ET3-EDNRB Signaling in Enterocolitis Associated with Colonic Aganglionosis

描述(由申请人提供)：患有无神经节段结肠的儿童出现一系列症状(例如，严重便秘、腹泻、肠道炎症)，容易发生脓毒症，并可能危及生命。手术矫正在大多数情况下可以消除这些症状。但在高达30%的病例中，手术未能治愈这种疾病，此后，儿童患上了慢性小肠结肠炎，可能导致菌血症、彻底的败血症，甚至死亡。对于这种术后小肠结肠炎的潜在机制几乎一无所知。作为一种进行机制研究的手段，我们使用患有结肠性无神经节细胞增多症的内皮素受体B缺失的小鼠，建立了与无神经节细胞增多症相关的术后小肠结肠炎的动物模型。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，并基本上治愈了EDNRB-/-小鼠的无神经节细胞巨结肠，就像它在人类患者身上所做的那样。我们发现，尽管手术结果令人满意，但EDNRB-/-小鼠仍有相当一部分发生术后小肠结肠炎，与临床上发生在人类患者中的情况相似。在我们的研究过程中，我们偶然发现了EDNRB-/-小鼠一系列显著的免疫异常。这些异常发生在出生时或出生后不久，似乎随着年龄的增长而恶化，包括脾大小减小，脾中淋巴细胞绝对数减少，脾边缘带B细胞和B细胞/T细胞比率显著降低，胸腺退缩和骨髓异常。对缺乏内毒素-3(ET3；EDNRB受体的首选配体)的小鼠进行检查后，我们发现了非常相似的免疫异常。到目前为止，我们的初步研究与以下解释是一致的，即通过EDNRB的ET3信号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。因此，我们认为1)ET3/EDNRB信号对免疫系统的正常发育是必不可少的；2)当被破坏时，会出现两个不同的表型特征：结肠性无神经节细胞增多症，即使在成功的手术矫正后仍有明显的发展趋势，以及免疫系统表现出明显的结构和功能异常。我们怀疑这两个表型特征确实是相互关联的，我们的最终目标是理解和描述分子和细胞术语中相互关系的确切基础。这份K08申请提出了一项详细、全面的培训计划，允许申请者进行与中断的ET3/EDNRB信号相关的免疫学研究。一支优秀的导师团队将直接监督研究、培训和调查的各个方面。最终目标是申请者利用在这项拟议的调查期间获得的数据来成功地申请R01。这将允许申请者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，以及这些信号通路和基因如何与结肠神经节病和这些疾病中发生的相关小肠结肠炎有关。

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.