Dissecting anti-ceramide scFv vascular mitigation of the Radiation GI Syndrome

项目摘要/摘要 目前还没有针对急性放射性胃肠道综合征(RGS)的有效对策。在此建议的研究 将为我们正在进行的开发抗神经酰胺抗体的努力提供支持，作为一种基于机制的方法 减轻RGS的致命性。RGS的原因是隐窝/绒毛单位的破坏，粘膜完整性的丧失，以及 肠道细菌菌群感染。最近的数据表明，RGS的病理生理学涉及一种 位于Lieberkühn称为隐窝底部的小肠干细胞(ISCs)池 柱状细胞(CBCs)。我们最近发现CBCs比转运扩增的祖细胞更耐辐射。 或分化的绒毛细胞，通过同源重组有效地保护自己。此外，CBC 耗竭是双相电离后辐射(IR)，因为细胞凋亡发生在生长停滞的前24小时， 紧随其后的是有丝分裂死亡，在24-48小时内，一旦CBCs开始重新启动，就会进入快速再生期 骑自行车。在2.5天之间，由于细胞凋亡和有丝分裂死亡导致的CBC耗尽先于隐窝的物理溶解。 3.0发布IR。此外，第2天的CBC存活率预测两个隐窝在第3.5天再生，由 威瑟斯和艾尔金德的克隆形成试验和RGS致死率。除了对ISC的直接破坏外，我们的实验室还建议 IR对小肠微血管的损伤对预后有显著影响。具体来说，我们 建议IR使酸性鞘磷脂酶(ASMase)在分钟内释放到外层内皮细胞血浆 膜，在那里它找到底物鞘磷脂并产生促凋亡的第二信使 神经酰胺。神经酰胺在内皮细胞表面组装一个信号平台，介导细胞凋亡，并且 微血管损伤和直接损伤共同决定了ISC的存活率。此外，我们 最近报道了我们的抗神经酰胺2a2抗体与照射内皮细胞表面的神经酰胺结合，防止 形成内皮细胞死亡所需的富含神经酰胺的平台，从而保护小鼠免受RGS的伤害 杀伤力。在未发表的研究中，我们发现血管内皮细胞凋亡的第二波延长发生在高密度脂蛋白之后。 持续4天的剂量IR伴随ASMase分泌进入小鼠的体循环 表明持续的血管损伤/功能障碍。在这里，我们将在三个具体目标中详细说明这一血管 综合征，确定其在CBC死亡IR后的作用，并表明单链可变区(ScFv)的 皮下注射抗神经酰胺抗体。减轻它，减轻CBC的消耗，提高小鼠的存活率。vbl.使用 新的遗传和生物工具，我们将正式展示单链抗体通过抑制神经酰胺来减轻RGS 在内皮细胞上生成，但不在肿瘤实质细胞上生成。一旦ScFv缓解的机制是 我们将把它与IL-22结合起来，IL-22是我们最近展示的一种促进IR后CBC再生的药物。 目前缺乏关于小肠辐射后立即环境的信息。 粘膜。这里的研究应该填补关于微血管损伤对演变中的粘膜损伤的影响的空白 并增加我们的知识，以优化抗神经酰胺策略，以减轻RGS的致命性。

Project Summary/Abstract There are no effective countermeasures for the acute Radiation GI Syndrome (RGS). Studies proposed here will provide support for our ongoing effort to develop an anti-ceramide Ab as a mechanism-based approach to mitigate RGS lethality. The RGS results from destruction of crypt/villus units, loss of mucosal integrity, and infection by resident enterobacterial flora. Recent data indicate RGS pathophysiology involves depletion of a pool of small intestinal stem cells (ISCs) residing at the bottom of the Crypts of Lieberkühn termed crypt base columnar cells (CBCs). We recently showed CBCs to be more radioresistant than transit amplifying progenitors or differentiated villus cells, protecting themselves effectively by homologous recombination. Further, CBC depletion is biphasic post-ionizing radiation (IR) as apoptosis occurs in the first 24h during growth arrest, followed by mitotic death at 24-48h during the rapid regenerative phase that ensues once CBCs begin re- cycling. CBC depletion by apoptosis and mitotic death precede physical dissolution of crypts between Day 2.5- 3.0 post IR. Further, CBC survival at Day 2 predicts both crypt regeneration at Day 3.5 measured by the Clonogenic Assay of Withers and Elkind, and RGS lethality. In addition to direct ISC damage, our lab proposed that IR-induced injury to small intestinal microvasculature plays a prominent role in outcome. Specifically, we propose IR causes release within min of acid sphingomyelinase (ASMase) to the outer endothelial plasma membrane, where it finds its substrate sphingomyelin and generates the pro-apoptotic second messenger ceramide. Ceramide assembles a signaling platform on the endothelial surface that mediates apoptosis, and the coupling of microvessel injury to direct ISC damage coordinately determines ISC survival. Further, we recently reported our anti-ceramide 2A2 Ab binds ceramide on the irradiated endothelial surface, preventing formation of ceramide-rich platforms required for endothelial death, thereby protecting mice against RGS lethality. In unpublished studies, we show a 2nd prolonged wave of endothelial apoptosis occurs post high dose IR accompanied by ASMase secretion into the systemic circulation of mice that persists for 4 days indicative of ongoing vascular damage/dysfunction. Here, in 3 specific aims we will detail this vascular syndrome, determine its role in CBC demise post IR, and show that a single chain variable fragment (scFv) of anti-ceramide Ab injected s.c. attenuates it, mitigating CBC depletion and improving mouse survival. Using new genetic and biologic tools we will formally show that the scFv mitigates the RGS by inhibiting ceramide generated on endothelial but not tumor parenchymal cells. Once the mechanism of scFv mitigation is delineated we will combine it with IL-22, an agent recently shown by us to improve CBC regeneration after IR. There is currently a dearth of information regarding the immediate post-radiation milieu of the small intestinal mucosa. Studies here should fill in gaps regarding impact of microvascular damage on evolving mucosal injury and increase our knowledge to optimize anti-ceramide strategies to mitigate RGS lethality.