这项研究由 NIH 授予 HL66299、RB、SL、HL60024 到 ts 和 HL136869 到 MF 的部分资金。

所有动物程序都由南阿拉巴马大学的机构和动物护理委员会审查和批准, 并按照所有联邦、州和地方条例进行。主要培养的大鼠肺微血管内皮细胞 (PMVECs) 是从细胞培养核心设施, 在南阿拉巴马大学的肺生物学中心。细胞是用先前描述的步骤16编写的。
1. 细胞毒性上清液的产生
注: 在这里, 我们使用两种不同菌株的铜绿假单胞菌: PA103, 它有一个完整的 III 型分泌系统能够转移 exoenzymes ExoU 和 ExoT 的目标细胞在感染期间, ∆PcrV, 这是缺乏第三类分泌系统, 并在接种后无法将 exoenzymes 转移到靶细胞。
<ol><li>条纹细菌上伏地尔-博纳 (VB) 琼脂板17和增长隔夜在37摄氏度。<ol>
<li>要准备板材, 请做以下库存解决方案 (伏戈尔-博纳盐; 10x 库存): 测量2克 MgSO4, 20 克柠檬酸 (游离酸), 100 克 K2po4, 35 克 NaH2po 4。添加 ddH2O 到1升和高压釜为15分钟与慢排气。</li>
		<li>放置7.5 克琼脂在450毫升 ddH2O 和高压釜如上所述。</li>
		<li>跟随热处理, 安置两个解答入50°c 水浴冷却。</li>
		<li>添加50毫升的 10x VB 库存盐溶液的琼脂。</li>
		<li>添加羧苄青霉素到400µg/毫升 (对于使用的铜绿假单胞菌), 并通过旋转烧瓶混合良好。</li>
		<li>将 VB 琼脂溶液的5毫升放入单独的无菌培养皿中, 使琼脂凝固, 然后贮存在4摄氏度直到细菌播种的那天。</li>
		<li>在细菌播种的当天, 预热一盘到37摄氏度, 然后用无菌循环将细菌纹在盘子上。把盘子放在37摄氏度的恒温箱里过夜。</li>
	</ol>
</li>
	<li>用荧光加纳单叶蔓荆凝集素和阴性染色与荧光螺旋蜗牛凝集素 (动脉内皮细胞的标记) 进行阳性染色, 验证 PMVEC 纯度。整除细胞并在液氮中冷冻。</li>
	<li>在实验中, 维持 Dulbecco 的改良鹰培养基 (DMEM) 中的细胞补充10% 胎牛血清, 并在37摄氏度孵化器中生长细胞, 含 5% CO2。使用 5-15 通道上的细胞。<ol>
<li>对于生成的细胞毒性上清液, 板 2 x 106细胞成单独的 150 cm 菜肴和成长为 3-4 天, 直到汇合达到。每个实验使用两个盘子 (见下文)。</li>
		<li>为分析细胞毒性上清液, 板 1 x 105细胞成一个6井盘的个体井并且增长四天直到汇合达到。</li>
	</ol>
</li>
	<li>要生产上清, 洗一 PMVECs 的两个150厘米菜肴之一从步骤1.3.1 与磷酸盐缓冲盐水, 胰蛋白酶处理, 然后计数的细胞 (我们使用一个自动单元计数器, 并按照制造商的协议; 见材料表)。用汉克的平衡盐溶液 (HBSS) 洗另一盘 PMVECs, 然后用任一株铜绿假单胞菌感染20:1。实现方式如下:<ol>
<li>对于铜绿假单胞菌, OD540 0.25 = 2 x 108 CFUs/毫升。要准备这种稀释, 添加1毫升 HBSS 到1毫升一次性试管。在步骤1.1 中所准备的板上刮出足够的细菌, 直到使用分光光度计测量540的0.25。下一步将提供一个计算多少细菌将需要。</li>
		<li>一旦确定了培养皿中的 PMVECs 数量 (上面的步骤 1.4), 确定了适当数量的细菌添加到第二个, 非 trypsinized 培养皿中含有 PMVECs。例如, 如果确定 PMVECs 的培养皿包含 1.3 x 107细胞, 则准备 1.3 x 107细胞 x 20 细菌/细胞 x 1 mL/2 x 108 CFUs = 1.3 毫升细菌。</li>
		<li>将1.3 毫升的细菌稀释成 HBSS 的最后体积20毫升, 使整个表面的150厘米的盘子可以被液体覆盖, 然后将稀释的细菌添加到 PMVECs 的盘子里。</li>
		<li>孵化的 PMVECs 接种的细菌在 5% CO2孵化器37°c 为 4-5 h, 直到缝隙可以看到形成在细胞单层时, 显微镜观察 (见图 1为适当水平的差距形成)。</li>
		<li>收集上清, 然后离心机10分钟在 2000 x g 在一个台式离心机去除细胞碎片。 注: 任何能够产生足够的 g 力的表顶离心机 unlysed 细胞和大细胞碎片可用于这一步骤。</li>
		<li>将上清液倒入装有0.2 µm 过滤器的注射器上, 然后通过过滤器将上清液移除细菌。</li>
