这项工作是由该基金VOOR Wetenschappelijk Onderzoe​​k（FWO）VLAANDEREN G0466.10，11I0113N支持; “埃迪墨克斯研究基金”和“Sporta研究经费”为小儿心脏病，UZ比利时鲁汶（JC）;该中心分子和血管生物学由Programmafinanciering鲁汶（PF / 10/014）的支持下，由鲁汶大学的“Geconcentreerde Onderzoe​​ksacties”（GOA一十三分之二千零九）和勃林格殷格翰研究经费。

动物实验批准了鲁汶的伦理委员会。 体外灌注和体内实验1.准备细菌<ol><li>我们使用S.金黄色葡萄球菌菌株Newman在这个手稿中描述的所有实验。S.金黄色葡萄球菌Newman贮存在脑心浸液（BHI），用10％甘油在-80℃。 </li><li>使用无菌环刮冷冻细菌关闭并接种在5毫升的胰蛋白酶大豆肉汤（TSB）中O / N在37℃（OD 600&gt; 3）。 </li><li>通过离心（2600克，室温，5分钟）洗涤细菌和悬浮细菌沉淀在5ml PBS（磷酸盐缓冲盐水）。 </li><li>准备一个1mg / ml的溶液的5（6） - 羧基荧光素的N-羟基琥珀酰亚胺基酯（羧基荧光素）的乙醇。稀释的1mg / ml的羧基荧光素的溶液，以150微克/毫升在实验室级水（ 例如，的MilliQ水）。避光管用铝箔和储存在-20℃。 </li><li>离心细菌（2600×g离心，室温，5分钟）。重悬细菌沉淀在800微升PBS中，并加入200微升或150微克/毫升羧基荧光素溶液的400微升（终浓度50μg/ ml的为进行体内实验）（30微克/毫升为灌注实验的最终浓度）。保护管从光用铝箔和孵育在室温30分钟在振荡器上。 </li><li>标记后，用在PBS中的6％的牛血清白蛋白（BSA）溶液块。 </li><li>稀释使用光学密度菌（OD）的OD用于体外实验600 0.65（相当于约3×10 8菌落形成单位（CFU）/ ml的金黄色葡萄球菌 ）和一个外径为进行体内实验600 1.8（对应于在PBS中约1×10 9 CFU / ml的金黄色葡萄球菌 ）。从光用铝箔保护管，置于冰上。 </li></ol> 2. 体外灌流实验<ol><li>盖玻片涂层<ol><li>在实验室级水稀von Willebrand因子（vWF）（Haemate磷，库存浓度2400微克/毫升）（用去离子），以50微克/毫升的最终浓度。 </li><li>在等渗葡萄糖溶液（SKF溶液，pH值2.7-2.9，由制造商提供）至160微克/ ml的终浓度稀释胶原。 </li><li>涂层的玻璃盖玻片（24×50毫米）的VWF或胶原滴加200μl的上封口膜涂层并放置盖玻片上的液滴的顶部。液滴将沿盖玻片的表面上扩散。 </li><li>孵育在加湿容器盖玻片为4小时，在室温。小心地提起从封口膜盖玻片用钝针头。安装盖玻片在流动室的底部部分。 </li></ol></li><li>塑料涂布泥釉与内皮细胞<ol><li>外套塑料单（1阱的PCA细胞培养室，Sarstedt的，德国）用1ml在PBS中的1％明胶溶液并孵育30分钟，在37℃。在明胶包被的塑料单种子的人脐静脉内皮细胞（HUVECs）和成长他们70-80％汇合。装入塑料滑移在流动室的底部部分。 </li></ol></li><li>灌注试验 注： 在体外灌注模型的示意图表示在图1。 <ol><li>执行体外细菌粘附研究中微平行板流动室在层流剪切应力2.5达因/厘米2和20达因/厘米2之间，以模拟不同的生理流动条件。 </li><li>流室（室内设计）由金属框架，并提出了有机玻璃的灌注腔（聚（甲基）丙烯酸甲酯（PMMA））的。通过将其连接到一个高精度输注泵（PHD 2000输液，哈佛仪器，USA）中，我们可以生成流岭0.0001微升/分钟和220.82毫升/分钟之间TES。 </li><li>的管路连接到流动室的上部，并在管道注入介质。轻轻放置在底部部件的顶流室的上部和组装流室。要小心，以避免产生气泡。注射1毫升培养基通过腔室以确保该室是不渗漏，并除去多余的涂覆溶液。避免气泡。 </li><li>鼠标放置在一个热控制的加热垫在37℃下在显微镜托盘。由于这是一个终端的程序，也没有必要为严格asceptic程序。使靠近颈静脉切开，轻轻取出右侧颈肌和从周围组织分离出颈静脉。 </li><li>设置输液泵和荧光显微镜。输液泵设置取决于注射器直径和所需的流量（参见2.4节）。从现在起，工作在一个黑暗的房间。 </li><li> VWF涂层： <ol><li>填的注射器用荧光标记的细菌，并将其连接到入口管。避免气泡。启动输液泵10分钟。输注时间取决于所用的剪切速率和涂层，细菌和介质，并应代表粘连的稳定状态。 </li><li> 10分钟后，洗去未结合的细菌用PBS注射器连接到入口管和启动输液泵。 </li><li>至少要花15幅图像或电影在洗涤过程后不同的位置。细菌是小的和潜在的难以集中。在此之前的体外流动的实验中，合适的焦平面可以将一滴荧光标记的细菌对盖玻片和放置盖玻片在流室进行检索。然后，搜索相应的焦平面并保存设置。 注：在体外流动实验，在清洗步骤期间捕获的图像（开始后±5分钟）确保只信号附着的细菌被捕获。 </li></ol></li><li>胶原蛋白涂层： <ol><li>添加60微克/毫升VWF的荧光标记的细菌刚开始灌注前。填的注射器用补充有60微克/毫升的VWF荧光标记的细菌或荧光标记的细菌，并将其连接到入口管。避免气泡。重复步骤2.3.5.2 2.3.5.3至</li></ol></li><li>内皮细胞： <ol><li>激活的内皮细胞通过灌注用的Ca 2+ -ionophore A23187的0.1毫溶液（原液的10mM溶解在二甲基亚砜（DMSO））的DMEM以相同的剪切速率为细菌灌注10分钟通过灌注用0.1毫米。重复步骤2.3.5.2 2.3.5.3来。 </li></ol></li></ol></li><li>计算剪切速率和剪切应力如下。 剪切速率= 6Q / WH 2 其中：Q：流速在毫升/分钟，瓦特：在厘米宽度中，h：高度厘米 剪切应力（τ）=剪鼠前粘度（μ） 其中μ：媒体：0.01达因x秒/厘米2，全血：0.04达因x秒/厘米2 </li><li>图像分析<ol><li>使用带有黑白摄像机倒置荧光显微镜获取实时图像，并使用图像处理软件开发。用1.5秒的曝光时间。采取多个快照（至少15）随机分布在流室的涂层表面，并将其保存在相应的文件格式。 </li><li>与ImageJ的图像进行分析。减去背景来从图像中删除（处理过程 - Substract背景）平滑连续的背景和定义的阈值来设置下限和上限阈值，分割灰度图像转换成所关注的特征。测量限于阈的区域。 </li><li>比较细菌粘附，表示为荧光区域， 例如，使用统计分析软件。比较使用单向ANOVA或叔基团WO尾学生的t检验。报告所有值平均值±标准平均（SEM）的错误。考虑&lt;0.05显著一个P值（* P &lt;0.05; ** P &lt;0.01; *** P &lt;0.001）。 </li></ol></li></ol> 3. 体内肠系膜灌注模型<ol><li>鼠标的制备/手术<ol><li>实验前禁食的小鼠的夜晚，以限制排便。 </li><li>通过皮下注射丁丙诺啡（0.1毫克/公斤体重（BW））20-30之前的手术分钟得到6-8周龄小鼠（C57BL / 6）的手术前镇痛。 </li><li>通过腹膜内注射氯胺酮（125毫克/千克体重）和甲苯噻嗪（12.5毫克/千克体重），麻醉鼠标。通过检查反射踏板。适用兽医药膏，以防止干燥。 </li><li>鼠标放置在一个热控制的加热垫在37℃下在显微镜托盘。由于这是一个终端的程序，也没有必要为严格asceptic程序。使靠近颈静脉切开，轻轻取出右侧颈肌和从周围组织分离出颈静脉。 </li><li>插入一个2法国静脉导管插入右颈静脉输液荧光标记的细菌或其它解决方案。通过中线腹部切口打开腹腔，并用棉签传播系膜并以可视化的肠系膜动脉和小静脉循环。 </li><li>将鼠标在右侧上的透明板和固定套管用胶带。使用热敷，以防止体温过低。为了防止组织脱水，降500微升0.9％氯化钠的肠子。 </li></ol></li><li>细菌粘附的荧光显微镜肠系膜循环<ol><li>在黑暗的房间工作。用棉签来固定船只和可视化他们倒置显微镜下。 </li><li>局部应用5微升10毫米的解决方案邻f显示的Ca 2+ -ionophore A23187溶解在DMSO中。 10秒后，注入100微升标记的细菌（参见步骤1）通过颈静脉导管。花时间推移图像。后的实验结束后，根据机构批准的指南安乐死的小鼠。 </li></ol></li><li>图像分析<ol><li>获得使用倒置荧光显微镜，采用了黑白摄像机和使用任何图像软件开发捕获的实时图像。应用自动曝光时间和特定于所使用的设备的对比度优化。 </li><li>获得使用工具栏中的“收购”工具（多维收购 - 时间）时间推移图像使用1000张/秒的40个循环。将图像保存在一个合适的图像文件格式。 </li><li>使用ImageJ分析软件来测量每幅图像的荧光信号的区域处理的图像。定义阈值来设置下限和上限阈值，分割灰度图像到f中感兴趣的功能设定。标识感兴趣（血管）的区域，并测量限于阈值和感兴趣的区域的面积。比较细菌粘附，使用任何统计或制图软件表示为荧光区域。 </li></ol></li></ol>

