Pathobiology of Cystic Fibrosis-Related Diabetes in a Ferret Model

雪貂模型中囊性纤维化相关糖尿病的病理学

• 批准号: 8468701
• 负责人: Zoe Stewart
• 金额: $ 14.75万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-08 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8468701
• 关键词: Abdomen; Address; Adenoviruses; Adult; Age; Alkaline Phosphatase; Animal Model; Animals; Antibiotics; Attenuated; Award; Basic Science; Bicarbonates; Birth; Caucasians; Caucasoid Race; Cell Culture Techniques; Cell Lineage; Cell physiology; Cells; Characteristics; Chloride Channels; Clinical; Commit; Complementary DNA; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Defect; Development; Diabetes Mellitus; Disease; Disease Progression; Doctor of Philosophy; Ductal; Duodenum; Effectiveness; Endocrine; Epithelial Cells; Epithelium; Excision; Exhibits; Exposure to; Fellowship; Ferrets; Fibrocystic Disease of Pancreas; Foundations; Functional disorder; Funding; Gallbladder; Gene Delivery; Gene Expression; Gene Transduction Agent; Genetic; Glucagon; Glucose; Goals; Growth; Health; Histopathology; Human; Hyperglycemia; IL8 gene; Infection; Inflammation Mediators; Insulin; Interleukin-1; Intestines; Islets of Langerhans; K-Series Research Career Programs; Knock-out; Laboratories; Laboratory Research; Lead; Leadership; Learning; Life; Life Expectancy; Liver; Luciferases; Lung; Lung diseases; Measures; Mediating; Medical; Mentors; Mentorship; Methods; Modeling; Molecular; Molecular Genetics; Mus; Neonatal; Nutritional; Onset of illness; Operative Surgical Procedures; Oral; Organ; Outcome Study; Pancreas; Pancreatic Diseases; Pancreatic duct; Pancreatic enzyme; Pathway interactions; Patients; Perinatal mortality demographics; Phenotype; Physicians; Physiology; Predisposition; Process; Proton Pump Inhibitors; Quality of life; Recombinants; Records; Regulator Genes; Reporter Genes; Research; Research Personnel; Residencies; Satellite Viruses; Scientist; Secure; Serotyping; Serum; Severities; Symptoms; Systems Development; Techniques; Testing; Therapeutic; Time; Training; Transplantation; Tropism; Variant; Viral Vector; Weight Gain; Work; Writing; adeno-associated viral vector; apical membrane; authority; base; blood glucose regulation; career; cell type; clinical care; cohort; cystic fibrosis patients; effective therapy; gastrointestinal; gastrointestinal function; gene replacement; gene replacement therapy; gene therapy; glucose metabolism; improved; in vivo; inhibitor/antagonist; intestinal epithelium; lipid metabolism; nutrition; programs; response; skills; success; therapy development; tool; undergraduate student; vector

