EXOSOMES: ROLE IN ALLOGRAFT REJECTION AND POTENTIAL AS A BIOMARKER

 描述（由申请人提供）：肺移植（LTx）是终末期肺部疾病的可行治疗选择。虽然短期存活率有所提高，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。经支气管肺活检是诊断急性排斥反应的金标准。然而，观察者之间存在显著的差异，高达40%的活检不足以评估 排斥反应Ab介导的排斥反应的诊断特征也有很大差异。最后，LTx后的慢性排斥反应在组织学上以闭塞性细支气管炎（OB）为特征，这是一种涉及终末和呼吸性细支气管的纤维增生性病变。经支气管肺活检是一种不敏感的检测OB的方法。因此，慢性肺同种异体移植排斥反应的诊断和分期根据肺功能测定法测量的递减，下游事件，诱导气道纤维化和闭塞。因此，进一步了解LTx后排斥反应的机制并确定排斥反应的特异性生物标志物至关重要 移植肺的长期功能。使用人LTxR和慢性排斥、闭塞性气道疾病（OAD）的鼠模型，我们证明：1）在人LTxR中，在急性细胞排斥期间可检测到循环外泌体（A1和A2）。2)外泌体可以在急性细胞排斥反应之前在血清中检测到。3)DSA的从头发展伴随着支气管肺泡灌洗液（BAL）中的外来体。4)外泌体含有胶原V（Col-V），其已显示在肺实质中选择性表达。5)外泌体在OAD病变的临床证据之前很久就存在于局部部位（BAL）中，并且6）来自诊断为BOS的LTxR的血清也具有在BOS之前很久在血清中可检测到的含有Col-V的外泌体。基于这些发现，我们提出确定：1）供体特异性免疫应答后外泌体诱导导致肺同种异体移植物排斥的机制;和2）确定外泌体发育的动力学，目标是采用血清中的外泌体检测作为排斥和排斥治疗的潜在生物标志物。为此，我们将使用超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，并将结果与急性细胞、急性Ab介导和慢性排斥反应相关联。后续实验将; a）确定外来体的组成; B）确定对外来体有贡献的细胞;和c）确定存在于外来体中的HLA特异性信使RNA（mRNA）是否将供体HLA转移到受体上皮和内皮细胞。这是基于我们的前提，即外泌体将包含参与免疫调节的几种microRNA和供体HLA mRNA。此外，外泌体中HLA的mRNA可以将信息转移到受体的上皮或内皮细胞，导致针对供体HLA的持续免疫应答，导致人LTx后的慢性排斥。

 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.