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 &#916;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&#160;</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&#160;</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

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

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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 ...