Isolation, Identification and Characterization of a Toxin Causing Biliary Atresia

描述（由申请人提供）：胆道闭锁（BA）是一种胆道炎性疾病，是新生儿胆汁淤积的主要原因，也是儿科人群中最常见的肝移植适应症。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，和最知名的动物模型的疾病，感染新生BALB/c小鼠与恒河猴轮状病毒（RRV），提供了重要的信息，免疫方面的疾病，但一直不太有用的研究快速进行性纤维化的特点，疾病。对BA发病机制的了解来自对该疾病的自然发生的动物模型的研究。 在过去的40年里，澳大利亚新生牲畜中发生了三次BA流行病，与怀孕的绵羊和奶牛摄入植物球孢花有关。受影响的羔羊和小牛的临床和病理学结果显示与人BA惊人的相似性，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一般的胆管细胞损伤途径，最终导致BA和脊椎动物纤维化。 因此，我们收集并进口了D。glomulifera从澳大利亚牧场受最近的流行病，并采用了斑马鱼生物测定，以连续subfractionate植物和鉴定活性馏分。我们有几个馏分的复杂性为2-20，显着，造成BA样损伤模式（与闭锁的胆囊和肝外胆管树）在斑马鱼幼虫暴露后，胆道形态发生已经发生，模仿人类BA。因此，本提案的总体目标是确定在Dysphania胆毒素，表征它们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。这将通过三个具体目标来实现：1.识别D. glomulifera胆毒素使用体内斑马鱼生物测定~ 2.目的：鉴定D. glomulifera毒素~和3.鉴定和表征D. glomulifera毒素在哺乳动物模型。 公共卫生相关性：本提案的目的是确定植物球囊困难中的毒素，这些毒素是造成牲畜胆道闭锁的原因。 胆道自流病是儿科人群中一种主要但知之甚少的肝病。Dysphania毒素将用于开发新的胆道闭锁模型，包括斑马鱼和小鼠模型，并鉴定胆道闭锁中受损细胞中毒素诱导的信号通路。

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.