Defining bacterial virulence, cAMP and PKA in necrotizing enterocolitis

坏死性小肠结肠炎中细菌毒力、cAMP 和 PKA 的定义

• 批准号: 9115585
• 负责人: Catherine Jane Hunter
• 金额: $ 14.95万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115585
• 关键词: Adherence; Advisory Committees; Affect; Apical; Apoptosis; Apoptotic; Award; Bacteria; Bacterial Infections; Binding; Biological Assay; Biological Models; Calendar; Caspase; Cell Line; Cells; Child; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; Data; Disease; Disease Outbreaks; Dose; Enteral; Environment; Enzyme-Linked Immunosorbent Assay; Epithelial; Evaluation; Event; Experimental Models; Failure; Figs - dietary; Functional disorder; Funding; Genetic; Goals; Health; Human; Immunofluorescence Immunologic; Immunofluorescence Microscopy; In Situ Nick-End Labeling; In Vitro; Infant; Infection; Inflammatory disease of the intestine; Injury; Intestines; Knowledge; Lead; Link; Location; Long-Term Survivors; Measurement; Measures; Mediating; Mediator of activation protein; Mentors; Mentorship; Microbiology; Mission; Modeling; Molecular Biology; Mutagenesis; Necrotizing Enterocolitis; Neonatal Intensive Care Units; Neurologic; Operative Surgical Procedures; Oral Administration; Pathogenesis; Pathway interactions; Patients; Permeability; Phosphorylation; Premature Infant; Production; Protein Kinase; Protein Kinase Inhibitors; Pseudomonas aeruginosa; Publishing; Rattus; Research; Research Design; Research Methodology; Research Personnel; Resistance; Rodent; Rodent Model; Role; Sampling; Scientist; Sepsis; Serum; Short Bowel Syndrome; Signal Pathway; Signal Transduction; Small Interfering RNA; Specimen; Staging; Supervision; Surface; Surgeon; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Training; Translational Research; United States National Institutes of Health; Virulence; Virulence Factors; Western Blotting; Work; Yersinia pestis; apical membrane; base; career; career development; established cell line; fluorescein isothiocyanate dextran; improved; in vitro Model; in vivo; innovation; intestinal epithelium; knock-down; meetings; microbial; mutant; novel; novel strategies; pathogen; prevent; programs; protein activation; protein kinase inhibitor; pup; response; responsible research conduct; skills; success; therapeutic protein; tool; transcription factor

 DESCRIPTION (provided by applicant): 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.

 描述(由申请人提供)：此申请定义了一个计划，以促进一个有前途的初级调查员在指导环境下的研究生涯。成功完成研究将使研究人员开始作为一名独立的NIH资助的外科医生兼科学家的职业生涯，进行旨在识别坏死性小肠结肠炎(NEC)和其他导致肠道败血症的原因的关键途径的转译研究。一旦确定了与NEC致病相关的特定途径，就可能开发出更好的治疗方法。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NEC暴发的细菌。根据初步数据和已发表的工作，我们假设CS附着在肠上皮的顶膜上，是增加细胞内cAMP、PKA活性和上皮细胞凋亡所必需的， 导致肠道屏障衰竭和NEC。研究设计和方法：目的1确定CS是否刺激实验性NEC中cAMP、PKA和CREB的激活。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。手术采集的NEC患儿肠道标本中的cAMP、PKA和CREB水平将与对照组进行比较。结果将在模型系统之间进行比较。此外，还将确定NEC过程中激活的PKA的亚细胞位置。目的2确定PKA介导的途径是否诱导实验性NEC上皮细胞凋亡和肠屏障功能丧失。体外模型对药物PKA抑制剂和激活剂的凋亡和屏障反应，以及利用siRNA对PKA的遗传抑制。用免疫印迹分析和免疫荧光法检测细胞凋亡的标志物(半胱氨酸天冬氨酸氨基转移酶和TUNEL)。我们将确定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抑制剂在NEC幼鼠模型中的作用将通过组织显微镜、免疫荧光和蛋白质印迹分析来评估。肠道损伤评分将在不同组之间进行比较，以及幼崽的存活率。FITC-葡聚糖组间屏障功能的比较将通过基于血清的检测来实现。目的3明确CS毒力因子(S)在实验性NEC中的作用。缺乏促进宿主细胞结合的毒力因子的CS突变体将被评估其诱导上皮细胞凋亡和实验性NEC的能力。转座子诱变可以产生更多的突变体。这个项目是新颖的，因为以前没有研究过PKA在NEC中的作用。本课题的创新之处在于提出了CS触发cAMP释放，改变PKA介导的活性，导致细胞凋亡和NEC的机制。此前还没有研究探讨cAMP、PKA和CREB在NEC中的作用，也没有定义可能触发NEC的毒力因子。研究环境：应聘者建议在培养初级调查人员的职业生涯方面取得成功的环境下开发该项目。在一支杰出的导师团队的监督下，该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、细胞信号通路、免疫染色、肠道通透性评估和微生物突变方面的培训。该提案中的职业发展活动包括分子生物学、细胞信号机制和微生物学的教学课程，职业咨询委员会的定期评估，以及负责任的研究行为培训。候选人有75%(9个日历月)的受保护研究时间。