Isolation, Identification and Characterization of a Toxin Causing Biliary Atresia

描述(申请人提供)：胆道闭锁(BA)是一种纤维炎症性疾病，是新生儿胆汁淤积的主要原因，也是儿科人群中肝移植最常见的适应症。虽然流行病学数据表明BA是由遗传风险因素和环境暴露相互作用引起的，但其病因尚不清楚。动物模型有限，而最著名的动物模型，即新生BALB/c小鼠感染恒河猴轮状病毒(RRV)，提供了关于该疾病免疫学方面的重要信息，但对研究该疾病快速进展的纤维化特征用处较小。对BA发病机制的洞察来自于对这种疾病的自然发生的动物模型的研究。在过去的40年里，澳大利亚发生了三次与怀孕的绵羊和奶牛摄入植物Dysphania glomulifera有关的新生家畜BA疫情。受影响的羔羊和小牛的临床和病理结果与人类BA有惊人的相似之处，特别是在诊断时明显的纤维化。我们假设，确定致病毒素将能够识别最终导致脊椎动物BA和纤维化的一般胆管细胞损伤途径。因此，我们从受最近疫情影响的澳大利亚牧场收集和进口了金线莲，并采用了斑马鱼生物测定方法来连续对该植物进行细分和鉴定有效部位。我们有几个组分的复杂性为2-20，值得注意的是，导致斑马鱼幼体在胆管形态发生后暴露的类似BA的损伤模式(胆道闭锁和肝外胆管树)，类似于人类BA。因此，这项建议的总体目标是确定胆汁毒素在呼吸困难中的作用，表征其诱导的胆管细胞损伤途径，并建立新的动物模型，作为了解人类BA发病机制的一种手段。这将通过三个特定的目的来实现：1.利用斑马鱼体内生物测定来鉴定金线虫胆汁毒素；2.鉴定斑马鱼毒素处理的斑马鱼仔鱼肝外胆管损伤的分子标记和基因；3.在哺乳动物模型中鉴定和表征金线虫毒素诱导的损伤途径。 公共卫生相关性：该提案的目标是确定导致家畜胆道闭锁的植物中的毒素。胆道闭锁是儿科人群中一种主要但知之甚少的肝病。Disphania毒素将被用于开发新的胆道闭锁模型，包括斑马鱼和小鼠的模型，并确定毒素诱导的胆道闭锁细胞受损的信号通路。

DESCRIPTION (provided by applicant): Biliary atresia (BA) is a fibro inflammatory disorder that is the leading cause of neonatal cholestasis and the most common indication for liver transplant in the pediatric population. Although epidemiologic data suggest that BA arises from the interplay of genetic risk factors coupled with environmental exposures, the etiology is unknown. Animal models are limited, and the best-known animal model of the disease, the infection of newborn BALB/c mice with rhesus rotavirus (RRV), has provided important information about immunological aspects of the disease but has been less useful for studying the rapidly progressive fibrosis characteristic of the disease. Insight into the pathogenesis of BA comes from the study of a naturally-occurring animal model of the disease. Over the last 40 years, there have been three epidemics of BA in newborn livestock in Australia associated with ingestion of the plant Dysphania glomulifera by pregnant sheep and cows. Clinical and pathological findings from the affected lambs and calves show striking similarities with human BA, in particular marked fibrosis at the time of diagnosis. We hypothesize that determining the causative toxin will enable the identification of general cholangiocyte damage pathways that lead ultimately to BA and fibrosis in vertebrates. We have thus collected and imported D. glomulifera from the Australian pasture affected by the most recent epidemic and have employed a zebrafish bioassay to successively subfractionate the plant and identify active fractions. We have several fractions with a complexity of 2-20 that, remarkably, cause a BA-like pattern of injury (with atresia of the gallbladder and extrahepatic biliary tree) in zebrafish larvae exposed after biliary morphogenesis has occurred, mimicking human BA. The overall goal of this proposal is therefore to identify the biliary toxins in Dysphania, characterize the cholangiocyte damage pathways they induce, and establish new animal models as a means of understanding the pathogenesis of human BA. This will be achieved through three specific aims: 1. to identify D. glomulifera biliary toxins using the in vivo zebrafish bioassay~ 2. To identify molecular markers and genes that regulate extrahepatic biliary injury in zebrafish larvae treated with D. glomulifera toxins~ and 3. To identify and characterize damage pathways induced by D. glomulifera toxins in mammalian models. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to identify the toxins in the plant Dysphania glomulifera that are responsible for biliary atresia in livestock. Biliary artesian is a major but poorly understood liver disease in the pediatric population. Dysphania toxins will be used to develop new models of biliary atresia, including models in zebrafish and mice, and to identify the toxin-induced signaling pathways in cells that are damaged in biliary atresia.