		<li>使用整除的无菌上清液测试细胞毒性 (见 2.1), 并冻结其余的上清在-80 °c, 以供将来使用。</li>
	</ol>
</li>
</ol>2. 细胞毒性上清液分析
<ol><li>细胞毒性试验<ol>
<li>在6井的菜肴中生长 PMVECs 直到汇合。用 HBSS 洗井一次, 然后添加1.5 毫升的过滤灭菌上清 (从 PA103 或∆PcrV 接种的细胞收集) 到个别水井。将无菌 HBSS 添加到另一个井中作为阴性对照。</li>
		<li>将该板块放入 CO2孵化器中 21-24 小时, 然后观察细胞杀伤/细胞毒性 (见下一步)。使用上清液的细胞毒性进行进一步的实验, 并丢弃那些不显示细胞杀戮。</li>
		<li>细胞杀伤的定量<ol>
<li>利用商用 LDH 检测试剂盒和制造商推荐的程序, 测定死细胞中乳酸脱氢酶 (LDH) 的释放。</li>
			<li>或者, 用 ImageJ 软件量化细胞杀伤。为此, 记录了含有完整细胞的培养皿的显微图像和区域, 然后使用自定义 ImageJ (国家卫生研究院) 宏量化缺乏细胞的区域 (用单层表示细胞死亡) (参见补充编码文件).如果需要, 请手动执行宏步骤, 如下所示:<ol>
<li>输入图像 (RGB 或 Tiff 格式) 到宏中, 并调整对比度为15% 饱和像素。复制对比度调整图像, 并执行 "减去背景" 命令, 以获得在视野内的细胞和间隙区域的高对比度图像。</li>
				<li>从原始图像中减去此图像, 并使用图像计算器 "和" 功能将生成的图像与原始图像相结合。将结果图像转换为黑色 (空白) 和白色 (单元格) 掩码, 具有阈值函数 (最小 0, 最大值 5), 并使用 "二进制腐蚀" 函数从图像中移除噪音。</li>
				<li>使用 "区域分数" 度量测量生成图像内的黑色和白色像素的比例。绘制和表达每个处理时间点的分数区域作为最大间隙面积的百分比。</li>
				<li>计算方法和比较使用单向方差分析与 Tukey 的后专项测试, p值小于0.05 被认为是重要的。</li>
			</ol>
</li>
		</ol>
</li>
		<li>利用应用免疫印迹分析建立淀粉样蛋白、寡聚头和 Aβ在培养上清液中的存在。使用标准程序10、11、12进行应用免疫印迹分析, 但在电泳前至少有10倍的浓缩培养上清液。<ol>
<li>要集中上清液, 将 1-2 毫升的上清液放入离心过滤器单元, 其膜的分子量为 10 kDa。然后, 将过滤单元放入工作台顶部离心机和离心机 2000 x 克, 直到达到所需浓度 (通常为 45-60 分钟)。</li>
			<li>确定浓缩清液11中的蛋白质含量, 并将等量的样品装入凝胶的单个水井中。</li>
			<li>运行凝胶, 转移蛋白质到硝化棉, 然后探针印迹与 A11 抗淀粉样蛋白抗体, T22 抗寡聚头抗体, 或 MOAB2 抗 Aβ抗体, 其次是适当的二级抗体。使用标准化学发光程序来开发印迹。</li>
		</ol>
</li>
		<li>为了进行 thioflavin t 检测, 使用 thioflavin t 荧光法对上清液中的 amyloids 进行量化。对于这些测量, 使用过滤灭菌上清液。<ol>
<li>通过将8毫克 thioflavin t (thioflavin) 悬浮到10毫升磷酸盐缓冲盐水 (PBS) 中, 准备一份库存溶液 (50x)。混合后, 通过0.22 µm 过滤器过滤溶液以去除微粒。</li>
			<li>对于测量, 在1毫升分光光度计试管中添加20µL 的股票到1毫升 PBS。将稀释后的样品放入 spectrofluorimeter。</li>
			<li>测量基线荧光发射使用425毫微米激发和扫描荧光发射从450-575 毫微米在2毫微米增量。</li>
			<li>使用 425 nm 激发和 482 nm 发射进行时间推移扫描, 数据在六十年代每0.2 秒获得一次。</li>
			<li>初始二十年代的时间推移扫描措施荧光在空白试管。在二十年代, 暂停扫描, 并添加10µL 过滤消毒上清到试管。通过反转混合试管, 然后再放回 spectrofluorimeter。</li>
			<li>恢复基于时间的扫描, 获取数据的最后四十年代。</li>
			<li>完成定时扫描后, 使用相同的设置进行最终的荧光发射频谱扫描, 如2.1.5.3 中详细说明。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

