Differentiation Of Acute Rejection From Infection In A R

AR 中急性排斥反应与感染的鉴别

• 批准号: 6825058
• 负责人: Michael A 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/6825058
• 关键词: Escherichia coli infections; artificial immunosuppression; bacterial pneumonia; cardiovascular disorder chemotherapy; cyclosporines; disease /disorder model; disease /therapy duration; heart transplantation; laboratory rat; lymphocyte; microarray technology; nonhuman therapy evaluation; respiratory infections; transplant rejection; transplantation immunology

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 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). Currently we are enrolling animals in 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 will undergo heart transplantation on day 0 in conjunction with daily CSA (10 mg/kg subcutaneous) to suppress rejection. After transplant, animals will be randomized at day 4 to have CSA discontinued, in order to initiate rejection, or continued, in order to further suppress rejection. After discontinuing CSA the animals will again be randomized on day 13 to receive intrabronchial E. coli inoculation or saline inoculation. Consequently, four groups will be 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 will be 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 will also be procured in the primary study and preserved for potential future analysis.

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，我们还使用基因微阵列技术来研究手术后炎症变化的时间过程，以确定收获移植心脏的最合适时间（即当手术炎症变化导致的基因微阵列特征消失时）。目前我们正在将动物纳入主要研究方案。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。所有大鼠将在第 0 天接受心脏移植，并结合每日 CSA（10 mg/kg 皮下注射）以抑制排斥反应。移植后，动物将在第 4 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，动物将在第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，将研究四组：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，将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，动物的肺、脾、肝和胸腺也将在初步研究中获得并保存以供将来分析。