Differentiation Of Acute Rejection From Infection In Rat Heart Transplant Model

心脏移植后急性排斥反应和感染仍然是心脏移植后发病率和死亡率的主要来源，占报告死亡人数的近50%。通常很难在临床上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案是必不可少的。找出能够安全而简明地早期区分移植患者的排斥反应和感染的实验室方法将会改善结果。我们建立了ACUC方案，使我们能够研究外周血单个核细胞(PBMC)的基因芯片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议定书还允许我们进行必要的试点研究，以支持主要议定书。我们建立了成功实施和维持大鼠移植模型所必需的外科技术。我们在这个模型中确定了环孢素(CsA)的剂量，它在给药过程中可靠地抑制了排斥反应，但在停药后将允许出现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，足以在接受CsA的移植大鼠中引起肺炎和全身炎症反应，而不会立即致死。此外，我们还利用基因芯片技术研究了动物品系对排斥反应期间基因表达的影响(BMC基因组学10：280,2009)以及术后炎症变化的时间进程，以确定获取移植心脏的最佳时机(即手术炎症变化导致的基因微阵列信号消散的时间)。我们还完成了主要的研究方案。我们的主要方案结合了两个成熟的大鼠模型，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。所有大鼠在第0天接受心脏移植，同时每日皮下注射环孢素A(CsA)10 mg/kg以抑制排斥反应。移植后，动物在第6天随机停用CsA，以启动排斥反应，或继续进行，以进一步抑制排斥反应。停用环孢素A后，动物在第13天再次随机接受支气管内接种大肠杆菌或生理盐水接种。因此，研究了四组(2×2设计)：无排斥反应(即接受CsA)而无感染、无排斥反应(即接受CsA)并感染、排斥反应(即未接受CsA)无感染、排斥反应(即未接受CsA)有感染。第14天处死所有动物，取出血液和心脏进行基因芯片分析。其他可能使用的分析工具包括：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，动物的心脏、肺、脾、肝和胸腺都是在初步研究中获得的，并保存下来以供将来进行潜在的分析。在该方案于2012年关闭的整个过程中，总共使用了124只老鼠。然而，我们正在继续处理和分析与以下相关的数据：排斥对细胞能量代谢的代谢影响(JHLT 36(4S)：S372-S373,2017)；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。 在2016-17报告期间，我们与NHLBI移植基因组学实验室(LTG)进行了合作。这项合作的主要目标是开发一种处理来自福尔马林固定组织的DNA的协议，用于甲基原子测序。作为常规移植护理的一部分，肺和心脏移植受者接受定期和临床预定的活检，以监测急性排斥反应，多余的活检组织储存在福尔马林中。我们为LTG提供了10块福尔马林固定的大鼠心脏移植组织的石蜡切片。用1 mg的组织提取DNA，获得了适合下游分析的DNA产量和质量。该计划是使用脱氧核糖核酸进行亚硫酸氢盐治疗和甲基化测序，然后将表观遗传学图景与已知的心脏特异信号(https://www.genboree.org/epigenomeatlas/index.rhtml).进行比较如果这项分析产生了显著的相关性，那么使用福尔马林固定的同种异体移植心脏组织进行表观遗传学分析是可能的。最近，表观遗传学图谱已经成为研究移植相关并发症的发病机制和进展的有用工具。

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.