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.

急性心脏同种异体移植排斥和感染仍然是心脏移植后发病和死亡率的重要来源，占报告死亡的近50％。通常很难在临床上区分排斥和感染，因为它们都是具有相似，非特异性症状的炎症过程。但是，这种差异对于确定治疗至关重要。识别实验室方法，可以使移植患者的排斥和感染之间的安全和简洁的早期分化将大大改善结果。我们已经建立了一种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会可靠地区分移植大鼠的急性心脏排斥与感染。 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执行和维护大鼠移植模型所需的手术技术。 我们已经在该模型中建立了一定剂量的环孢菌素（CSA），可可靠地抑制其在给药过程中的排斥，但会允许在停用后出现3级排斥。 我们还确定了适当的支气管内大肠杆菌细菌的接种，足以引起肺炎和全身性炎症反应，而不会立即在接受CSA的移植大鼠中致命。 此外，我们已经使用了基因微键技术来研究动物菌株对排斥反应和手术后炎症后的基因表达的影响，以确定收获移植心脏的最适当时间（即，由于手术炎症而导致的基因微单次签名是消散的）。 我们还完成了主要研究方案。 我们的主要协议结合了两个完善的大鼠模型，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。 所有大鼠在第0天接受心脏移植，并与每日CSA（10 mg/kg皮下）一起抑制排斥反应。移植后，在第6天随机分配动物，以使CSA停用，以引发排斥或继续，以进一步抑制排斥。停用CSA后，在第13天将动物再次随机分组，以接收临时的大肠杆菌接种或接种盐水。因此，研究了四个组：没有感染没有拒绝（即接收CSA），没有拒绝（即接受感染的CSA），拒绝（即未接受感染的CSA），而没有感染，拒绝（即未接受感染）被拒绝（即未接受CSA）。在第14天，所有动物被处死，并去除血液和心脏进行基因微阵列分析。可以使用的其他分析工具包括：RT-PCR，Western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，动物肺，脾脏，肝脏和胸腺正在主要研究中采购，并保存以进行未来的分析。 迄今为止，该方案上有124只动物。 在2009年接受并发表了一种建立大鼠菌株对基因表达的影响的手稿。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作用相关的数据；随着时间的流逝，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。