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: 10391067
• 负责人: Catherine Jane Hunter
• 金额: $ 5.97万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-30 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391067
• 关键词: Adherence; Advisory Committees; Affect; Apoptosis; Apoptotic; Bacteria; Binding; Biological Assay; Biological Models; CREB1 gene; Calendar; Caspase; Cell Line; Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Disease Outbreaks; Dose; Environment; Enzyme-Linked Immunosorbent Assay; Epithelial; Evaluation; Failure; Funding; Genetic; Human; Immunofluorescence Immunologic; In Situ Nick-End Labeling; In Vitro; Infant; Intestinal permeability; Intestines; Location; Measurement; Measures; Mediating; Mentors; Mentorship; Microbiology; Microscopy; Mission; Modeling; Molecular Biology; Mutagenesis; Necrotizing Enterocolitis; Neonatal Intensive Care Units; Operative Surgical Procedures; Oral Administration; PKA inhibitor; Pathogenesis; Pathway interactions; Pharmacologic Substance; Premature Infant; Publishing; Rattus; Research; Research Design; Research Methodology; Research Personnel; Resistance; Role; Scientist; Sepsis; Serum; Signal Pathway; Signal Transduction; Small Interfering RNA; Specimen; Supervision; Surgeon; Testing; Therapeutic; Time; Tissues; Training; Translational Research; United States National Institutes of Health; Virulence; Virulence Factors; Western Blotting; Work; apical membrane; base; career; career development; fluorescein isothiocyanate dextran; in vitro Model; innovation; intestinal barrier; intestinal epithelium; intestinal injury; microbial; mutant; novel; pathogen; programs; pup; response; responsible research conduct; success

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.

项目摘要 目的：本申请书定义了一个项目，以促进一个有前途的初级研究员的研究生涯 在一个有指导的环境中。成功完成将允许研究者开始职业生涯， 独立的NIH资助的外科医生-科学家，进行转化研究，旨在确定关键的 坏死性小肠结肠炎（NEC）和其他肠败血症的原因所涉及的途径。一旦具体 如果确定了NEC病因中涉及的途径，则可以开发更好的治疗剂。 背景：NEC影响所有住院早产儿的5%，并且在其最严重的形式中可能是致命的。 细菌与疾病的发病机制有关，坂崎克罗伊氏菌（CS）已被确定为引起 NEC的爆发。基于初步数据和已发表的工作，我们假设CS对 肠上皮细胞的顶膜，对于增加细胞内cAMP、PKA活化和 上皮细胞凋亡，导致肠屏障衰竭和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NEC。在CS的不同剂量和浓度后，通过ELISA测定cAMP水平。 将测试NEC的体外肠细胞系模型和大鼠幼仔模型。cAMP、PKA和CREB 将从患有NEC的婴儿取得的手术肠样本中的水平与对照进行比较。结果将 在模型系统中进行比较。此外，在NEC过程中激活的PKA的亚细胞定位将被确定。 鉴定目的2将确定是否上皮细胞凋亡和肠屏障功能的丧失是由 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的PKA遗传抑制。标志物 细胞凋亡（半胱天冬酶和TUNEL）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 通过体外跨上皮电阻测量来测量。PKA抑制剂在NEC大鼠幼仔中的作用 将通过组织显微镜、免疫荧光和蛋白质印迹分析来评估模型。肠损伤 将比较各组之间的评分以及幼仔存活率。屏障功能将在 通过基于血清的测定，通过口服施用FITC-葡聚糖对各组进行测定。目标3将定义CS毒力的作用 实验NEC中的因素。缺乏促进宿主细胞结合的毒力因子的CS突变体将是 评估它们诱导上皮细胞凋亡和实验NEC的能力。另外的突变体可以是 通过转座子诱变产生。这个项目是新颖的，因为以前没有研究调查的作用， PKA在NEC。该项目在提出CS触发cAMP释放的机制方面是新颖和创新的， 改变PKA介导的活性，导致细胞凋亡和NEC。以前没有研究探讨过 cAMP、PKA和CREB在NEC中的作用以及可能触发NEC的毒力因子尚未明确。 研究环境：候选人建议在既定的环境中开发该项目 成功培养初级调查员的职业生涯。在一位杰出导师的指导下 该项目将为候选人的背景增加新的专业知识，包括分子生物学方面的培训， 细胞信号传导途径、免疫染色、肠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、细胞信号传导 机制和微生物学，职业咨询委员会的定期评估， 负责任地进行研究。候选人有75%（9个日历月）的受保护的研究时间。

项目成果

Urinary Claudin-2 Measurements as a Predictor of Necrotizing Enterocolitis: A Pilot Study

尿液 Claudin-2 测量作为坏死性小肠结肠炎的预测因子：一项初步研究

• DOI: 10.21699/jns.v4i4.282
• 发表时间: 2015
• 期刊: Journal of Neonatal Surgery
• 作者: Brian P Blackwood M.D.;Douglas R Wood B.S.;Carrie Y Yuan B.S.;Joseph D Nicolas;Anne Griffiths M.D.;Karen Mestan M.D.;Catherine J Hunter M.D.
• 通讯作者: Catherine J Hunter M.D.

The science and necessity of using animal models in the study of necrotizing enterocolitis.

• DOI: 10.1053/j.sempedsurg.2017.11.006
• 发表时间: 2018-03
• 期刊: Seminars in pediatric surgery
• 影响因子: 1.7
• 作者: Ares GJ;McElroy SJ;Hunter CJ
• 通讯作者: Hunter CJ

Experimental Modeling of Necrotizing Enterocolitis in Human Infant Intestinal Enteroids.

• DOI: 10.1080/08941939.2020.1829755
• 发表时间: 2022-01
• 期刊: Journal of investigative surgery : the official journal of the Academy of Surgical Research
• 作者: Buonpane C;Ares G;Yuan C;Schlegel C;Liebe H;Hunter CJ
• 通讯作者: Hunter CJ

Apical-Out Enteroids as an Innovative Model for Necrotizing Enterocolitis.

• DOI: 10.1016/j.jss.2022.11.048
• 发表时间: 2023-03
• 期刊: The Journal of surgical research
• 作者: Heather L. Liebe;Camille Schlegel;Xue Cai;A. Golubkova;Christopher Loerke;Tyler Leiva;C. Hunter

Pediatric Intussusception: Decreased Surgical Risk with Timely Transfer to a Children's Hospital.

• DOI: 10.21767/2471-805x.100018
• 发表时间: 2016-01-01
• 期刊: Pediatric care (Wilmington, Del.)
• 作者: Blackwood, Brian P;Theodorou, Christina M;Hunter M, Catherine J
• 通讯作者: Hunter M, Catherine J