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没有报告。

在这里, 概述了简单的体外方法, 可以证明在感染时产生细胞毒性 amyloids 的生物。这些方法包括细胞培养毒性试验、免疫印迹法、定量的细胞杀死使用一种新的显微方法, 并具有约束力。对细胞毒性剂的分析表明, 它们是淀粉样蛋白 (图 2 和图 4), 并表现出朊病毒12的特征。在肺炎期间, 细胞毒性朊病毒分子的生成为肺...

在出院1、2、3、4、5、6之后的几个月里, 患肺炎的患者死亡率升高。在大多数情况下, 死亡发生的一些类型的最终器官衰竭, 包括肾脏, 肺, 心脏, 或肝脏事件, 以及中风5,6。这个病人的死亡率上升的原因从来没有建立。
肺炎被归类为社区获得的或医院获得的 (医院), 可以引起肺炎的药物包括细菌、病毒、真菌和化学物质。细菌性铜绿假单胞菌是医院内肺炎的主要病因之一。铜绿假单胞菌是一种革兰阴性有机体, 使用 III. 型分泌系统将各种效应分子 (称为 exoenzymes) 直接转移到靶细胞7、8的细胞质中。在肺内皮细胞感染期间, exoenzymes 靶向各种胞内蛋白, 包括微管相关蛋白头9、10、11、12 的内皮形态., 导致内皮屏障破裂导致严重肺水肿, 肺功能减退, 常常死亡。
如前所述, 在出院后的头12月中, 幸存于最初肺炎的病人的死亡率升高。解释这种现象的一个潜在机制是, 在最初的感染过程中产生了某种长期存在的毒素, 导致了长期的不良结果。两项意见支持这种可能性。首先, 在细菌被抗生素杀死13之后, 最初用铜绿假单胞菌治疗的培养的肺内皮细胞不能增殖长达一周。第二, 长期存活的普利子和具有朊病毒特征的特工已经在各种人类和动物疾病中表现出来, 特别是与神经系统有关的疾病14,15。
在肺部感染过程中, 研究长期存活的细胞毒性药物的潜在生产方法从未被描述过。这里概述了一系列简单的体外检测方法, 可用于调查使用常见肺炎引起的铜绿假单胞菌感染后毒素的生产和活动。这些化验应易于适应, 以调查可能的毒素诱导后, 使用其他药物引起肺炎的感染, 并产生的上清液也应有助于调查细胞毒素的影响, 整个器官或动物。最后, 这里概述的化验结果最有可能会适应于在肺炎期间生产细胞毒素的动物和人体生物液。

