Differentiation Of Acute Rejection From Infection In Rat Heart Transplant Model

急性同种异体心脏移植排斥反应和感染仍然是心脏移植术后发病率和死亡率的重要来源，占报告死亡人数的近50%。临床上通常很难区分排斥反应和感染，因为它们都是炎症过程，具有相似的非特异性症状。然而，这种差异对于确定治疗方法至关重要。确定实验室方法，允许安全和简洁的早期区分移植患者的排斥反应和感染，将大大改善结果。我们已经建立了ACUC方案，使我们能够研究外周血单个核细胞（PBMC）基因微阵列分析是否能够可靠地区分移植大鼠的急性心脏排斥反应和感染。ACUC协议还允许我们进行必要的试点研究，以支持主要协议。到目前为止，我们已经建立了成功实施和维持大鼠移植模型所需的手术技术。我们在该模型中建立了环孢素（CSA）的剂量，在给药期间可靠地抑制排斥反应，但在停药后会允许出现3级排斥反应。我们还确定了适当的支气管内大肠杆菌接种剂量，足以引起肺炎和全身炎症反应，而不会立即致死接受CSA的移植大鼠。此外，我们利用基因微阵列技术研究了动物品系在排斥反应过程中对基因表达的影响以及术后炎症变化的时间过程，以确定移植心脏的最佳收获时间（即手术炎症变化引起的基因微阵列特征消失的时间）。我们也完成了主要的研究方案。我们的主要方案结合了两种成熟的大鼠模型，第一种是异位心脏移植模型，第二种是大肠杆菌肺部感染模型。所有大鼠在第0天接受心脏移植，同时每日皮下注射CSA （10mg /kg）以抑制排斥反应。移植后，动物在第6天随机选择停用CSA以引发排斥反应，或继续CSA以进一步抑制排斥反应。停用CSA后，动物再次在第13天随机接受支气管内大肠杆菌接种或生理盐水接种。因此分为四组：无感染的无排斥反应（即接受CSA）、感染的无排斥反应（即接受CSA）、感染的不排斥反应（即不接受CSA）和感染的不排斥反应（即不接受CSA）。第14天处死所有动物，取血和心脏进行基因芯片分析。其他可能采用的分析工具包括：RT-PCR， western blot，原位杂交，蛋白质组学，免疫组织化学和组织病理学。此外，在初步研究中获得了动物的肺、脾、肝和胸腺，并保存下来以供将来分析。迄今为止，已有124只动物被用于该方案。2009年，一篇建立大鼠毒株对排斥反应中基因表达影响的论文被接受并发表。我们正在继续处理和分析与排斥对细胞能量代谢的代谢影响相关的数据；手术炎症对基因表达的影响；以及急性细胞排斥反应和/或感染对基因表达的影响。

Acute cardiac allograft rejection and infection remain significant sources of morbidity and mortality after heart transplantation, accounting for nearly 50% of reported deaths. It is often difficult to clinically distinguish between rejection and infection because they are both inflammatory processes with similar, nonspecific symptoms. However, this differential is essential for determining therapy. Identifying laboratory methods that will permit safe and concise early differentiation between rejection and infection in the transplant patient will improve outcome substantially. We have established an ACUC protocol that allows us to study whether gene microarray analysis of peripheral blood mononuclear cells (PBMC) will reliably differentiate acute heart rejection from infection in the transplanted rat. The ACUC protocol also allows us to do pilot studies necessary to support the main protocol. To date, we have established the surgical techniques necessary to successfully perform and maintain the rat transplant model. We have established a dose of cyclosporin (CSA) in this model that reliably suppresses rejection during its administration, but will permit the emergence of Grade 3 rejection upon its discontinuation. We have also determined the appropriate inocula of intra-bronchial E. coli bacteria that is sufficient to cause a pneumonia and a systemic inflammatory response without being immediately lethal in transplanted rats receiving CSA. In addition, we have used gene microarry technology to study the impact of animal strain on gene expression during rejection and the time course of post surgical inflammatory changes in order to determine the most opportune time to harvest the transplanted hearts (i.e. when gene microarry signatures due to surgical inflammatory changes are dissapating). We have also completed the main study protocol. Our main protocol combines two well-established rat models, the first is a heterotopic heart transplantation model and the second is an E. coli pulmonary infection model. All rats underwent heart transplantation on day 0 in conjunction with daily CSA (10 mg/kg subcutaneous) to suppress rejection. After transplant, animals were randomized at day 6 to have CSA discontinued, in order to initiate rejection, or continued, in order to further suppress rejection. After discontinuing CSA the animals were again randomized on day 13 to receive intrabronchial E. coli inoculation or saline inoculation. Consequently, four groups were studied: No rejection (i.e. receiving CSA) without infection, no rejection (i.e. receiving CSA) with infection, rejection (i.e. not receiving CSA) without infection, and rejection (i.e. not receiving CSA) with infection. On day 14, all animals were sacrificed and the blood and heart removed for gene microarray analysis. Other analytic tools that may be employed include: RT-PCR, western blot, in-situ hybridization, proteomics, immunohistochemistry, and histopathology. In addition, the animals lungs, spleen, liver, and thymus are being procured in the primary study and preserved for potential future analysis. There have been 124 animals used on this protocol to date. A manuscript establishing the impact of rat strain on gene expression in rejection was accepted and published in 2009. We are continuing to process and analyze data related to the metabolic effects of rejection on cellular energy metabolism; the effects of surgical inflammation over time on gene expression; and the effects of acute cellular rejection and/or infection on gene expression.