University of Maryland SOM, Baltimore, MD, USA.
GlycoMimetics, Inc, Rockville, MD, USA.
Xenotransplantation. 2018 Mar;25(2):e12381. doi: 10.1111/xen.12381. Epub 2018 Jan 23.
Alongside the need to develop more effective and less toxic immunosuppression, the shortage of human organs available for organ transplantation is one of the major hurdles facing the field. Research into xenotransplantation, as an alternative source of organs, has unveiled formidable challenges. Porcine lungs perfused with human blood rapidly sequester the majority of circulating neutrophils and platelets, which leads to inflammation and organ failure within hours, and is not significantly attenuated by genetic modifications to the pig targeted to diminish antibody binding and complement and coagulation cascade activation.
Here, we model the interaction of freshly isolated human leukocytes with xenotransplanted vasculature under physiologic flow conditions using microfluidic channels coated with porcine endothelial cells. Both isolated human neutrophils and whole human blood were perfused over transgenic pig aortic endothelial cells that had been activated with rhTNF-α or rhIL-4 using the BioFlux system. Novel compounds GMI-1271 and rPSGL1.Fc were tested as E- and P- selectin antagonists, respectively. Cellular adhesion and rolling events were tracked using FIJI (imageJ).
Porcine endothelium activated with either rhTNF-α or rhIL-4 expressed high amounts of selectins, to which isolated human neutrophils readily rolled and tethered. Both E-and P-selectin antagonism significantly reduced the number of neutrophils rolling and rolling distance in a dose-dependent manner, with near total inhibition at higher doses (P < .001). Similarly, with whole human blood, selectin blocking compounds exhibited dose-dependent inhibition of prevalent leukocyte adhesion and severe endothelial injury (Untreated: 394 ± 97 PMNs/hpf, 57 ± 6% loss EC; GMI1271+rPSGL1.Fc: 23 ± 9 PMNs/hpf, 8 ± 6% loss EC P < .01).
Selectin blockade may be useful as part of an integrated strategy to prevent neutrophil-mediated organ xenograft injury, especially during the early time points following reperfusion.
除了需要开发更有效和毒性更低的免疫抑制药物外，可供器官移植的人体器官短缺也是该领域面临的主要障碍之一。作为器官的替代来源，异种移植的研究揭示了巨大的挑战。用人血灌注的猪肺会迅速吞噬循环中的大多数中性粒细胞和血小板，导致数小时内发生炎症和器官衰竭，而针对减少抗体结合、补体和凝血级联激活的猪基因修饰并不能显著减弱这种情况。
在这里，我们使用涂有猪内皮细胞的微流控通道，在生理流动条件下模拟新鲜分离的人白细胞与异种移植血管的相互作用。使用 BioFlux 系统，将分离的人中性粒细胞和全血分别灌注到经过 rhTNF-α 或 rhIL-4 激活的转基因猪主动脉内皮细胞上。新型化合物 GMI-1271 和 rPSGL1.Fc 分别作为 E- 和 P-选择素拮抗剂进行测试。使用 FIJI（imageJ）跟踪细胞黏附和滚动事件。
用 rhTNF-α 或 rhIL-4 激活的猪内皮细胞表达大量选择素，分离的人中性粒细胞很容易在其上滚动和附着。E- 和 P-选择素拮抗剂均以剂量依赖性方式显著减少中性粒细胞滚动的数量和滚动距离，高剂量时几乎完全抑制（P <.001）。同样，用全血，选择素阻断化合物表现出剂量依赖性抑制常见白细胞黏附和严重内皮损伤（未处理：394 ± 97 PMNs/hpf，57 ± 6% EC 丢失；GMI1271+rPSGL1.Fc：23 ± 9 PMNs/hpf，8 ± 6% EC 丢失 P <.01）。
选择素阻断可能是预防中性粒细胞介导的器官异种移植损伤的综合策略的一部分，特别是在再灌注后的早期时间点。
