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: 9549442
• 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/9549442
• 关键词: Accounting; Acute; Animals; Bacteria; Biopsy; Blood; Cardiac; Caring; Cessation of life; Clinical; Collaborations; Cyclosporins; DNA; Data; Dose; Energy Metabolism; Epigenetic Process; Escherichia coli; Formalin; Future; Gene Expression; Genes; Genomics; Goals; Harvest; Heart; Heart Transplantation; Histopathology; Human; In Situ Hybridization; Infection; Inflammation; Inflammatory; Inflammatory Response; Laboratories; Liver; Lung; Lung Transplantation; Metabolic; Methods; Methylation; Microarray Analysis; Modeling; Monitor; Morbidity - disease rate; National Heart, Lung, and Blood Institute; Operative Surgical Procedures; Paraffin; Pathogenesis; Peripheral Blood Mononuclear Cell; Pilot Projects; Pneumonia; Process; Proteomics; Protocols documentation; Randomized; Rattus; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Saline; Schedule; Source; Spleen; Symptoms; Techniques; Technology; Thymus Gland; Time; Tissues; Transplant Recipients; Transplantation; Transplanted tissue; Western Blotting; allograft rejection; allotransplant; analytical tool; bisulfite; design; epigenetic profiling; heart allograft; improved outcome; indexing; 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 outcomes. We established an ACUC protocol that allowed us to study whether gene microarray analysis of peripheral blood mononuclear cells (PBMC) would reliably differentiate acute heart rejection from infection in the transplanted rat. The ACUC protocol also allowed us to do pilot studies necessary to support the main protocol. We established the surgical techniques necessary to successfully perform and maintain the rat transplant model. We determined the 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 inoculant 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 (BMC Genomics 10:280, 2009) 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 dissipating). 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 (2 by 2 design) 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, immunehistochemistry, and histopathology. In addition, the animals' hearts, lungs, spleen, liver, and thymus were procured in the primary study and preserved for potential future analysis. A total of 124 rats were used over the duration of the protocol, which was closed in 2012. However we are continuing to process and analyze data related to the metabolic effects of rejection on cellular energy metabolism (JHLT 36 (4S):S372-S373, 2017); the effects of surgical inflammation over time on gene expression; and the effects of acute cellular rejection and/or infection on gene expression. During the 2016-17 reporting period we entered into a collaboration with the Laboratory of Transplantation Genomics (LTG) of the NHLBI. The main objective of the collaboration is to develop a protocol for processing DNA derived from formalin-fixed tissue for methylatomic sequencing. As part of regular transplant care, lung and heart transplant recipients undergo regular and clinically-scheduled biopsies to monitor for acute rejection with excess biopsy tissue being stored in formalin. We provided LTG with 10 paraffin blocks of formalin-fixed rat cardiac transplant tissues from this protocol. One mg of this tissue was used for DNA isolation and suitable yields and quality of DNA has been obtained for down-stream analysis. The plan is to use the DNA for bisulfite treatment and methylation sequencing and then compare the epigenetic landscape with known cardiac specific signatures (https://www.genboree.org/epigenomeatlas/index.rhtml). If this analysis yields a significant correlation, then it may be possible to use formalin fixed human allotransplant cardiac tissue for epigenetic profiling. Recently, epigenetic profiling has emerged as a useful tool to study the pathogenesis and progression of transplant-related complications.

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化的早期区分将改善预后。我们建立了一个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可以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。 我们建立了成功执行和维护大鼠移植模型所需的手术技术。 我们在该模型中确定了环孢菌素（CSA）的剂量，该模型可靠地抑制其在给药过程中的排斥，但将允许3级拒绝在其中断时出现。我们还确定了适当的支气管内大肠杆菌细菌的接种剂，足以引起肺炎和全身性炎症反应，而不会立即在接受CSA的移植大鼠中致死。此外，我们还使用了基因微荷晶技术来研究动物菌株对拒绝期间基因表达的影响（BMC基因组学10：280，2009）和手术后炎症性变化的时间过程，以确定收集移植心脏的最适当的时间（即，何时由于经表面炎症而导致的基因微型识别何时会导致散发性易变的变化。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（2乘2设计）：没有感染没有拒绝（即接收CSA），没有感染的拒绝（即接收CSA），拒绝（即未接受CSA）没有感染和拒绝（即注意到感染的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，在初级研究中采购了动物的心，肺，脾脏，肝脏和胸腺，并保存以进行潜在的未来分析。在2012年关闭的方案期间，总共使用了124只大鼠。但是，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有关的数据（JHLT 36（4S）：S372-S373，2017）;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。 在2016 - 17年的报告期间，我们与NHLBI的移植基因组学（LTG）进行了合作。该协作的主要目的是制定一种处理源自福尔马林固定组织的DNA的方案，以进行甲基化的测序。作为常规移植护理的一部分，肺部和心脏移植受者进行了规则和临床安排的活检，以监测急性排斥，并在福尔马林中存储过多的活检组织。我们从该方案中为LTG提供了10个石蜡固定大鼠心脏移植组织的石蜡块。该组织用于DNA分离，并获得了合适的产率和DNA质量进行下游分析。该计划是使用DNA进行亚硫酸盐治疗和甲基化测序，然后将表观遗传景观与已知的心脏特异性特异性特异性（https://www.genboree.org/epigegenomeatlas/index.rhtml进行比较）。如果该分析产生显着的相关性，则可以使用福尔马林固定的人类同种异体养殖心脏组织进行表观遗传分析。最近，表观遗传分析已成为研究与移植相关并发症的发病机理和进展的有用工具。