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A., Lehrer, S., Sohn, H. Y., Conzen, P., Pohl, U., Kr&#246;tz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+cyclooxygenase-2+enhances+platelet+adhesion+in+hamster+arterioles+in+vivo.">Selective inhibition of cyclooxygenase-2 enhances platelet adhesion in hamster arterioles in vivo.</a> Circulation. 110 (14), 2053-2059 (2004).</li><li>Kim, K. H., Barazia, A., Cho, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+heterotypic+platelet-neutrophil+interactions+on+the+activated+endothelium+during+vascular+inflammation+and+thrombus+formation+in+live+mice.">Real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice.</a> J Vis Exp. 2 (74), (2013).</li></ol>

作者什么都没有透露。

剪应力是为早期细菌粘附于血管壁和用于随后产生的血管内或心内膜赘生物和转移性感染4,5的一个关键因素。我们的体外和体内模型中所述互补研究生理剪切应力下的血管内感染的发病机制。这些模型使我们能够确定血管性血友病因子结合蛋白（vWbp）作为主要的S.金黄色葡萄球菌蛋白流下互动与一个受伤的血管壁露出VWF 4。 血管内感染，尤其是感染性心内膜炎，值得关注，不仅是因为败血症所致的器官衰竭而死亡，但由于也本地和遥远（&#39;转移&#39;）的并发症。引起感染性心内膜炎和转移性感染，细菌有附着​​到容器壁上，从而抗蚀剂流动的血液的剪切应力。最对细菌的研究毒力因子已经在静态条件下被执行。然而，这些相互作​​用建立可能无法承受剪切力，并研究流量条件下，可以揭示新的，以前没有认识到的因素在细菌宿主相互作用。 使用微平行流动室，我们和其他人已经表明的VWF对血管粘附的重要性。在剪切应力，VWF逐步展现出来的，从它的休息球状结构，并公开通过其受体GPIb的血小板6交互的A1域。流动腔室已被广泛用来研究血小板功能7。 值得注意的是，还S.下流动葡萄球菌粘附需要的VWF，以及尤其是在剪切露出的A1结构域。我们确定vWbp调解VWF结合。 vWbp是凝固酶，有助于S.金黄色葡萄球菌病理生理通过激活宿主的凝血酶原。 Staphylothrombin，水库ulting细菌凝固酶和凝血酶原复合物，纤维蛋白原转变成不溶性纤维蛋白8,9。我们的研究表明，vWbp不仅激活凝血酶原，但触发细菌血纤维蛋白-血小板聚集，这提高下流动4,10,11的粘附到血管的形成。 体外流室模型允许不同的球员 ​​在细菌粘附研究细胞或基质成分。细菌毒力因子可以通过使用突变体或无害的细菌表达特定表面蛋白进行研究。可替代地，药理抑制剂或阻断抗体可以被添加到在流动室中的介质。的宿主因素如细胞外基质的不同成分的作用可以通过使用盖玻片与不同涂层进行研究。盖玻片也可覆盖有内皮细胞，其中的活化状态可以通过添加特异性刺激进行调制。阿帕室温从血管壁，宿主血细胞和血浆蛋白质的贡献可以通过加入这些因素，流动介质进行研究。因此，增加了复杂性不同的条件可以在层流的标准条件进行研究，以解开，允许细菌附着于血管壁体内的相互作用。 在体外模型中鉴定相互作用随后研究的动物模型，以测试其相关性在一个复杂的有机体。其它体内模型来研究下流动的动态相互作用已被描述，如仓鼠背皮褶腔12和提睾模型13。相比较而言，这里所描述的肠系膜灌注模型提供，因为它易于使用的几个优点，有可​​能以改变承载小鼠的遗传背景，并评价药物干预。 总之，所描述的模型提供研究表面蛋白不仅S的可能性金黄色葡萄球菌 ，但在不同的主机背景许多其它微生物，以更好地理解血管感染的发病机制。

