在作者的实验室的工作是由自然科学和工程研究理事会，加拿大，加拿大卫生研究院的研究所，加拿大研究教席计划的拨款支持。 JL支持通过全宗RECHERCHE EN桑特魁北克的资金由集团的练习曲DES Protéines Membranaires（GEPROM）的奖学金。

1。初步：菌株和上皮细胞。 无菌层流罩下进行细胞和细菌的操纵。 <ol><li>新鲜分离的大肠杆菌大肠杆菌 2787，2787Δ 阿依达 ，和C600从甘油溶原性肉汤（LB）琼脂平板（1％蛋白胨，0.5％氯化钠，0.5％酵母提取物，1.5％琼脂）和成长在37 ° C。为了尽量减少检测的可变性，这是应始终使用新鲜镀株，并保持在4 ° C，在密封的Petri板最大的几个星期的菌株。 </li><li>文化HEp - 2细胞在高糖贝科的改良Eagle培养基（DMEM）（ATCC CCL - 23），辅以10％热灭活小牛血清（热灭活是在56℃30分钟）。在常规培养中，我们还添加了10 U / ml青霉素和10μg/ mL链霉素。 </li><li> Hep - 2细胞增长在37 °与大气中含有5％的CO 2细胞培养箱中的C。使用标准细胞培养过程，以维持细胞，这是生长在75厘米2瓶和传代培养，每次他们到达汇合。我们用我们的，直到他们达到30至40代的培养细胞，然后将它们丢弃。 </li><li> HEp - 2细胞在75厘米2烧瓶时，几乎达到汇合，因此坚持实验的准备，准备一个试验。 </li></ol> 2。第一天：准备接种和上皮细胞<ol><li>洗烧瓶中的HEP - 2细胞一旦与温暖的贝科的磷酸盐缓冲生理盐水（DPBS：8 g / L的氯化钠，0.2克/ L的KCl，0.2 g / L的KH 2 PO 4，0.21 g / L的钠2 HPO 4：7H 2 O） </li><li>细胞培养与EDTA的0.05％胰蛋白酶 - 5分钟，然后加入新鲜温暖的完全培养基。添加新鲜培养基后，细胞悬浮吹打向上和向下。 </li><li>在5分钟2000转离心细胞悬液，并重新悬浮颗粒DPBS离心一次。 </li><li>辅以10％的血清，但没有抗生素，含有2 × 10 5细胞/毫升的浓度在新鲜培养基重悬细胞。 </li><li>种子毫升细胞悬液三套复孔在24孔板中心（每一株），并在细胞培养孵化器，孵化盘一夜之间。 注意：重要的是彻底decomplemented血清和抗生素后这个阶段省略。如果不这样做，可能会杀死细菌感染。 </li><li>接种5毫升LB培养基（1％蛋白胨，0.5％氯化钠，0.5％酵母提取物）在每个菌株的一个孤立的殖民地“（27872787Δ阿依达和C600）和成长一夜之间在37 °剧烈摇晃C（ 180 RPM ）。 </li></ol> 3。第2天：细胞的感染<ol><li>检查HEp - 2细胞，倒置显微镜下，以确定他们至少有90％的融合，而不是污染。 </li><li>洗净，用温暖的DPBS，每口井的细胞，辅以10％血清的新鲜培养基中添加1毫升，但不含有抗生素。另外补充新鲜培养基三无细胞井。这将是用来确定每一株接种中的细菌总数。 </li><li>测量在600 nm（外径0.600纳米 ）的细菌培养的光密度。每个细菌培养等分一套含有Hep - 2细胞中的重复井和一个不包含细胞。通常，我们使用了相应的10 6个菌落形成单位（CFU）过夜培养量。这代表了感染复数（MOI），以5:1（细菌细胞）。虽然我们直接使用我们的过夜培养，它有时是最好离心机的细菌和重悬在DPBS他们在一夜之间文化分泌的分子，以避免有害影响（如细胞毒素，例如） 注：内政部可以100:1和1:10之间变化。高等教学语言的收益率更高的变异性和背景和细菌往往会坚持的塑料板。下的教学语言还具有高可变性。一旦MOI选择，当务之急是要一致，并保持这种教学语言。 </li><li>感染的细胞在细胞培养孵化器孵育3小时，在37℃，5％的CO 2 。 注：也有可能短暂离心板在低速（如1000 XG为1-2分钟），直接与细胞接触，以便使所有的细菌。这同步感染的增值收益，并允许更短的孵育时间（15-30分钟低至）。 </li><li>从被感染的细胞中取出，和洗脸用温水DPBS细胞的3倍。在这一步，附着细菌通常可以用一个标准的显微镜，我们经常执行此检查。 </li><li>裂解细胞分离坚持BACteria，加100μL1％Triton X - 100的的每个以及含有细胞。可以用其他洗涤剂（如例如皂甙）。 </li><li>为在室温下10分钟孵育细胞，然后加入900μLLB培养基。 </li><li>轻轻的同质化和重复吹打悬浮液。不含有细胞接种的水井也同样轻轻均匀。 注：有些细菌可能是过于敏感，洗涤剂，在这种情况下，我们孵化上皮细胞分离与0.05％胰蛋白酶- 20分钟EDTA在37 ° C。坚持细菌细胞聚集，也可以用枪头井刮掉。 </li><li>准备坚持细菌和使用3稀释（通常为1:1,000，1:10,000和1:100,000稀释）的LB琼脂LB肉汤和板100μL接种悬浮系列10倍稀释，孵育过夜，于37 ° C 。 </li></ol> 4。第3天：菌落计数和数据演示。 <ol><li>计数板的殖民地和坚持细菌的接种人数计算，平均每个系列稀释的CFU。应该算只有10-300殖民地之间的板。 </li></ol> 5。代表性的成果： 下表显示了典型的菌落计数坚持细菌接种在不同的日子进行了3个实验的结果： <img src="/files/ftp_upload/2783/2783table1.jpg" alt="表1" /> 结果可直接作为坚持CFU，如在图1A所示。由于接种的大小可以不同菌株之间由于增长率的差异或移液错误，它往往是最好的报告坚持细菌的百分比的结果。坚持细菌的百分比，可以计算的CFU的接种人数除以坚持细菌的CFU数量，如图1B所示。通过执行一个重复测量的方差分析和Bonferroni后测试比较图1B中的所有列，我们可以看到，有显着差异（P &lt;0.05）之间的2787和2787Δ 阿依达 ，以及2787和C600之间，但无显着差异2787Δ 阿依达和C600之间。 <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="图1"/> 图1 CFU坚持细菌（A）或（二）坚持细菌的百分比结果的代表性。 观察坚持的百分比往往是非常依赖对实验设置（以及在较小程度上，实验者）。尤其重要的是教学语言和清洗，这样的比例不应该从字面上解释。 有时在接种细菌可以增长速度远远比在细菌细胞的存在，比较真实的细菌总数与细胞井倾斜接种量的大小。为了缓解这一问题，建议两种：（i）使用与细胞井，以确定接种：HEp - 2细胞接种于附加以及和感染。在检测结束，而不是丢弃，上清，洗涤，100μL10％的Triton X - 100添加到井里。细胞裂解和裂解液含有细菌坚持和非粘贴轻轻反复向上和向下移液匀浆;或（ii）在实验结束收集感染的细胞培养上清，以及上清DPBS清洗并确定这些汇集上清对CFU。这将产生非坚持细菌的CFU数量。坚持细菌的百分比，然后可以坚持细菌的CFU数量除以除了坚持和非坚持的细菌菌落数计算。 为了说明检测的稳健性，我们可以比较与S4074进行了检测， 粘合剂猪胸膜肺炎放线杆菌7致病株，与最近成立的气管仔猪细胞株 ，NPTr 8细胞培养的这个协议。除了从媒体中的细菌和细胞生长所需的差异，唯一的区别是感染细菌的大幅增长是从文化，从一个固定相的过夜培养。 与 A 胸膜肺炎 ，它也是非常重要的，尊重MOI为10:1，并以不超过3个小时的感染，否则分泌的毒素会导致细胞死亡和偏见的结果。此外，这株A 的胸膜肺炎可以很容易地坚持以塑料的，因此重要的是使用融合细胞，并验证视觉细胞不塌。

