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 目标：本应用程序定义了一个计划，以进一步发展有前途的初级研究员的研究职业 在重要的环境中。成功完成将使调查员能够从事职业 独立的NIH资助的外科医生 - 科学家，进行转化研究，旨在识别密钥 涉及坏死性小肠结肠炎（NEC）和其他肠道脓毒症的途径。一旦具体 鉴定出参与NEC病原体的途径，可以开发更好的治疗剂。 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。 疾病发病机理中隐含细菌，sakazakii（CS）已被确定为导致 NEC的爆发。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，对于增加细胞内营地，PKA激活和 上皮凋亡，导致肠屏障衰竭和NEC。 研究设计和方法：AIM 1将确定CS是否刺激CAMP，PKA和CREB激活 在实验性NEC中。 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。营地，PKA和Creb 将从患有NEC的婴儿手术的手术肠道标本的水平与对照组进行比较。结果将是 比较模型系统。此外，NEC期间活化PKA的亚细胞位置将为 确定。 AIM 2将确定上皮凋亡和肠道屏障功能的丧失是由 PKA介导的实验NEC中的途径。体外模型对 使用siRNA对PKA的PKA抑制剂和活化剂以及PKA的遗传抑制。标记 凋亡（caspase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激活的时机，并定义其与凋亡的关系。屏障功能的变化将 可以通过体外的旋转耐药性测量进行测量。 PKA抑制剂在NEC大鼠幼犬中的影响 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。肠损伤 将比较组之间的分数以及幼崽的存活率。将比较障碍功能 通过基于血清的测定法对FITC – dextran进行口头施用组。 AIM 3将定义CS病毒的作用 实验NEC中的因子。缺乏促进宿主细胞结合的病毒因子的CS突变体将是 评估了它们诱导上皮凋亡和实验性NEC的能力。其他突变体可能是 由转座子诱变产生。该项目是新颖的，因为没有先前的研究调查了 NEC中的PKA。该项目在提出一种机制方面是新颖和创新的，CS触发营地释放， 改变PKA介导的活性，导致凋亡和NEC。以前没有研究检查过 NEC中的CAMP，PKA和CREB以及可能触发NEC的病毒因素的定义。 研究环境：在具有既定的环境中开发该项目的候选提案 成功培养初级调查人员的职业。在杰出心态的监督下 团队，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 该提案中的开发活动包括分子生物学的教学课程，细胞信号传导 机制和微生物学，职业咨询委员会定期评估以及培训 负责任的研究。候选人有75％（9个日历月）的受保护时间。

项目成果

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

Outcomes and associated ethical considerations of long-run pediatric ECMO at a single center institution.

在单一中心机构长期运行儿科 ECMO 的结果和相关伦理考虑。

• DOI: 10.1007/s00383-019-04443-y
• 发表时间: 2019
• 期刊: Pediatric surgery international
• 影响因子: 1.8
• 作者: Ares,GuillermoJ;Buonpane,Christie;Helenowski,Irene;Reynolds,Marleta;Hunter,CatherineJ
• 通讯作者: Hunter,CatherineJ