反应的生物标志物方法外，表达谱还具有鉴定免疫调节途径的潜力，这些免疫调节途径可以作为免疫抑制治疗的新靶点（合理药物开发）。

Acute cardiac allograft cellular rejection remains a significant source of morbidity and mortality within the first year after heart transplantation. Afterwards, cardiac allograft vasculopathy (CAV), as a result of chronic vascular rejection, is the major cause of morbidity and mortality. Within the first year post-transplantation, almost two-thirds of recipients will experience at least one rejection episode. At five years post-transplantation, nearly 50% of survivors will have CAV. In the first year after transplantation, nearly 63% of patients experience at least one episode of cardiac rejection and approximately one third of these patients will have multiple episodes. The clinical symptoms of acute cardiac rejection are relatively nonspecific (fatigue, dyspnea, low grade fever). Most CAV patients remain asymptomatic until they develop serious problems such as myocardial infarction, heart failure, ventricular dysrhythmias or sudden cardiac death. No noninvasive method exists for the diagnosis of acute or chronic cardiac rejection. Noninvasive methods such as electrocardiography, echocardiography, and nuclear studies all have been studied, but have been unsuccessful, thus far, for either condition. Several methodologies have been studied including electrocardiography, echocardiography, nuclear imaging, and phosphorus spectroscopy without success. The current gold standard for the diagnosis of acute cellular rejection remains right ventricular endomyocardial biopsy. This is an invasive method of diagnosis subject to morbidity and random sampling and interpretation error. Likewise, the gold standard for diagnosing CAV is cardiac catheterization with intravascular ultrasound, an invasive procedure also subject to morbidity. We are applying functional genomics to study acute cardiac allograft cellular rejection and CAV. Our group has developed and tested standard laboratory procedures for sample processing and if necessary amplification. We have established laboratory and bioinformatics infrastructure to support oligonucleotide microarray investigations. Two major local transplant programs (Johns Hopkins University and INOVA-Fairfax) have agreed to collaborate. We have an IRB approved protocol and are currently actively enrolling patients (77 patients enrolled to date). We hypothesize that large scale expression profiling of circulating peripheral blood mononuclear cells (PBMC, predominantly T lymphocytes) will identify genes that can serve as reliable biomarkers of acute and chronic cardiac cellular rejection. In the initial bench phase of the project, PBMCs are obtained from heart transplant recipients during periods of immunological tolerance of the allograft (no acute rejection) and immunologic intolerance of the allograft (acute rejection) and from heart transplant recipients with and without CAV to determine whether unique gene expression patterns are associated with each state. Other analytic tools that may be employed include proteomics, RT-PCR, Western blot, insitu-hybridization, immunohistochemistry, and histopathology. In the latter phase of the project we hope to translate these profiles into an acceptable bedside test for acute and chronic cardiac allograft cellular rejection. In addition to developing a biomarker approach to the diagnosis of rejection in cardiac transplant patients, expression profiling has the potential to identify immunoregulatory pathways that can serve as new targets for immunosuppressive therapy (rational drug development).