<ol>
	<li>Kline, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesins+in+host-microbe+interactions.">Bacterial adhesins in host-microbe interactions.</a> Cell Host Microbe. 5, 580-580 (2009).</li><li>Cossart, P., Boquet, P., Normark, S., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+microbiology+emerging.">Cellular microbiology emerging.</a> Science. 271, 315-315 (1996).</li><li>Elsinghorst, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasion+by+gentamicin+resistance.">Measurement of invasion by gentamicin resistance.</a> Methods Enzymol. 236, 405-405 (1994).</li><li>Pizarro-Cerda, J., Lecuit, M., Cossart, P., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cellular Microbiology. , 161-161 (2002).</li><li>Srivastava, A., Isberg, R. R., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cellular Microbiology. , 179-179 (2002).</li><li>Benz, I., Schmidt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+an+adhesin+(AIDA-I)+involved+in+diffuse+adherence+of+enteropathogenic+Escherichia+coli.">Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.</a> Infect Immun. 57, 1506-1506 (1989).</li><li>Charbonneau, M. E., Berthiaume, F., Mourez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+processing+is+not+essential+for+multiple+functions+of+the+Escherichia+coli+autotransporter+adhesin+involved+in+diffuse+adherence+(AIDA-I).">Proteolytic processing is not essential for multiple functions of the Escherichia coli autotransporter adhesin involved in diffuse adherence (AIDA-I).</a> J Bacteriol. 188, 8504-8504 (2006).</li><li>Jacques, M., Paradis, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesin-receptor+interactions+in+Pasteurellaceae.">Adhesin-receptor interactions in Pasteurellaceae.</a> FEMS Microbiol Rev. 22, 45-45 (1998).</li><li>Auger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+of+Actinobacillus+pleuropneumoniae+with+porcine+lung+and+tracheal+epithelial+cells.">Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells.</a> Infect Immun. 77, 1426-1426 (2009).</li><li>Jacques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipo-oligosaccharides+and+lipopolysaccharides+in+bacterial+adherence.">Role of lipo-oligosaccharides and lipopolysaccharides in bacterial adherence.</a> Trends Microbiol. 4, 408-408 (1996).</li><li>Nataro, J. P., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diarrheagenic+Escherichia+coli.">Diarrheagenic Escherichia coli.</a> Clin Microbiol Rev. 11, 142-142 (1998).</li><li>Jallat, C., Darfeuille-Michaud, A., Rich, C., Joly, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+clinical+isolates+of+diarrhoeogenic+Escherichia+coli:+diffusely+adhering+E.+coli+strains+with+multiple+adhesive+factors.">Survey of clinical isolates of diarrhoeogenic Escherichia coli: diffusely adhering E. coli strains with multiple adhesive factors.</a> Res Microbiol. 145, 621-621 (1994).</li></ol>

