PDF(Pubmed)
Abstract：
UNASSIGNED: The standard treatment for preventing rejection in vascularized composite allotransplantation (VCA) currently relies on systemic immunosuppression, which exposes the host to well-known side effects. Locally administered immunosuppression strategies have shown promising results to bypass this hurdle. Nevertheless, their progress has been slow, partially attributed to a limited understanding of the essential mechanisms underlying graft rejection. Recent discoveries highlight the crucial involvement of innate immune components, such as neutrophil extracellular traps (NETs), in organ transplantation. Here we aimed to prolong graft survival through a tacrolimus-based drug delivery system and to understand the role of NETs in VCA graft rejection.
UNASSIGNED: To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus.
UNASSIGNED: Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus.
UNASSIGNED: Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target.
UNASSIGNED: To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus.
UNASSIGNED: Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus.
UNASSIGNED: Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target.
摘要：
■在血管化复合同种异体移植（VCA）中预防排斥反应的标准治疗目前依赖于全身免疫抑制，这会使宿主暴露于众所周知的副作用。局部施用的免疫抑制策略已经显示出绕过这一障碍的有希望的结果。然而，他们进展缓慢，部分归因于对移植物排斥的基本机制的有限理解。最近的发现强调了先天免疫成分的关键参与，如中性粒细胞胞外诱捕网(NET)，在器官移植中。在这里，我们旨在通过基于他克莫司的药物递送系统延长移植物存活，并了解NETs在VCA移植物排斥中的作用。
■为了防止脱靶毒性并促进移植物存活，我们在多重MHC不匹配的猪VCA模型中测试了局部给药的他克莫司加载的按需给药系统(TGMS-TAC).在组织和血液中评估脱靶毒性。移植排斥是宏观评估的，而补体系统，T细胞,通过免疫荧光和/或蛋白质印迹分析移植组织中的中性粒细胞和NETs。使用Luminex磁珠猪小组测量炎症细胞因子的血浆水平，使用DNA-MPOELISA测量血浆和组织中的NETs。最后,为了评估他克莫司对网状结构的影响，在与他克莫司孵育后,在猪和人外周嗜中性粒细胞中体外诱导NETs。
■TGMS-TAC的移植物内重复给药可使全身毒性最小化并延长移植物存活。然而，在终点观察到排斥反应的迹象.系统地，细胞因子水平没有增加，补体过敏毒素，T细胞亚群,或排斥时的嗜中性粒细胞。然而，组织分析显示T细胞和中性粒细胞的局部浸润，连同中性粒细胞胞外陷阱（NET）在排斥移植物中。有趣的是,移植物内施用他克莫司有助于减少T细胞浸润和NETs。事实上,体外NETosis评估显示,他克莫司治疗刺激中性粒细胞后,NETs减少62-84%.
■我们的数据表明，在多MHC-错配VCA模型中，拟议的免疫抑制局部递送避免了脱靶毒性，同时延长了移植物的存活时间。此外，发现NETs在移植物排斥中起作用，因此可能是潜在的创新治疗靶标。
■为了防止脱靶毒性并促进移植物存活，我们在多重MHC不匹配的猪VCA模型中测试了局部给药的他克莫司加载的按需给药系统(TGMS-TAC).在组织和血液中评估脱靶毒性。移植排斥是宏观评估的，而补体系统，T细胞,通过免疫荧光和/或蛋白质印迹分析移植组织中的中性粒细胞和NETs。使用Luminex磁珠猪小组测量炎症细胞因子的血浆水平，使用DNA-MPOELISA测量血浆和组织中的NETs。最后,为了评估他克莫司对网状结构的影响，在与他克莫司孵育后,在猪和人外周嗜中性粒细胞中体外诱导NETs。
■TGMS-TAC的移植物内重复给药可使全身毒性最小化并延长移植物存活。然而，在终点观察到排斥反应的迹象.系统地，细胞因子水平没有增加，补体过敏毒素，T细胞亚群,或排斥时的嗜中性粒细胞。然而，组织分析显示T细胞和中性粒细胞的局部浸润，连同中性粒细胞胞外陷阱（NET）在排斥移植物中。有趣的是,移植物内施用他克莫司有助于减少T细胞浸润和NETs。事实上,体外NETosis评估显示,他克莫司治疗刺激中性粒细胞后,NETs减少62-84%.
■我们的数据表明，在多MHC-错配VCA模型中，拟议的免疫抑制局部递送避免了脱靶毒性，同时延长了移植物的存活时间。此外，发现NETs在移植物排斥中起作用，因此可能是潜在的创新治疗靶标。