To establish endovascular infections, pathogens require a mechanism to adhere to the endothelium, which lines the vessel wall and the inner surface of the heart, and to persist and establish an infection despite being exposed to the shear stress of rapidly flowing blood. The most frequent pathogen causing life-threatening endovascular infections and infective endocarditis is Staphylococcus aureus (S. aureus)1. 
Various bacterial surface-bound adhesive molecules mediate adhesion to host tissue by interacting with extracellular matrix components. These MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) recognize molecules such as fibronectin, fibrinogen, collagen and von Willebrand factor (VWF). MSCRAMMs are important virulence factors of S. aureus and are implicated in the colonization and invasion of the host2. Most studies on these virulence factors have been performed in static conditions, and thus may not be representative for human infections where initial adhesion of the bacteria occurs in flowing blood. 
In the case of bloodstream infections, bacteria need to overcome the shearing forces of flowing blood in order to attach to the vessel wall. Models that investigate the interaction between bacteria and endothelium or subendothelium under flow conditions are therefore of particular interest. 
A recent study showed that the adhesion of S. aureus to blood vessels under shear stress is mediated by VWF3. VWF, a shear stress-operational protein, is released from endothelial cells upon activation. Circulating VWF binds to collagen fibers of the exposed subendothelial matrix. Our group reported that the von Willebrand factor-binding protein (vWbp) of S. aureus is crucial for shear-mediated adhesion to VWF4. 
In this article, we present an in vitro flow chamber model where bacterial adhesion to different components of the extracellular matrix or to endothelial cells can be evaluated. To validate the findings from in vitro data, we have developed an in vivo model that visualizes and quantifies the direct interaction of bacteria with the vessel wall and the formation of bacteria-platelet thrombi in the mesenteric circulation of mice, using real-time intravital vascular microscopy.