DESCRIPTION (provided by applicant): I have been committed to a career in basic science since my first exposure to research as an undergraduate student. After completing an MD/PhD program, I went on to pursue surgical residency and then a fellowship in abdominal transplant surgery. To achieve my goal of becoming a successful independent researcher, I recently entered the laboratory of Dr. John Engelhardt, a national authority on the molecular pathophysiology of cystic fibrosis (CF) and strategies to utilize recombinant adenovirus-associated viruses (rAAV) as gene therapy vectors. Dr. Engelhardt has proven track records in both mentoring physician- scientists and securing research funding, and his guidance will be a critical determinant of my success in developing my independent research laboratory. Under Dr. Engelhardt's mentorship, I am focused on combining my basic science training with my medical background to tackle an important clinical question that directly impacts my patients, namely studying the pathophysiology of cystic fibrosis-related diabetes (CFRD). This is a clinically important question, as CF is the most common lethal autosomal recessive disease in Caucasians, resulting from defects in the cystic fibrosis transmembrane conductance regulator (CFTR) channel. This K-award will allow critical protected time for me to fully develop my independent research career. During the period of this award, I intend to: 1) expand my skills in molecular genetics, 2) master the techniques relevant to CF research and learn to generate pathophysiologically important hypotheses about CFRD, 3) develop skill in the implementation of rAAV gene therapies, 4) strengthen my writing and leadership/mentoring skills, and 5) solidify preliminary data for my first R01 application. In CF patients, pancreatic disease leads to diabetes mellitus in up to 50% of adult patients, resulting in reduced life expectancy. Progress in elucidating the pathophysiology of CFRD has been hindered by the lack of an animal model. Dr. Engelhardt's laboratory has recently developed a CF-knockout ferret - the first animal model that develops CFRD. However, CF ferrets have high perinatal mortality from abnormal intestinal and pancreatic function, altered glucose and lipid metabolism, and lung infections. Elevating GI pH appears to attenuate nutritional abnormalities, as the use of proton-pump inhibitors (PPIs) normalizes weight gain during the neonatal period. The CF ferret model appears to be a robust system for the development of CF pancreatic therapies and is the only animal model available for the study of CFRD. We hypothesize that effective treatment of CFRD will lead to reduced nutritional complications in CF ferrets, increased long-term survival, and reduced severity of lung disease. We will test this hypothesis by carrying out the following specific aims: 1) Identify the defects underlying the pancreatic and intestinal pathophysiologies associated with CFRD in the ferret CF model and 2) Establish a CFTR gene replacement therapy that will improve pancreatic function in CF ferrets. To address the goals of Aim 1, we will use standard pancreatic endocrine functional tests to confirm that a subset of CF ferrets develop CFRD and characterize the disease pathophysiology. We will determine if CFTR-mediated bicarbonate (HCO3-) secretion by the pancreatic duct and/or duodenal epithelial cells is reduced, and if so, how this impacts CFRD phenotypic severity. We will also evaluate the activity of compensatory, non-CFTR-based HCO3- secretory pathways using CFTR inhibitors to determine if these channels might influence the severity and/or disease onset of CFRD. To address the goals of Aim 2, we will adapt methods developed for the mouse (application of rAAV vectors encoding the WT ferret CFTR cDNA) for in vivo gene delivery to the pancreas in CFTR-/- ferrets. We will assess the effectiveness of pancreatic CFTR gene replacement in the CFTR-/- ferrets by evaluating serum glucose, insulin, glucagon, endocrine pancreatic function, and pancreatic duct cell physiology. We will also assess the impact of pancreatic CFTR gene therapy by evaluating ferret growth and nutritional parameters in the presence and absence of PPIs and pancreatic enzymes. Finally, we will evaluate the impact of pancreatic CFTR gene therapy on the progression of early pulmonary disease progression in CF ferrets following removal of antibiotics. These studies will assess lung inflammatory mediators (IL-1, IL-8, and TNFa), lung bacterial colonization, and lung histopathology. The major expected outcomes of this study are: 1) a more comprehensive characterization of endocrine pancreas abnormalities in the new CF ferret model, 2) an understanding of the basis of phenotypic variation in CFTR-associated pancreatic disease observed in both the CF ferrets and humans, and 3) the development of a pancreatic gene replacement approach that will aid in dissecting pathophysiology and treatment of CFRD. These results are expected to have a significant positive impact on the field of CF research.

