Differentiation Of Acute Rejection From Infection In Rat Heart Transplant Model

大鼠心脏移植模型中感染急性排斥反应的鉴别

• 批准号: 8565289
• 负责人: Michael Solomon
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8565289
• 关键词: Accounting; Acute; Animals; Bacteria; Blood; Cessation of life; Cyclosporins; Data Analyses; Dose; Energy Metabolism; Escherichia coli; Future; Gene Expression; Genes; Goals; Harvest; Heart; Heart Transplantation; Histopathology; Immunohistochemistry; In Situ Hybridization; Infection; Inflammation; Inflammatory; Inflammatory Response; Laboratories; Liver; Lung; Manuscripts; Metabolic; Methods; Microarray Analysis; Modeling; Morbidity - disease rate; Operative Surgical Procedures; Outcome; Peripheral Blood Mononuclear Cell; Pilot Projects; Pneumonia; Process; Proteomics; Protocols documentation; Publishing; Randomized; Rat Strains; Rattus; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Saline; Source; Spleen; Symptoms; Techniques; Technology; Thymus Gland; Time; Transplant Recipients; Transplantation; Western Blotting; allograft rejection; heart allograft; improved; mortality; subcutaneous; tool

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.

急性同种异体心脏移植排斥和感染仍然是心脏移植后发病和死亡的重要来源，占报告死亡的近 50%。临床上通常很难区分排斥和感染，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。确定能够安全、简洁地早期区分移植患者排斥和感染的实验室方法将大大改善结果。我们建立了 ACUC 方案，使我们能够研究外周血单核细胞 (PBMC) 的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和感染。 ACUC 协议还允许我们进行支持主要协议所需的试点研究。 迄今为止，我们已经建立了成功进行和维持大鼠移植模型所需的手术技术。 我们在该模型中确定了环孢素 (CSA) 的剂量，该剂量可以在给药期间可靠地抑制排斥反应，但在停药后会出现 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物，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，我们还利用基因微阵列技术来研究动物品系对排斥过程中基因表达的影响以及术后炎症变化的时间进程，以确定收获移植心脏的最佳时机（即当基因微阵列特征发生时）。手术炎症变化正在消失）。 我们还完成了主要研究方案。 我们的主要方案结合了两种成熟的大鼠模型，第一个是异位心脏移植模型，第二个是大肠杆菌肺部感染模型。 所有大鼠均在第 0 天接受心脏移植，并每日联合使用 CSA（10 mg/kg 皮下注射）以抑制排斥反应。移植后，动物在第 6 天随机停止 CSA，以引发排斥反应，或继续 CSA，以进一步抑制排斥反应。停止CSA后，动物在第13天再次随机接受支气管内大肠杆菌接种或盐水接种。因此，研究了四组：无感染的无排斥（即接受 CSA）、无感染的排斥（即接受 CSA）、无感染的排斥（即不接受 CSA）以及感染的排斥（即不接受 CSA）。第14天，处死所有动物并取出血液和心脏用于基因微阵列分析。可以使用的其他分析工具包括：RT-PCR、蛋白质印迹、原位杂交、蛋白质组学、免疫组织化学和组织病理学。此外，初步研究中还获取了动​​物的肺、脾、肝和胸腺，并保存起来以供将来分析。 迄今为止，该方案已使用 124 只动物。 一份确定大鼠品系对排斥反应中基因表达影响的手稿已于 2009 年被接受并发表。我们正在继续处理和分析与排斥反应对细胞能量代谢的代谢影响相关的数据；随着时间的推移，手术炎症对基因表达的影响；以及急性细胞排斥和/或感染对基因表达的影响。