ET3-EDNRB Signaling in Enterocolitis Associated with Colonic Aganglionosis

描述（由申请人提供）：患有神经节结的儿童表现出一系列症状（如严重便秘、腹泻、肠道炎症），易患败血症，并可能危及生命。手术矫正在大多数情况下可以根除这些症状。但在高达30%的病例中，手术无法治愈这种疾病，此后，儿童患上慢性小肠结肠炎，可能导致菌血症、彻底败血症，甚至死亡。关于这种术后小肠结肠炎的潜在机制几乎一无所知。作为一种机制研究的手段，我们使用内皮素受体b缺失的结肠神经节病小鼠建立了术后小肠结肠炎伴神经节病的动物模型。我们开发了一种新的显微外科手术，基本上与临床上进行的手术相同，基本上治愈了Ednrb-/-小鼠的神经节巨结肠，就像在人类患者身上一样。我们发现，尽管手术结果令人满意，但仍有相当比例的Ednrb- /-小鼠发生术后小肠结肠炎，类似于临床发生在人类患者中的情况。在我们的研究过程中，我们在Ednrb-/-小鼠中偶然发现了一系列惊人的免疫异常。这些异常发生在出生时或出生后不久，随着年龄的增长而恶化，包括脾脏体积缩小，脾脏淋巴细胞绝对数量减少，脾脏边缘区B细胞和B细胞/T细胞比例明显减少，胸腺退化和骨髓异常。在检查遗传缺乏内皮素-3 （ET3； Ednrb受体的首选配体）的小鼠时，我们发现了非常相似的免疫异常。到目前为止，我们的初步研究与通过Ednrb的ET3信号传导是免疫系统正常发育和/或成年动物免疫系统正常功能所必需的解释一致。因此，我们认为1)ET3/Ednrb信号对免疫系统的正常发育至关重要；2)当被破坏时，出现两种明显的表型特征：结肠神经节病，即使在成功的手术矫正后也有明显的发展成小肠结肠炎的倾向；免疫系统表现出明显的结构和功能异常。我们怀疑这两种表型特征确实是相互关联的，我们的最终目标是从分子和细胞的角度理解和描述这种相互关系的精确基础。该K08申请提出了一个详细、全面的培训计划，允许申请人进行与ET3/Ednrb信号中断相关的免疫学研究。一个优秀的导师团队将直接监督研究培训和调查的各个方面。申请人的最终目标是将在此建议调查期间获得的数据利用到成功的R01应用程序中。这将允许申请人成为一名独立资助的外科科学家，研究特定信号通路和基因在免疫发育和功能中的作用，以及它们如何与这些疾病中发生的结肠神经节病和相关小肠结肠炎联系起来。

DESCRIPTION (provided by applicant): Children with aganglionic colon present with a constellation of symptoms (e.g., severe constipation, diarrhea, intestinal inflammation) that predispose to sepsis, and can be life-threatening. Surgical correction eradicates these symptoms in most cases. But in up to 30% of cases, surgery fails to cure the disorder, and thereafter, children suffer from chronic enterocolitis that can lead to bacteremia, outright sepsis, and even death. Almost nothing is known about the underlying mechanisms of this postoperative enterocolitis. As a means to pursue mechanistic studies, we created an animal model of postoperative enterocolitis associated with aganglionosis using the Endothelin Receptor B-null mouse with colonic aganglionosis. We developed a novel microsurgical procedure that is essentially identical to the operation performed clinically, and essentially cures aganglionic megacolon in Ednrb-/- mice, just as it does in human patients. We found that a significant proportion of Ednrb- /- mice develop postoperative enterocolitis despite satisfactory surgical outcome, similar to what occurs clinically in human patients. In the course of our studies, we serendipitously discovered a spectrum of striking immune abnormalities in Ednrb-/- mice. These abnormalities occur at or shortly after birth, appear to worsen with age, and include reduced spleen size, reduced absolute numbers of lymphocytes in the spleen, marked reduction in marginal zone B cells and B cell/T cell ratio in the spleen, thymic involution, and bone marrow abnormalities. Upon examination of mice with a genetic lack of entdothelin-3 (ET3; the preferred ligand for the Ednrb receptor), we found very similar immune abnormalities. Our preliminary studies thus far are consistent with the interpretation that ET3 signaling through Ednrb is required for normal development of the immune system, and/or for normal function of the immune system in the adult animal. We therefore propose that 1) ET3/Ednrb signaling is essential for normal development of the immune system; 2) when disrupted, two distinct phenotypic features emerge: colonic aganglionosis, with a marked propensity to develop enterocolities even after successful surgical correction, and an immune system that displays marked structural and functional abnormalities. We suspect that these two phenotypic features are indeed related to one another, and our ultimate goal is to understand and describe in molecular and cellular terms the precise basis of the interrelationships. This K08 application proposes an a detailed, comprehensive training plan that will allow the applicant to pursue immunologic studies related to disrupted ET3/Ednrb signaling. An outstanding team of Mentors will directly oversee the research training and the all aspects of the investigation. The ultimate goal is for the applicant to leverage the data obtained during this proposed investigation into a successful R01 application. This will then allow the applicant to become an independently-funded surgeon-scientist investigating the role of specific signaling pathways and genes in immune development and function, and how these are linked to colonic ganglionosis and the associated enterocolitis that occurs in these diseases.