EXOSOMES: ROLE IN ALLOGRAFT REJECTION AND POTENTIAL AS A BIOMARKER

 描述(申请人提供)：肺移植(LTX)是终末期肺部疾病的一种可行的治疗选择。尽管短期存活率有所改善，但急性和慢性排斥反应仍然是器官长期功能的障碍。诊断人类LTX后的急性和慢性排斥反应仍然是一个重要的挑战。经纤维支气管镜肺活检已成为诊断急性排斥反应的金标准。然而，观察者之间有显著的可变性，高达40%的活检不足以评估 拒绝。抗体介导的排斥反应的诊断特征也有很大的不同。最后，LTX后慢性排斥反应的组织学特征是闭塞性毛细支气管炎(OB)，这是一种累及终末和呼吸性细支气管的纤维增生性病变。经纤维支气管镜肺活检是检测OB的一种不敏感的方法。因此，慢性同种异体肺移植排斥反应的诊断和分期是根据肺活量测量的减少来进行的，这是一种引起呼吸道纤维化和闭塞的下游事件。因此，进一步了解排斥反应的发生机制和确定排斥反应的特定生物标志物是至关重要的。 用于移植肺的长期功能。利用人的LTxR和慢性排斥、闭塞性呼吸道疾病(OAD)的小鼠模型，我们证明：1)在人的LTxR中，在急性细胞排斥反应(A1和A2)中可以检测到循环外切体。2)在急性细胞排斥反应早期即可在血清中检测到外切体。3)DSA的新生发育伴随着支气管肺泡灌洗液(BAL)中的外切体。4)外体含有V型胶原(Col-V)，其在肺实质中选择性表达。5)早在OAD的临床证据出现之前，局部部位(BAL)就存在外切体；6)诊断为BOS的LTxR患者的血清中也存在含有Col-V的外切体，这些外切体在BOS之前就可以在血清中检测到。基于这些发现，我们建议确定：1)供者特异性免疫反应后的外切体诱导导致肺移植排斥反应的机制；2)确定外切体发育的动力学，目的是利用血清中的外切体检测作为潜在的排斥和排斥治疗的生物标志物。为此，我们将使用超速离心法和蛋白质分离方法来确定LTX后BAL和血清中存在的外切体的动力学，并将研究结果与急性细胞排斥、急性抗体介导的和慢性排斥反应相关联。随后的实验将：a)确定外切体的组成；b)确定构成外切体的细胞；c)确定外切体中存在的人类白细胞抗原特异性信使RNA(MRNAs)是否会将供体的人类白细胞抗原转移到受体上皮细胞和内皮细胞。这是基于我们的假设，即外体将包含几个参与免疫调节的microRNAs和供体HLAmRNAs。此外，外切体中的人类白细胞抗原的信使核糖核酸可能将信息传递给受体的上皮细胞或内皮细胞，导致针对供体人类白细胞抗原的持续免疫反应，从而导致人类LTX后的慢性排斥反应。

 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.