Defining bacterial virulence, cAMP and PKA in necrotizing enterocolitis

项目总结 目标：本申请明确了一项计划，旨在促进有前途的初级调查员的研究生涯 在有指导的环境下。成功完成调查将使调查员开始作为一名 由美国国立卫生研究院资助的独立外科医生兼科学家，进行转译研究，旨在确定关键 坏死性小肠结肠炎(NEC)和其他引起肠道败血症的途径。一旦具体 NEC的致病途径被确定，更好的治疗方法可能被开发出来。 背景：NEC影响所有住院早产儿的5%，在最严重的情况下可能是致命的。 细菌与疾病的发病机制有关，阪崎慢杆菌(CS)已被确定为引起疾病的原因 NEC的暴发。根据初步数据和已发表的工作，我们假设CS遵守 肠上皮的顶膜，对于增加细胞内cAMP，PKA活性和 上皮细胞凋亡，导致肠屏障功能衰竭和NEC。 研究设计和方法：目的1确定CS是否刺激cAMP、PKA和CREB的激活 在实验中的NEC。在不同剂量和浓度的CS作用下，用ELISA法测定cAMP水平。 NEC的体外肠道细胞系模型和大鼠幼鼠模型都将进行测试。CAMP、PKA和CREB 从患有NEC的婴儿的外科肠道样本中提取的水平将与对照组进行比较。结果将是 在模型系统之间进行了比较。此外，在NEC期间激活的PKA的亚细胞位置将是 已确认身份。目的2将确定是否诱导肠上皮细胞凋亡和肠屏障功能丧失 实验性NEC中PKA介导的通路。体外模型的细胞凋亡和屏障反应 药物PKA抑制剂和激活剂，以及使用siRNA对PKA的遗传抑制。标记： 用免疫印迹和免疫荧光法检测细胞凋亡率(半胱氨酸天冬氨酸氨基转移酶和TUNEL)。我们会 确定PKA激活的时间，并确定其与细胞凋亡的关系。屏障功能的变化将 通过体外跨上皮阻力测量来测量。PKA抑制剂对NEC仔鼠的影响 模型将通过组织显微镜、免疫荧光和蛋白质印迹分析进行评估。肠道损伤 分数将在不同组之间进行比较，以及幼崽的存活率。屏障函数将在以下情况下进行比较 组口服FITC-葡聚糖，以血清为基础测定。目标3将定义CS毒力的作用 实验用NEC中的S因素。缺乏促进宿主细胞结合的毒力因子的CS突变体将是 评估其诱导上皮细胞凋亡和实验性NEC的能力。其他突变体可能是 由转座子诱变产生。这个项目是新颖的，因为之前没有任何研究调查过 NEC中的PKA。本项目创新性地提出了CS触发cAMP释放的机制， 改变PKA介导的活性，导致细胞凋亡和NEC。在此之前，还没有任何研究考察过 CAMP、PKA和CREB在NEC中的表达，以及可能引发NEC的毒力因子尚未定义。 研究环境：应聘者建议在已建立的环境中开发此项目 成功培育初级调查员的职业生涯。在一位杰出导师的指导下 团队，这个项目将为候选人的背景增加新的专业知识，包括分子生物学方面的培训， 细胞信号通路、免疫染色、肠道通透性评估和微生物诱变。职业生涯 该提案中的开发活动包括分子生物学、细胞信号转导等教学课程 机制和微生物学，职业咨询委员会的定期评价，以及 负责任的研究行为。候选人有75%(9个日历月)的受保护研究时间。

PROJECT SUMMARY Objectives: This application defines a program to further the research career of a promising junior investigator within a mentored setting. Successful completion would allow the investigator to initiate a career as an independent NIH-funded surgeon-scientist, conducting translational research directed at identifying key pathways involved in necrotizing enterocolitis (NEC) and other causes of intestinal sepsis. Once specific pathways involved in the pathogenies of NEC are identified, better therapeutics may be developed. Background: NEC affects 5% of all hospitalized premature infants, and may be fatal in its most severe forms. Bacteria are implicated in disease pathogenesis, and Cronobacter sakazakii (CS) has been identified as causing outbreaks of NEC. Based on preliminary data and published work, we hypothesize that CS adherence to the apical membrane of the intestinal epithelium, is essential for increased intracellular cAMP, PKA activation and epithelial apoptosis, resulting in intestinal barrier failure and NEC. Research Design and Methods: Aim 1 will determine whether CS stimulates cAMP, PKA and CREB activation in experimental NEC. cAMP levels will be assayed by ELISA following various doses and concentrations of CS. Both in vitro intestinal cell line models and the rat pup model of NEC will be tested. cAMP, PKA and CREB levels in surgical intestinal specimens taken from infants with NEC will be compared to controls. Results will be compared among model systems. Furthermore, the subcellular location of activated PKA during NEC will be identified. Aim 2 will determine whether epithelial apoptosis and loss of intestinal barrier function is induced by PKA-mediated pathways in experimental NEC. The apoptotic and barrier responses of the in vitro models to pharmaceutical PKA inhibitors and activators, as well as genetic inhibition of PKA using siRNA. Markers of apoptosis (caspase and TUNEL) will be measured by western blot analysis and immunofluorescence. We will determine the timing of PKA activation, and define its relationship to apoptosis. Changes in barrier function will be measured by transepithelial resistance measurement in vitro. The effect of PKA inhibitors in the NEC rat pup model will be assessed by tissue microscopy, immunofluorescence and western blot analysis. Intestinal injury scores will be compared between groups, as well as pup survival. Barrier function will be compared between groups by oral administration of FITC–Dextran by serum based assay. Aim 3 will define the role of CS virulence factor(s) in experimental NEC. CS mutants lacking virulence factors that facilitate host cell binding will be assessed for their ability to induce epithelial apoptosis and experimental NEC. Additional mutants may be generated by transposon mutagenesis. This project is novel because no prior study has investigated the role of PKA in NEC. This project is novel and innovative in proposing a mechanism by which CS trigger cAMP release, alter PKA mediated activity, resulting in apoptosis and NEC. No study has previously examined the role of cAMP, PKA and CREB in NEC, and the virulence factors that may trigger NEC are not defined. Research environment: The candidate proposes to develop this project within an environment with established success at nurturing the careers of junior investigators. Under the supervision of an outstanding mentorship team, this project will add new expertise to the candidate's background, including training in molecular biology, cell signaling pathways, immunostaining, intestinal permeability assessment, and microbial mutagenesis. Career development activities within the proposal include didactic coursework in molecular biology, cell signaling mechanisms and microbiology, regular evaluations by a career advisory committee, and training in the responsible conduct of research. The candidate has 75% (9 calendar months) of protected research time.