Expression Profiling In Acute and Chronic Cardiac Allograft Rejection

心脏移植后第一年内，急性同种异体心脏移植的细胞排斥反应仍然是发病率和死亡率的重要来源。随后，慢性血管排斥反应所致的心脏移植物血管病(CAV)成为发病率和死亡率的主要原因。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%的幸存者将感染CAV。在移植后的第一年，近63%的患者至少经历一次心脏排斥反应，其中约三分之一的患者将发生多次排斥反应。急性心脏排斥反应的临床症状相对非特异性(乏力、呼吸困难、低热)。大多数CAV患者直到出现严重问题，如心肌梗死、心力衰竭、室性心律失常或心脏性猝死，才会出现症状。目前尚无诊断急性或慢性心脏排斥反应的非侵入性方法。非侵入性方法，如心电图术、超声心动图和核研究，都已被研究过，但到目前为止，对于这两种情况都没有成功。已经研究了几种方法，包括心电图学、超声心动图、核成像和磷光谱，但均未成功。目前诊断急性细胞排斥反应的金标准仍然是右室心内膜心肌活检。这是一种侵入性的诊断方法，容易出现发病率和随机抽样和解释错误。同样，诊断CAV的金标准是血管内超声心导管插入术，这是一种有创的手术，也容易发生并发症。 我们正在应用功能基因组学来研究急性同种异体心脏移植细胞排斥反应和CAV。我们小组已经开发和测试了用于样品处理和必要时放大的标准实验室程序。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-Fairfax)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(迄今已登记115人)。我们假设，循环外周血单个核细胞(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。在项目的初始阶段，从同种异体移植免疫耐受(无急性排斥)和免疫耐受(急性排斥)期间的心脏移植受者以及有和没有CAV的心脏移植受者获得PBMC，以确定每种状态是否有独特的基因表达模式。其他可能使用的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。在该项目的后期阶段，我们希望将这些特征转化为可接受的急性和慢性心脏移植细胞排斥反应的床边测试。除了开发一种生物标记物方法来诊断心脏移植患者的排斥反应外，表达谱还有可能识别免疫调节途径，这些途径可以作为免疫抑制治疗(合理的药物开发)的新靶点。

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 (115 individuals 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).