本协议描述了一个标准的细菌黏附实验研究入侵（如使用庆大霉素保护法 3）可以修改。菌落形成单位计数允许量化，如姬姆萨染色，在显微镜下，依靠标准的可视化技术的方法相比。后者只给出了一个定性的附着力，但往往是一个有益的补充，因为它可以区分各种图案的附着力和mechanistical 见解 9 。例如，一个姬姆萨染色进行接种10 7 cfu的2787，如图2所示，表明整个细胞?...

<table border="1"><tbody><tr><th>试剂</th><th>公司</th><th>目录编号</th></tr><tr><td>高糖DMEM </td><td> GIBCO，Invitrogen公司</td><td> 12430-054 </td></tr><tr><td> DDPBS </td><td> GIBCO，Invitrogen公司</td><td> 14190-144 </td></tr><tr><td>牛生长血清</td><td> Hyclone公司</td><td> SH3054 </td></tr><tr><td>青霉素/链霉素</td><td> GIBCO，Invitrogen公司</td><td> 15140-148 </td></tr><tr><td> 24孔板</td><td>康宁</td><td> 3337 </td></tr><tr><td> TRITON X - 100 </td><td> Fisher Scientific则</td><td> BP151 - 100 </td></tr></tbody></table>

in vitro assay of bacterial adhesion onto mammalian epithelial cells

导致细菌感染，细菌必须去占领他们的主人。细菌病原体表达的不同的分子或结构，能够促进宿主细胞的附件1。这些粘附依靠与宿主细胞表面的受体或可溶性蛋白质充当细菌和主机之间的桥梁的相互作用。附着力是关键的第一步之前，入侵和/或分泌的毒素，因此，它是在细菌的发病机制研究的一个重要事件。此外，坚持细菌往往诱发精美微调的细胞反应，其中已生育的“细胞微生物学”2领域的研究。因此，乐百氏细菌粘附在宿主细胞和它们的入侵检测细菌的发病机制研究中发挥关键作用，并一直在许多先驱实验室 3,4 。这些检测现在实行的大多数细菌的发病工作实验室。 在这里，我们描述了一个标准的遵守实验说明了一个具体黏附的贡献。我们利用大肠杆菌 2787 5，人类致病株表达autotransporter弥漫遵守（AIDA）的黏附。作为对照，我们使用了一个突变株缺乏2787Δ 阿依达 （F. Berthiaume和M. Mourez，未发表）， 阿依达的基因，和E.商业实验室应变大肠杆菌 ，C600（New England Biolabs公司）。这种细菌是留给坚持从常用的HEP - 2人类上皮细胞株的细胞。此实验已经越来越广泛地描述前 6 。

Watch this Scientific Journal Video about In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells at JoVE.com

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

此协议是一个简单的细菌粘附实验，计数菌落形成单位，坚持到培养细胞的数量。该检测是可靠的，独立的黏附研究，并有众多的变化在大多数实验室中使用细菌发病机制的工作。

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

in-vitro-assay-of-bacterial-adhesion-onto-mammalian-epithelial-cells

Research

JoVE Journal

Immunology and Infection

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

25.2K 次观看.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. 此协议是一个简单的细菌粘附实验，计数菌落形成单位，坚持到培养细胞的数量。该检测是可靠的，独立的黏附研究，并有众多的变化在大多数实验室中使用细菌发病机制的工作。

视频: In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

肠道细菌侵入肠上皮细胞<em&gt;体外</em&gt;使用垂直扩散室模型，显着增强

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

人中性粒细胞流动室粘附实验

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...