Amphotericin B Restoration of Anion Secretion in Cystic Fibrosis Airways

项目2 项目总结 囊性纤维化(CF)是由编码囊性纤维化的基因功能丧失突变引起的 跨膜电导调节器(CFTR)顶膜阴离子通道。CFTR的损失- 呼吸道上皮细胞介导的HCO3-和Cl-分泌损害呼吸道宿主防御，导致细菌 感染、炎症、粘液堆积和呼吸衰竭。恢复阴离子的一种策略 对顶膜的通道功能是增加突变的CFTR通道的功能。那就是 事实证明，对于具有特定CFTR突变的人来说，这种方法是成功的。然而，约10%的CF患者患有 对cftr调节器没有反应的突变，或者它们不能接受调节器。另一个 战略是提供一种替代渠道。为此，我们研究了两性霉素B(Amb)； 研究表明，AMB形成阴离子通道。我们发现心尖Amb增加了HCO3-和 培养的CF呼吸道上皮细胞的CL分泌，包括CFTR零突变的细胞。AMB 提高了ASL的pH值、身高和抗菌活性。AMB被临床批准用于治疗真菌感染， 而标签外的肺部雾化治疗有着令人印象深刻的安全记录。因此，AMB可能会提供一种 CF肺部疾病的新突变-不可知性治疗。然而，许多问题仍然没有得到回答， 我们缺乏重要的活体数据。因此，我们的首要目标是了解 AMB诱导呼吸道上皮细胞阴离子分泌及验证AMB可恢复的假说 Cf宿主在体内的防御。为了实现这一目标，我们将从三个方面回答问题。目标1.什么 Amb介导的阴离子分泌的分子和细胞机制？没有cAMP依赖 AMB对阴离子的选择性只有CFTR的一半，不是CFTR的理想替代品。我们会 测试有关Amb如何在呼吸道上皮细胞中发挥作用的关键假设。目标2.哪些上皮细胞起作用 AMB目标以及AMB如何改变它们？这些研究将揭示Amb在大小方面的功能 呼吸道上皮和不同类型的细胞，检测炎症重塑的呼吸道上皮中的Amb，并检测 进行补偿性的改变。目的3.在体内，Amb能逆转CF宿主防御缺陷吗？使用CF猪， 我们将测试雾化Amb是否会增加ASL的pH值，增加ASL的抗菌活性和高度， 增加粘液纤毛运输，并加强金黄色葡萄球菌从肺部的根除。结果是 这些研究将对开发一种新的基因不可知的治疗方法有直接的影响 Cf，并将在项目1和3中通知其他疗法的开发。我们的提案的成功是 通过威尔士(爱荷华州)和伯克(伊利诺伊州)实验室之间的持续合作得到加强，通过 未完成核心的利用以及方案项目之间的合作环境 和调查人员。有了这个背景，我们的记录，以及我们的承诺，我们相信这项研究 可以改变慢性萎缩性胃炎患者的生活。

PROJECT 2 PROJECT SUMMARY Cystic fibrosis (CF) is caused by loss-of-function mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) apical membrane anion channel. Loss of CFTR- mediated HCO3- and Cl- secretion by airway epithelia impairs respiratory host defenses, causing bacterial infection, inflammation, mucus accumulation, and respiratory failure. One strategy for restoring anion channel function to the apical membrane is to increase the function of mutant CFTR channels. That has proven successful for people with specific CFTR mutations. However, ~10% of people with CF have mutations that do not respond to CFTR modulators or they are not able to take modulators. Another strategy is to provide an alternative channel. Toward this end, we studied amphotericin B (AmB); earlier work showed that AmB forms anion channels. We discovered that apical AmB increased HCO3- and Cl- secretion in cultured CF airway epithelia, including those with CFTR-null mutations. AmB increased ASL pH, height, and antibacterial activity. AmB is clinically approved to treat fungal infections, and off-label lung aerosolization has an impressive safety record. Thus, AmB could potentially provide a new mutation-agnostic therapy for CF lung disease. However, many questions remain unanswered and we lack important in vivo data. Therefore, our overarching goal is to understand the mechanisms of AmB-induced anion secretion in airway epithelia and to test the hypothesis that AmB can restore CF host defenses in vivo. To achieve this goal, we will answer questions in three aims. Aim 1. What molecular and cellular mechanisms elicit AmB-mediated anion secretion? Without cAMP-dependent regulation and with half the anion selectivity of CFTR, AmB is an imperfect substitute for CFTR. We will test key hypotheses about how AmB functions in airway epithelia. Aim 2. Which epithelial cells does AmB target and how does AmB alter them? These studies will reveal AmB function in large and small airway epithelia and diverse cell-types, test AmB in airway epithelia remodeled by inflammation, and test for compensatory changes. Aim 3. Does AmB reverse CF host defense defects in vivo? Using CF pigs, we will test if nebulized AmB increases ASL pH, increases ASL antimicrobial activity and height, increases mucociliary transport, and enhances S. aureus eradication from the lungs. The results of these studies will have direct implications for developing a new genotype agnostic approach to treating CF, and will inform development of other therapies in Projects 1 and 3. Our proposal’s success is enhanced by an ongoing collaboration between the labs of Welsh (Iowa) and Burke (Illinois), by utilization of outstanding cores, and by an environment of cooperation between the Program’s projects and investigators. With this background, our track record, and our commitment, we believe this research can change the lives of people with CF.