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

基本信息

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
项目类别：
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

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

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项目摘要

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（由应用程序提供）：本应用程序定义了一个计划，以进一步在设置问题的情况下进一步发展有前途的初级调查员的研究职业。成功的完成将使研究人员能够从事独立的NIH资助的外科医生科学家的职业，进行转化研究，旨在识别涉及坏死性小肠结肠炎（NEC）和其他肠道脓毒症原因的关键途径。一旦确定了NEC病原体涉及的特定途径，就可以发展出更好的治疗。背景：NEC影响所有住院过早的婴儿的5％，并且最严重的形式可能是致命的。细菌在疾病发病机理中暗示，sakazakii（CS）已被确定为导致NEC爆发。根据初步数据和已发表的工作，我们假设肠上皮的CS顶膜对于增加细胞内营地，PKA激活和上皮凋亡至关重要，即导致肠壁障碍和NEC。研究设计和方法：AIM 1将确定CS在实验NEC中是否刺激CAMP，PKA和CREB激活。 CAMP级别将由ELISA分配，因为CS各种剂量和浓度。都将测试体外肠道细胞系模型和NEC的大鼠幼崽模型。在模型系统中，将比较从具有NEC结果的婴儿的外科肠道标本中的CAMP，PKA和CREB水平。此外，将确定NEC期间活化的PKA的亚细胞位置。 AIM 2将确定在实验NEC中PKA介导的途径诱导上皮凋亡和肠屏障功能的丧失。体外模型对药物PKA抑制剂和激活剂的凋亡和障碍反应，以及使用siRNA对PKA的遗传抑制。细胞凋亡（caspase和tunel）的标记将通过蛋白质印迹分析和免疫荧光来测量。我们将确定PKA激活的时机，并确定其与凋亡的关系。势垒功能的变化将通过体外的经耐压测量来衡量。 PKA抑制剂在NEC大鼠幼犬模型中的影响将通过组织显微镜，免疫荧光和蛋白质印迹分析进行评估。将比较组之间的肠道损伤评分以及幼犬的存活率。通过基于血清的测定，将通过口服FITC-脱骨器进行口服给予障碍功能。 AIM 3将定义CS病毒因子在实验NEC中的作用。缺乏促进宿主细胞结合的病毒因子的CS突变体将根据其诱导上皮凋亡和实验性NEC的能力进行评估。可以通过转座子诱变产生其他突变体。该项目是新颖的，因为没有先前的研究研究了PKA在NEC中的作用。该项目在提出了一种机制方面是新颖和创新的，CS触发CAMP释放，改变PKA介导的活性，导致凋亡和NEC。以前没有研究检查CAMP，PKA和CREB在NEC中的作用，并且未定义可能触发NEC的病毒因子。研究环境：候选人提出的提案，在一个在护士的职业生涯中取得成功的环境中开发该项目的提议。在一个杰出的心态团队的监督下，该项目将为候选人的背景增添新的专业知识，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诱变的培训。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，职业咨询委员会定期评估的教学课程以及负责任的研究进行培训。候选人有75％（9个日历月）的受保护时间。