<table><tbody><tr><td>Rabbit anti beta amyloid</td><td>Thermo</td><td>71-5800</td><td></td></tr><tr><td>A11 amyloid oligomer antibody</td><td>StressMarq</td><td>SPC-506D</td><td></td></tr><tr><td>T22 anti-tau oligomer antibody</td><td>EMD Millipore</td><td>ABN454</td><td></td></tr><tr><td>Thioflavin T</td><td>Sigma Aldrich</td><td>T3516</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>0.22 micron syringe filters</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>HRP Goat antirabbit IgG</td><td>Abcam</td><td>ab6721-1</td><td></td></tr><tr><td>Strains PA103 and &amp;Delta;PcrV</td><td></td><td></td><td>These strains of P. aeruginosa were obtained from Dr. Dara Frank, University of Wisconsin Medical College, Milwaukee, WI</td></tr><tr><td>FITC Griffonia lectin</td><td>Sigma Aldrich</td><td>L9381</td><td></td></tr><tr><td>TRITC Helix pomatia lectin</td><td>Sigma Aldrich</td><td>L1261</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>0.22 micron nitrocellulose</td><td>BioRad</td><td>162-0112</td><td></td></tr><tr><td>Type 2 collagenase</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30898.03IH</td><td></td></tr><tr><td>PenStrep</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>Carbenicillin Disodium salt</td><td>Sigma&amp;nbsp;</td><td>C1389</td><td></td></tr><tr><td>Microcetrifugation concentrators- 10,000 MW cut-off</td><td>Millipore</td><td>UFC801008</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma&amp;nbsp;</td><td>795496-500G</td><td></td></tr><tr><td>Magnesium phosphate heptahydrate</td><td>MP Biomedicals</td><td>MP021914221</td><td></td></tr><tr><td>Citric Acid</td><td>G Biosciences</td><td>50-103-5801</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma</td><td>S9506-500G</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10277</td><td></td></tr></tbody></table>

methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by pseudomonas aeruginosa

在出院后的几个月里, 患有肺炎的病人死亡率升高。据推测, 肺炎期间肺部组织感染导致长期存活的细胞毒素的产生, 从而导致随后的最终器官衰竭。我们已经开发了体外检测, 以检验细胞毒素在肺部感染过程中产生的假说。将孤立大鼠肺内皮细胞和细菌铜绿假单胞菌作为模型系统, 用细胞培养法对 cytoxins 后的内皮细胞感染进行了分析, 然后用乳酸脱氢酶测定法直接定量, 并利用 ImageJ 技术进行了一种新的显微方法。通过 thioflavin T 结合试验, 免疫印迹法和 immunodepletion 采用 A11 抗淀粉样蛋白抗体, 证明了这些细胞毒素的淀粉样蛋白性质。进一步分析使用免疫印迹法表明, 寡聚头和 Aβ是由内皮细胞产生和释放后感染的铜绿假单胞菌。这些方法应易于适应对人体临床样品的分析。

本文建立了一种简单的体外检测方法, 用于检测上清液菌感染的细胞中是否存在细胞毒素. 基本上, 细菌添加后, 从感染细胞中采集到的培养基为4小时, 细菌通过培养基上清液的过滤杀菌去除, 然后将无菌上清液添加到新的细胞群中。细胞被观察 21-24 小时在增加上清和细胞杀害以后被定量。
将铜绿假单胞菌添...

Watch this Scientific Journal Video about Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa at JoVE.com

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

介绍了用铜绿假单胞菌感染肺内皮后细胞毒性 amyloids 的制备方法。

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

methods-for-detecting-cytotoxic-amyloids-following-infection

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

5.5K 次观看.  University of South Alabama. 介绍了用铜绿假单胞菌感染肺内皮后细胞毒性 amyloids 的制备方法。

视频: Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

科研

生物化学

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

免疫与感染

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

恒流中原发性人类内皮细胞的肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

自动、高通量检测细菌对宿主细胞的粘附

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

摘要

探索更多视频

此视频中的章节

制备及

单细胞测量液泡破裂引起细胞内病原体

绿脓杆菌引起的肺损伤模型

恒流中原发性人类内皮细胞的肺炎球菌感染

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

香烟烟雾对肺癫痫细胞伪多多多细胞的影响研究

囊性纤维化患者痰液中铜绿假单胞菌的可视化

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

自动、高通量检测细菌对宿主细胞的粘附

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

摘要

探索更多视频

此视频中的章节

制备及

单细胞测量液泡破裂引起细胞内病原体

绿脓杆菌引起的肺损伤模型

恒流中原发性人类内皮细胞的肺炎球菌感染

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

自动、高通量检测细菌对宿主细胞的粘附

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

香烟烟雾对肺癫痫细胞伪多多多细胞的影响研究

囊性纤维化患者痰液中铜绿假单胞菌的可视化