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			<td>Brain Heart Infusion (BHI)</td>
			<td>BD Plastipak&#160;</td>
			<td align="right">237500</td>
			<td></td>
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			<td>Tryptic Soy Broth (TSB)</td>
			<td>Oxoid</td>
			<td>CM0129</td>
			<td></td>
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			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190-169</td>
			<td>D-PBS</td>
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		<tr>
			<td>5(6)-carboxy-fluorescein N-hydroxysuccinimidyl ester&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>21878-25MG-F</td>
			<td>fluorescent labeling&#160;</td>
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		<tr>
			<td>Bovine Serum Albumin Fraction V (BSA)</td>
			<td>Roch</td>
			<td colspan="2">10 735 086 001</td>
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		<tr>
			<td>Haemate-P</td>
			<td>CSL Behring</td>
			<td>PL 15036/0010</td>
			<td>VWF</td>
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			<td>Horm collagen</td>
			<td>Takeda</td>
			<td align="right">10500</td>
			<td>collagen</td>
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			<td>1-well PCA cell culture chambers</td>
			<td>Sarstedt</td>
			<td align="center" class="xl63">########</td>
			<td>plastic slips</td>
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			<td>Temgesic</td>
			<td>Reckitt Benckiser</td>
			<td align="right">283716</td>
			<td>bruprenorphine</td>
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			<td>Anesketin (Ketamin hydrochloride 115 mg/ml (100 mg/ml ketaminum))</td>
			<td>Eurovet</td>
			<td>BE-V136516</td>
			<td>ketamin</td>
		</tr>
		<tr>
			<td>XYL-M 2% (xylazine hydrochloride 23.32 mg/ml (20 mg/ml xylazine))</td>
			<td>VMD Arendonk</td>
			<td>BE-V170581</td>
			<td>xylazine</td>
		</tr>
		<tr>
			<td>2 french intravenous catheter green</td>
			<td>Portex</td>
			<td colspan="2">200/300/010</td>
		</tr>
		<tr>
			<td>0,9% Sodium chloride (NaCl)</td>
			<td>Baxter Healthcare</td>
			<td>W7124</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton swabs</td>
			<td>International Medical Product</td>
			<td align="right">300230</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+-ionophore solution A23187</td>
			<td>Sigma-Aldrich</td>
			<td colspan="2">C7522-10 MG</td>
		</tr>
		<tr>
			<td>26 gauge 1 ml syringe&#160;&#160;</td>
