Genomic Research Alliance for Transplantation (GRAfT), Division of Intramural Research, National Institutes of Health, Bethesda, Maryland; Department of Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland; Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.
Cardiothoracic Surgery Research Program, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.
J Heart Lung Transplant. 2018 Aug;37(8):967-975. doi: 10.1016/j.healun.2018.04.009. Epub 2018 Apr 26.
Observational studies suggest that cell-free DNA (cfDNA) is a biomarker of tissue injury in a range of conditions including organ transplantation. However, the lack of model systems to study cfDNA and its relevance to tissue injury has limited the advancements in this field. We hypothesized that the predictable course of acute humoral xenograft rejection (AHXR) in organ transplants from genetically engineered donors provides an ideal system for assessing circulating cfDNA as a marker of tissue injury.
Genetically modified pig donor hearts were heterotopically transplanted into baboons (n = 7). Cell-free DNA was extracted from pre-transplant and post-transplant baboon plasma samples for shotgun sequencing. After alignment of sequence reads to pig and baboon reference sequences, we computed the percentage of xenograft-derived cfDNA (xdcfDNA) relative to recipient by counting uniquely aligned pig and baboon sequence reads.
The xdcfDNA percentage was high early post-transplantation and decayed exponentially to low stable levels (baseline); the decay half-life was 3.0 days. Post-transplantation baseline xdcfDNA levels were higher for transplant recipients that subsequently developed graft loss than in the 1 animal that did not reject the graft (3.2% vs 0.5%). Elevations in xdcfDNA percentage coincided with increased troponin and clinical evidence of rejection. Importantly, elevations in xdcfDNA percentage preceded clinical signs of rejection or increases in troponin levels.
Cross-species xdcfDNA kinetics in relation to acute rejection are similar to the patterns in human allografts. These observations in a xenotransplantation model support the body of evidence suggesting that circulating cfDNA is a marker of tissue injury.
观察性研究表明，无细胞 DNA（cfDNA）是包括器官移植在内的多种情况下组织损伤的生物标志物。然而，缺乏研究 cfDNA 及其与组织损伤相关性的模型系统限制了该领域的进展。我们假设，基因工程供体器官移植中可预测的急性体液性异种移植物排斥（AHXR）过程为评估循环 cfDNA 作为组织损伤标志物提供了理想的系统。
将基因修饰的猪供体心脏异位移植到狒狒（n=7）体内。从移植前和移植后狒狒血浆样本中提取无细胞 DNA，进行鸟枪法测序。在将序列读数与猪和狒狒参考序列对齐后，我们通过计算唯一对齐的猪和狒狒序列读数来计算相对于受者的异种移植物衍生的 cfDNA（xdcfDNA）的百分比。
移植后早期 xdcfDNA 百分比较高，并呈指数衰减至低稳定水平（基线）；衰减半衰期为 3.0 天。与未排斥移植物的 1 只动物相比，随后发生移植物丧失的移植受者的移植后基线 xdcfDNA 水平更高（3.2%比 0.5%）。xdcfDNA 百分比升高与肌钙蛋白升高和排斥的临床证据一致。重要的是，xdcfDNA 百分比升高先于排斥的临床迹象或肌钙蛋白水平升高。
与急性排斥相关的跨物种 xdcfDNA 动力学与人类同种异体移植物的模式相似。在异种移植模型中的这些观察结果支持了循环 cfDNA 是组织损伤标志物的证据。
