Isolation, Identification and Characterization of a Toxin Causing Biliary Atresia

描述（由申请人提供）：胆道闭锁（BA）是一种纤维性炎症性疾病，是新生儿胆汁淤滞症的主要原因，也是儿科人群中最常见的肝移植指征。虽然流行病学数据表明BA是由遗传风险因素与环境暴露的相互作用引起的，但病因尚不清楚。动物模型是有限的，该疾病最著名的动物模型，即新生BALB/c小鼠感染恒河轮状病毒（RRV），提供了有关该疾病免疫学方面的重要信息，但对于研究该疾病的快速进行性纤维化特征却不太有用。对BA发病机制的深入了解来自于对该疾病自然发生的动物模型的研究。在过去的40年里，澳大利亚的新生牲畜中发生了三次BA流行，这些流行与怀孕的羊和牛摄入植物球囊植物Dysphania glomerera有关。来自受影响羔羊和小牛的临床和病理结果显示与人类BA惊人的相似，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并进口了D. glomulifera，并采用斑马鱼生物测定法对该植物进行了连续的提取并鉴定了活性组分。我们有几个复杂程度为2-20的分数，值得注意的是，在胆道形态发生后暴露的斑马鱼幼虫中，它们会引起类似BA的损伤模式（胆囊和肝外胆道树闭锁），模仿人类BA。因此，本提案的总体目标是识别胆汁毒素在胆汁障碍中，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发病机制的手段。这将通过三个具体目标来实现：利用斑马鱼体内生物测定法鉴定肾小球胆毒素~ 2。目的：探讨经肾小球藻毒素~和3处理的斑马鱼肝外胆道损伤的调控分子标记和基因。在哺乳动物模型中鉴定和表征球孢霉毒素诱导的损伤途径。

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.