| Protocol for HeLa Cells Infection with Escherichia coli Strains Producing Colibactin and Quantification of the Induced DNA-damage
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Author:
Date:
2017-08-20
[Abstract] Strains of Escherichia coli bearing the pks genomic island synthesize the genotoxin colibactin. Exposure of eukaryotic cells to E. coli producing colibactin induces DNA damages, ultimately leading to cell cycle arrest, senescence and death. Here we describe a simple method to demonstrate the genotoxicity of bacteria producing colibactin following a short infection of cultured mammalian cells with pks+ E. coli.
[摘要] 具有pks基因组岛的大肠杆菌菌株合成基因毒素大肠杆菌素。 将真核细胞暴露于产生大肠杆菌的大肠杆菌诱导DNA损伤，最终导致细胞周期停滞，衰老和死亡。 在这里，我们描述了一种简单的方法来证明在用pks +大肠杆菌培养的哺乳动物细胞的短时间感染后，产生大肠杆菌素的细菌的遗传毒性。
【背景】大肠杆菌素是在大肠杆菌的肠外致病，共生和益生菌菌株中发现的基因毒素（Nougayrede等，2006）。大肠杆菌素也由其他肠杆菌科产生，包括肺炎克雷伯杆菌，产气肠杆菌和柠檬酸杆菌（Putze等人，2009）。大肠杆菌素是通过由多酮酶和非核糖体肽合酶（PKS和NRPS），定制和成熟酶以及外排泵组成的多酶机械合成的聚酮化合物/非核糖体肽杂化化合物（综述：Taieb等人。，2016）。该合成机制编码在52kb的基因组，即'pks'岛上。 Colibactin在感染pks +细菌的真核细胞中诱导DNA损伤。由大肠杆菌素诱导的遗传毒性作用需要活的pks +细菌与真核细胞的直接接触。事实上，杀死的细菌或细菌上清液或裂解物没有观察到遗传毒性作用。因此，为了证明产生大肠杆菌的大肠杆菌的基因毒性，培养的哺乳动物细胞（例如HeLa细胞）在4小时内用活的pks ...
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| Ex vivo Model of Human Aortic Valve Bacterial Colonization
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Author:
Date:
2017-06-05
[Abstract] The interaction of pathogens with host tissues is a key step towards successful colonization and establishment of an infection. During bacteremia, pathogens can virtually reach all organs in the human body (e.g., heart, kidney, spleen) but host immunity, blood flow and tissue integrity generally prevents bacterial colonization. Yet, patients with cardiac conditions (e.g., congenital heart disease, atherosclerosis, calcific aortic stenosis, prosthetic valve recipients) are at a higher risk of bacterial infection. This protocol was adapted from an established ex vivo porcine heart adhesion model and takes advantage of the availability of heart tissues obtained from patients that underwent aortic valve replacement surgery. In this protocol, fresh tissues are used ...
[摘要] 病原体与宿主组织的相互作用是成功定居和建立感染的关键步骤。在菌血症期间，病原体几乎可以达到人体内的所有器官（例如心脏，肾脏，脾脏），但是宿主的免疫力，血流和组织完整性通常防止细菌定植。然而，患有心脏病（例如先天性心脏病，动脉粥样硬化，钙化性主动脉狭窄，人工瓣膜受体）的患者处于较高的细菌感染风险。该方案从已建立的远端猪心脏粘连模型改编而成，并且利用从经历主动脉瓣置换手术的患者获得的心脏组织的可用性。在该方案中，使用新鲜组织来评估与心血管感染相关的细菌病原体（例如变形链球菌）与人主动脉瓣组织的直接相互作用。
背景 口腔病原体变形链球菌被认为是龋齿中的主要病原体，也可以与感染性心内膜炎（IE）等口腔外感染有关（Banas，2004）。 ...
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| Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)
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Author:
Date:
2017-05-05
[Abstract] Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence. With the use of a basic direct co-culture system, confocal microscopy and flow cytometry we visualised and effectively quantified MSC mitochondrial transfer to AM ...
[摘要] 间充质干/基质细胞（MSC）是成年干细胞，已被证明可以改善急性呼吸窘迫综合征（ARDS）和败血症临床前模型中的存活，增强细菌清除和减轻炎症。这些疾病的特征在于通常受细菌感染支持的不受控制的炎症。 MSC免疫调节作用的机制尚未完全了解。我们试图研究与肺泡巨噬细胞（AM），在肺炎症反应和抗菌防御中起重要作用的专业吞噬细胞的MSC细胞接触依赖性通信。通过使用基本的直接共培养系统，共聚焦显微镜和流式细胞术，我们通过隧道纳米管（TNT）显现并有效量化了MSC线粒体转移到AM。为了模拟人类AM，在粒细胞巨噬细胞集落刺激因子（GM-CSF）存在下，从人供体血液中分离原代单核细胞并分化成巨噬细胞（单核细胞衍生的巨噬细胞，MDM），从而允许适应AM样表型（de Almeida等人，2000; Guilliams等人，2013）。人骨髓衍生的MSC用线粒体特异性荧光染色标记，广泛洗涤，以1:20（MSC / ...
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