描述（由申请人提供）：自从我作为本科生第一次接触研究以来，我就一直致力于基础科学的职业生涯。完成医学博士/博士学位课程后，我继续进行外科住院医师实习，然后获得腹部移植手术的奖学金。为了实现成为一名成功的独立研究员的目标，我最近进入了 John Engelhardt 博士的实验室，他是囊性纤维化 (CF) 分子病理生理学和利用重组腺病毒相关病毒 (rAAV) 作为基因治疗策略的国家权威向量。恩格尔哈特博士在指导医师科学家和确保研究经费方面拥有良好的记录，他的指导将是我成功发展独立研究实验室的关键决定因素。在 Engelhardt 博士的指导下，我专注于将基础科学训练与医学背景相结合，解决直接影响患者的重要临床问题，即研究囊性纤维化相关糖尿病 (CFRD) 的病理生理学。这是一个临床上重要的问题，因为 CF 是白种人中最常见的致死性常染色体隐性遗传疾病，由囊性纤维化跨膜电导调节器 (CFTR) 通道缺陷引起。这个 K 奖将为我提供关键的受保护时间来充分发展我的独立研究生涯。在获得该奖项期间，我打算：1）扩展我在分子遗传学方面的技能，2）掌握与 CF 研究相关的技术并学习生成有关 CFRD 的病理生理学重要假设，3）培养实施 rAAV 基因疗法的技能，4) 加强我的写作和领导/指导技能，以及 5) 巩固我的第一个 R01 申请的初步数据。在 CF 患者中，胰腺疾病会导致高达 50% 的成年患者罹患糖尿病，从而导致预期寿命缩短。由于缺乏动物模型，阐明 CFRD 病理生理学的进展受到阻碍。 Engelhardt 博士的实验室最近开发了 CF 基因敲除雪貂，这是第一个出现 CFRD 的动物模型。然而，CF 雪貂因肠道和胰腺功能异常、葡萄糖和脂质代谢改变以及肺部感染而具有很高的围产期死亡率。提高胃肠道 pH 值似乎可以减轻营养异常，因为质子泵抑制剂 (PPI) 的使用可以使新生儿期的体重增加正常化。 CF 雪貂模型似乎是开发 CF 胰腺疗法的强大系统，也是唯一可用于 CFRD 研究的动物模型。我们假设 CFRD 的有效治疗将减少 CF 雪貂的营养并发症，提高长期存活率并降低肺部疾病的严重程度。我们将通过实现以下具体目标来检验这一假设：1) 确定雪貂 CF 模型中与 CFRD 相关的胰腺和肠道病理生理学缺陷；2) 建立 CFTR 基因替代疗法，以改善 CF 雪貂的胰腺功能。为了实现目标 1，我们将使用标准的胰腺内分泌功能测试来确认 CF 雪貂的一个子集会出现 CFRD 并描述该疾病的病理生理学特征。我们将确定 CFTR 介导的胰管和/或十二指肠上皮细胞的碳酸氢盐 (HCO3-) 分泌是否减少，如果是，这如何影响 CFRD 表型严重程度。我们还将使用 CFTR 抑制剂评估补偿性、非基于 CFTR 的 HCO3 分泌途径的活性，以确定这些途径是否可能影响 CFRD 的严重程度和/或疾病发作。为了实现目标 2，我们将采用为小鼠开发的方法（应用编码 WT 雪貂 CFTR cDNA 的 rAAV 载体），将体内基因递送至 CFTR-/- 雪貂的胰腺。我们将通过评估血清葡萄糖、胰岛素、胰高血糖素、内分泌胰腺功能和胰管细胞生理学来评估 CFTR-/- 雪貂中胰腺 CFTR 基因替代的有效性。我们还将通过评估雪貂在存在或不存在 PPI 和胰腺酶的情况下的生长和营养参数来评估胰腺 CFTR 基因治疗的影响。最后，我们将评估胰腺 CFTR 基因治疗对 CF 雪貂去除抗生素后早期肺部疾病进展的影响。这些研究将评估肺部炎症介质（IL-1、IL-8 和 TNFa）、肺部细菌定植和肺部组织病理学。本研究的主要预期结果是：1）新 CF 雪貂模型中内分泌胰腺异常的更全面表征，2）了解在 CF 雪貂和人类中观察到的 CFTR 相关胰腺疾病表型变异的基础，3) 胰腺基因替代方法的开发将有助于剖析 CFRD 的病理生理学和治疗。这些成果预计将对CF研究领域产生重大积极影响。

项目成果

Case report: successful treatment of recurrent focal segmental glomerulosclerosis with a novel rituximab regimen.

病例报告：采用新型利妥昔单抗方案成功治疗复发性局灶节段性肾小球硬化症。

• 发表时间: 2011-12
• 影响因子: 0.9
• 作者: Stewart, Z A;Shetty, R;Nair, R;Reed, A I;Brophy, P D
• 通讯作者: Brophy, P D

Pre-emptive eculizumab and plasmapheresis for renal transplant in atypical hemolytic uremic syndrome.

非典型溶血性尿毒症综合征肾移植的先发性依库丽单抗和血浆置换术。

• 发表时间: 2011-06
• 作者: Nester, Carla;Stewart, Zoe;Myers, David;Jetton, Jennifer;Nair, Ramesh;Reed, Alan;Thomas, Christie;Smith, Richard;Brophy, Patrick
• 通讯作者: Brophy, Patrick