Host-Pathogen Interactions From Measurements of Urinary Cell Free DNA in Kidney Transplantation

肾移植中尿细胞游离 DNA 测量的宿主-病原体相互作用

• 批准号: 9375180
• 负责人: Iwijn De Vlaminck
• 金额: $ 24.87万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-26 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9375180
• 关键词: Allografting; Antibiotic Therapy; Bacteria; Bacterial Genome; Biological Assay; Cells; Clinical; Complication; DNA Library; Data; Data Set; Development; End stage renal failure; Evaluation; Gene Expression; Genes; Genetic Fingerprintings; Genomics; Goals; Gold; Growth; Immune response; Immunocompromised Host; Immunologics; Incidence; Infection; Injury; Kidney Transplantation; Longevity; Measurement; Messenger RNA; Metagenomics; Monitor; Morbidity - disease rate; Multi-Drug Resistance; Natural Immunity; Nucleosomes; Pathogenesis; Patients; Pilot Projects; Population; Preparation; Recruitment Activity; Recurrence; Regulator Genes; Replication Origin; Resolution; Sampling; Sequence Analysis; Shotgun Sequencing; Shotguns; Single-Stranded DNA; Specimen; Structure; Study Section; Symbiosis; Techniques; Testing; Time; Tissues; Transcription Initiation Site; Transplant Recipients; Transplantation; Urinalysis; Urinary tract infection; Urine; Visit; Work; adaptive immunity; base; biobank; cell free DNA; cell injury; co-infection; cohort; diagnosis standard; differential expression; microbiome; microbiota; mortality; novel; pathogen; promoter; resistance mutation; response; tool; urinary

Kidney transplantation is the preferred treatment option for end stage kidney disease, but infectious and immunological complications limit the lifespan of kidney transplants. Urinary tract infections (UTI) occur at an alarmingly high rate after kidney transplantation and are associated with urosepsis, allograft loss, and mortality. The current gold standard for diagnosis of UTI is bacterial culture. Culture-based tests are, however, limited by their ability to detect only culturable bacteria, do not inform about co-infections and commensal microbiota, do not inform about bacterial growth dynamics, and do not inform about the host's response to infection. The goal of this proposal is to invent and apply genomic assays of urinary cell-free DNA that can simultaneously inform about the composition of the urinary microbiome, growth dynamics of pathogens associated with UTI, tissue injury in the graft, and the host's immune response to infection. A unique opportunity for this work is our large cohort of over 250 kidney transplant recipients in which we have serial biobanked urine specimens as well as routine conventional urine cultures in the first 3 months of transplantation. Using a newly developed single-stranded DNA library preparation technique, we will profile 290 urinary samples for cfDNA: i) serial samples from transplant recipients with UTI (prior to, during, and after UTI) ii) serial samples from recipients with recurrent UTIs; iii) samples from recipients with asymptomatic UTI and negative urinanalysis; and iv) samples from recipients who do not develop UTI in the first 3 months of transplantation. We will profile the dynamic urinary microbiome prior to, during, and after UTI in kidney transplantation. We will investigate whether cfDNA profiles are associated with UTI as well as whether they can anticipate UTI, UTI recurrence, and multi-drug resistant UTI. Based on the concept that bacterial genome replication rates and growth dynamics can be estimated from analyses of metagenomic sequence coverage, we will estimate growth dynamics over time and in response to antibiotic therapy in patients with UTI and recurrent UTI. We will monitor the host response to UTI via analyses of nucleosome footprints comprised within cfDNA and via donor specific cfDNA. Our pilot studies indicate that i) the structures of nucleosomal arrays in transcriptional regulatory elements are preserved and comprised within urinary cfDNA, and ii) that an analysis of nucleosomal occupancy in gene promoters can be used to quantify gene expression. We will investigate the differential host's response in subjects who have symptomatic UTI with positive urinalysis and asymptomatic UTI with negative urinalysis and the differential host's response in UTI patients after antibiotic treatment. We will assess tissue injury to the allograft in the scope of UTI, using donor specific cfDNA as a marker of allograft injury. Successful implementation of this proposal will provide a novel understanding of the pathogenesis of UTI as well as open new avenues for the utilization of cfDNA profiling as a sensitive and noninvasive transplant monitoring tool.

肾移植是终末期肾病的首选治疗选择，但具有传染性和 免疫并发症限制了肾移植的寿命。尿路感染（UTI）发生在 肾移植后的高发病率令人担忧，并与尿脓毒症、同种异体移植物丢失和死亡率相关。 目前诊断UTI的金标准是细菌培养。然而，基于文化的测试受到以下限制： 他们只检测可培养细菌的能力，不告知合并感染和肠道微生物群， 不告知细菌生长动力学，也不告知宿主对感染的反应。 本提案的目标是发明和应用尿游离DNA的基因组测定， 同时告知尿液微生物组的组成，病原体的生长动力学 与UTI、移植物中的组织损伤以及宿主对感染的免疫反应相关。一个独特 这项工作的机会是我们超过250名肾移植受者的大型队列，我们在其中进行了连续的 在移植的前3个月，生物库的尿液样本以及常规的常规尿液培养。 利用新开发的单链DNA文库制备技术，我们将对290份尿液样本进行分析 对于cfDNA：i）来自具有UTI的移植受体的系列样品（在UTI之前、期间和之后） iii）来自具有无症状UTI和阴性尿分析的接受者的样品; 和iv）来自在移植的前3个月内未发生UTI的受体的样品。 我们将分析肾移植中UTI之前、期间和之后的动态尿液微生物组。 我们将研究cfDNA谱是否与UTI相关以及它们是否可以预测UTI， UTI复发和多药耐药UTI。基于细菌基因组复制速率和 生长动力学可以从宏基因组序列覆盖率的分析中估计，我们将估计生长 在UTI和复发性UTI患者中随时间推移的动力学和对抗生素治疗的反应。 我们将通过分析cfDNA中包含的核小体足迹来监测宿主对UTI的反应。 以及通过供体特异性cfDNA。我们的初步研究表明，i）核小体阵列的结构， 转录调控元件被保存并包含在尿cfDNA内，和ii）分析 基因启动子中核小体占有率的测定可用于定量基因表达。我们将调查 尿分析阳性的症状性UTI受试者和无症状性UTI受试者的不同宿主应答 尿路感染患者抗生素治疗后尿常规阴性和不同的宿主反应。我们将 使用供体特异性cfDNA作为同种异体移植物损伤的标志物，在UTI范围内评估同种异体移植物的组织损伤。 这一建议的成功实施将提供一个新的理解尿路感染的发病机制以及 作为利用cfDNA谱分析作为敏感和非侵入性移植监测的新途径， 工具.