EXOSOMES: ROLE IN ALLOGRAFT REJECTION AND POTENTIAL AS A BIOMARKER

外泌体：在同种异体移植排斥中的作用以及作为生物标志物的潜力

• 批准号: 9007346
• 负责人: THALACHALLOUR MOHANAKUMAR
• 金额: $ 21.09万
• 依托单位: ST. JOSEPH'S HOSPITAL AND MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-15 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9007346
• 关键词: Acute; Antibodies; Area; Binding; Biological Markers; Biopsy; Biopsy Specimen; Blood Circulation; Body Fluids; Bronchiolitis; Bronchiolitis Obliterans; Bronchoalveolar Lavage Fluid; Cell Transplants; Cells; Characteristics; Chronic; Chronic Obstructive Airway Disease; Classification; Clinical; Collagen; Consensus; Cystic Fibrosis; Data; Detection; Development; Diagnosis; Diagnostic; Disease; Endothelial Cells; Epithelial; Epithelial Cells; Evaluation; Event; Extracellular Matrix Proteins; Fibrosis; Gene Expression; Goals; Gold; Hamman-Rich syndrome; Health; Histologic; Human; Immune; Immune response; Interobserver Variability; Intraobserver Variability; Kinetics; Lead; Lesion; Leukocytes; Lung; Lung Transplantation; Lung diseases; Lymphocyte; Measurement; Mediating; Messenger RNA; Methods; MicroRNAs; Molecular Profiling; Organ; Organ Transplantation; Pathology; Phenotype; Proteins; Reader; Regulation; Resistance; Role; Serum; Site; Spirometry; Staging; Structure of parenchyma of lung; Structure of respiratory bronchiole; Syndrome; Terminal Bronchiole; Testing; Transplant Recipients; Ultracentrifugation; allograft rejection; base; diagnosis standard; exosome; improved; insight; lung allograft; mouse model; novel marker; potential biomarker; research study; selective expression; specific biomarkers

 DESCRIPTION (provided by applicant): Lung transplantation (LTx) is a viable treatment option for end-stage lung diseases. Although short-term survival has improved, acute and chronic rejection remains as hurdles for long-term function of the organ. Diagnosis of both acute and chronic rejection following human LTx remains an important challenge. Transbronchial lung biopsy has been the gold standard for the diagnosis of acute rejection. However, there is significant interobserver variability, and up to 40% of biopsies are insufficient for evaluation of rejection. Diagnostic features for Ab mediated rejection also varies widely. Lastly, chronic rejection after LTx is characterized histologically by obliterative bronchiolitis (OB), a fibroproliferative lesion involving terminal and respiratory bronchioles. Transbronchial lung biopsy is an insensitive method for the detection of OB. Therefore, chronic lung allograft rejection is diagnosed and staged according to decrements in spirometry measurements, a downstream event that induces airway fibrosis and obliteration. Therefore, further insights into the mechanisms of rejection and identifying specific biomarkers for rejection after LTx is critical for long term function of the transplanted lungs. Using both human LTxR and a murine model of chronic rejection, obliterataive airway disease (OAD), we demonstrated that: 1) In human LTxR, circulating exosomes are detectable during acute cellular rejection (A1 and A2). 2) Exosomes can be detected in the sera earlier to acute cellular rejection. 3) De novo development of DSA is accompanied by exosomes in the bronchoalveolar lavage fluid (BAL). 4) Exosomes contained Collagen V (Col-V) which has been shown to be selectively expressed in the lung parenchyma. 5) Exosomes are present in the local site (BAL) much before clinical evidences of OAD lesions, and 6) Sera from LTxR diagnosed with BOS also have Col-V containing exosomes which are detectable in the sera much before BOS. Based on these findings, we propose to determine; 1) The mechanisms by which exosome induction following donor specific immune responses lead to lung allograft rejection; and 2) Determine the kinetics of exosome development with the goal to employ exosome detection in the sera as a potential biomarker for rejection and treatment of rejection. Towards this, we will determine the kinetics of exosomes present in the BAL and serum following LTx using ultracentrifugation and protein isolation methods and correlate the findings with acute cellular, acute Ab mediated, and chronic rejection. Subsequent experiments will; a) define the composition of exosomes; b) determine the cells contributing to the exosome; and c) determine whether HLA specific messenger RNA (mRNA) present in the exosomes will transfer the donor HLA to recipient epithelial and endothelial cells. This is based on our premise that the exosomes will contain several microRNAs involved in immune regulation and donor HLA mRNA. Further, mRNA for HLA in the exosomes may transfer the message to the recipient's epithelial or endothelial cells leading to continued immune responses against the donor HLA resulting in chronic rejection following human LTx.

 描述（由申请人提供）：肺移植（LTx）是终末期肺病的可行治疗选择。尽管短期生存有所改善，但急性和慢性排斥反应仍然是器官长期功能的障碍。人类 LTx 后急性和慢性排斥反应的诊断仍然是一个重要的挑战。经支气管肺活检一直是诊断急性排斥反应的金标准。然而，观察者之间存在显着差异，高达 40% 的活检不足以评估 拒绝。 Ab 介导的排斥反应的诊断特征也有很大差异。最后，LTx 后的慢性排斥在组织学上以闭塞性细支气管炎 (OB) 为特征，这是一种累及终末细支气管和呼吸细支气管的纤维增殖性病变。经支气管肺活检是一种不敏感的 OB 检测方法。因此，慢性肺同种异体移植排斥反应是根据肺活量测量的减少来诊断和分期的，肺活量测量是诱导气道纤维化和闭塞的下游事件。因此，进一步深入了解 LTx 后的排斥机制并确定排斥反应的特定生物标志物至关重要 为了移植肺的长期功能。使用人类 LTxR 和慢性排斥、闭塞性气道疾病 (OAD) 的小鼠模型，我们证明：1) 在人类 LTxR 中，在急性细胞排斥期间可检测到循环外泌体（A1 和 A2）。 2) 可以在急性细胞排斥反应发生之前在血清中检测到外泌体。 3）DSA的从头发育伴随着支气管肺泡灌洗液（BAL）中的外泌体。 4) 外泌体含有 V 型胶原蛋白 (Col-V)，已被证明在肺实质中选择性表达。 5) 外泌体早在 OAD 病变的临床证据之前就存在于局部部位 (BAL)，并且 6) 来自诊断为 BOS 的 LTxR 的血清也含有含有 Col-V 的外泌体，这些外泌体早在 BOS 之前就可以在血清中检测到。根据这些发现，我们建议确定； 1）供体特异性免疫反应后外泌体诱导导致肺同种异体移植排斥的机制； 2) 确定外泌体发育动力学，目标是利用血清中的外泌体检测作为排斥反应和排斥反应治疗的潜在生物标志物。为此，我们将使用超速离心和蛋白质分离方法确定 LTx 后 BAL 和血清中存在的外泌体的动力学，并将这些结果与急性细胞、急性 Ab 介导的和慢性排斥反应相关联。随后的实验将； a) 定义外泌体的组成； b) 确定对外泌体有贡献的细胞； c) 确定外泌体中存在的 HLA 特异性信使 RNA (mRNA) 是否会将供体 HLA 转移至受体上皮细胞和内皮细胞。这是基于我们的假设，即外泌体将含有几种参与免疫调节的 microRNA 和供体 HLA mRNA。此外，外泌体中 HLA 的 mRNA 可能会将信息传递给受体的上皮或内皮细胞，导致针对供体 HLA 的持续免疫反应，从而导致人类 LTx 后的慢性排斥反应。