Name: methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by pseudomonas aeruginosa
Uploaded: 2018-07-12T12:30:00
Description: Watch this Scientific Journal Video about Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa at JoVE.com

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, ...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+exoenzyme+S+regulon+of+Pseudomonas+aeruginosa.">The exoenzyme S regulon of Pseudomonas aeruginosa.</a> Mol Microbiol. 26 (4), 621-629 (1997).</li><li>Engel, J., Balachandran, P. <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+Pseudomonas+aeruginosa+type+III+effectors+in+disease.">Role of Pseudomonas aeruginosa type III effectors in disease.</a> Curr Opin Microbiol. 12 (1), 61-66 (2009).</li><li>Ochoa, C. 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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 ...

Research

JoVE Journal

Immunology and Infection

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

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

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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

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

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

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

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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

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

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

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

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

Pseudomonas aeruginosa Induced Lung Injury Model

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

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

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

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 ...