			<td>BD Plastipak&#160;</td>
			<td align="right">300013</td>
			<td></td>
		</tr>
		<tr>
			<td>26 gauge 1 ml syringe&#160; with needle</td>
			<td>BD Plastipak&#160;</td>
			<td align="right">300015</td>
			<td>intra-peritoneal injection</td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Centrifuge 5810-R</td>
			<td>Eppendorf</td>
			<td colspan="2">5811 000.320</td>
		</tr>
		<tr>
			<td>Glass cover slips (24x50)</td>
			<td>VWR</td>
			<td>BB02405A11</td>
			<td>Thickness No, 1</td>
		</tr>
		<tr>
			<td>PHD 2000 Infusion</td>
			<td>Harvard Apparatus</td>
			<td align="right">702100</td>
			<td>High-accuracy Harvard infusion pump</td>
		</tr>
		<tr>
			<td>Axio-observer DI</td>
			<td colspan="2">Carl-Zeiss</td>
			<td>Inverted fluorescence microscope</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td colspan="2">National Institute of Health</td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>Graphpad Prism 5,0</td>
			<td colspan="2">Graphpad Software</td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>AxioCam MRm</td>
			<td colspan="2">Carl-Zeiss&#160;</td>
			<td>Black and white camera</td>
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in vitro and in vivo model to study bacterial adhesion to the vessel wall under flow conditions

为了使血管内感染和感染性心内膜炎，细菌必须能够粘附于血管壁，同时被暴露于流动的血液的剪切应力。 要识别有助于微生物血管粘附的细菌和宿主因素，需要的研究生理剪切条件下，这些相互作​​用合适的模型。在这里，我们描述了一种在体外流动室模型，允许调查细菌粘附到细胞外基质的不同成分或与内皮细胞，并开发了一个活体显微镜模型直接可视化的细菌的初始粘合力，以在体内内脏循环。这些方法可用于鉴定所需细菌下流动的粘附的细菌和宿主因素。我们示出了剪切应力的相关性和血管性血友病因子为Staphy的粘合作用黄色葡萄球菌同时使用体外和体内模型。

金黄色葡萄球菌的粘合性的VWF，内皮下基质和内皮细胞是一种剪切应力依赖性现象要强调在间S的相互作用的剪切应力的作用金黄色葡萄球菌和VWF，我们进行灌注过的VWF在不同剪切速率涂覆的盖玻片（ 在体外灌注模型的示意图在图中给出的金黄色葡萄球菌 1。粘附到VWF从250秒的增加而增加的剪切速率-1〜2,000秒-1（ 图2），这表明高的剪切力不抑制，但加强细菌附着到VWF。 为了研究的VWF的细菌粘附于胶原的贡献，内皮下基质的主要成分，我们灌注荧光标记S.金黄色葡萄球菌在胶原中VWF的存在或不存在。在不存在的VWF，S的密合性一个ureus胶原随着剪切速率降低。然而，当VWF是存在于培养基中，S的粘着黄色葡萄球菌的增加而增加的剪切速率（ 图3）。 体外流模型还允许我们检查细菌的粘附下流动于内皮细胞。我们内皮细胞灌注与荧光标记的S.金黄色葡萄球菌在剪切速率为500〜2000秒-1。凡表示，内皮细胞被激活了的Ca 2+ -ionophore，造成VWF释放。内皮细胞的活化和随后的VWF释放，S的增加粘附金黄色葡萄球菌 （ 图4A），从而形成典型的“带状”图案的荧光标记在剪切力（ 图4B）的方向上排列的细菌集群，这表明沿着线性拉伸的VWF分子的细菌的结合。 最初在体内细菌附着在内脏静脉被VWF介由于S.金黄色葡萄球菌是能够坚持的VWF，我们用野生型小鼠（VWF + / +）和VWF缺陷型小鼠（VWF - / - ），调查细菌粘附到活化血管壁体内 。内脏静脉实时电视显微镜允许体内可视化的循环荧光标记S.黄色葡萄球菌 （ 在体内灌注模型的示意图概述示于图5）。 内皮由钙 -ionophore药理活化后，我们观察到个体的细菌和细菌的聚集体的快速局部堆积到WT小鼠的血管壁（补充视频1和2）。细菌几乎没有发生粘着上观察到VWF -deficie的活化的血管壁nt的小鼠（补充视频3）与WT小鼠的附着力相比（ 图6）。不存在的VWF降低S的能力金黄色葡萄球菌坚持以激活血管壁。 <img alt="图1" src="/files/ftp_upload/52862/52862fig1.jpg" /> 图1. 在体外流动模型的示意图。 在体外流动模型是一种多功能模型，它允许不同的剪切依赖性机制的研究，如细 ​​菌粘附于内皮下基质也是血栓形成。微平行流动室被放置在盖玻片（塑料或玻璃）与蛋白质和血管内皮细胞的不同的涂层。不同的细菌（橙色和灰色点）的密合性，可以分析和血浆蛋白，血小板和全血的存在的影响进行评估。荧光标记的血小板（蓝色椭圆形）或fibrinog烯（蓝色串）可以用在具有不同的抑制剂（黑色椭圆）组合来区分细菌和宿主因素。 S的 ​​细菌粘附的代表图像黄色葡萄球菌对VWF胶原涂层的存在（底部）或无（顶部）显示（比例尺为100微米）。 <a href="https://www.jove.com/files/ftp_upload/52862/52862fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52862/52862fig2.jpg" /> 图2.粘附S.黄色葡萄球菌对VWF增加而增加的剪切速率。微平行流动室灌注过涂覆的VWF（50微克/毫升）与荧光标记S.金黄色葡萄球菌Newman在250至2000秒-1（秒-1）在中等剪切速率（正&gt; 5）。所有结果表示为平均值±SEM表示。 * P &lt;0.05，** P &lt;0.01。 <img alt="图3" src="/files/ftp_upload/52862/52862fig3.jpg" /> 图3.粘附S.黄色葡萄球菌对内皮下膜是剪切和VWF依赖性。微平行流动室灌注过涂覆胶原（160微克/毫升）与荧光标记S.金黄色葡萄球菌Newman在250至2000秒-1在中等剪切速率（正&gt; 5）。 VWF（60微克/毫升）存在于培养基中，其中表示。所有结果表示为平均值±SEM表示。 ** P &lt;0.01。 <img alt="图4" src="/files/ftp_upload/52862/52862fig4.jpg" /> 图4.粘附S.黄色葡萄球菌对活化的内皮细胞是剪切依赖性的。微平行流动室灌注过的内皮细胞。 （ 一 ）人脐静脉内皮细胞的钙离子-ionophore一个激活23187（0.1毫摩尔），接着通过一个10分钟的灌注荧光标记S.金黄色葡萄球菌Newman在500至2000秒-1在中等剪切速率（正&gt; 5）。所有结果表示为平均值±SEM表示。 * P &lt;0.05。微平行流室灌注（B）在图像内皮细胞活化与S.金黄色葡萄球菌在1000秒的剪切速率-2 S。金黄色葡萄球菌形成了±200微米长字符串，暗示附着力VWF多（比例尺为100微米）。 <a href="https://www.jove.com/files/ftp_upload/52862/52862fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52862/52862fig5.jpg" /> 图5. 体内肠系膜灌注模型的示意图，一个右颈内静脉置管（黄线）插入氟的管理escently标记的细菌（橙色点），附加的麻醉剂或其它成分，如药物抑制剂和抗体。腹膜腔被打开，肠系膜铺展以可视化的血管（静脉和动脉）在荧光显微镜下。内皮由钙 -ionophore，其诱导的VWF的释放药理活化后，细菌可以通过颈静脉导管注射。实时视频血管显微允许循环荧光标记的细菌在体内的可视化和由此产生的细菌血小板血栓形成。 <a href="https://www.jove.com/files/ftp_upload/52862/52862fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52862/52862fig6.jpg" /> 图6的S的初始粘合金黄色葡萄球菌。 - / -小鼠对激活的内皮是在体内通过的VWF 的体内静脉肠系膜灌注之C57B1 / 6- VWF + / +和C57BL / 6- VWF模型介导的 。粘连的荧光标记S.金黄色葡萄球菌的局部活化血管壁是显著低于VWF - / -小鼠。所有结果表示为平均值±SEM表示。 *** P &lt;0.001，N&gt; 7。 <img alt="视频图1" src="/files/ftp_upload/52862/52862figV1.jpg" /> 视频1：S的实时粘连金黄色葡萄球菌活化血管壁VWF + / +小鼠。 <a href="https://www.jove.com/files/ftp_upload/52862/Video1JorienClaes.mov" target="_blank">请点击此处观看该视频。</a> 视频2：实时聚集形成和栓塞S.金黄色葡萄球菌在VWF + / +小鼠。 <a href="https://www.jove.com/files/ftp_upload/52862/Video2JorienClaes.mov" target="_blank">请点击此处观看该视频。</a> <img alt="视频图3" src="/files/ftp_upload/52862/52862figV3.jpg" /> 视频3：S的实时粘连黄色葡萄球菌对活化血管壁中VWF - - / -小鼠/ -与VWF + / +和VWF 小鼠 体内肠系膜灌注模型。五微升钙 -ionophore（10毫米）的是APPLI编到的可视化血管床的区域。羧基荧光素标记S的悬挂金黄色葡萄球菌是通过颈静脉导管注入。肠系膜循环被倒置显微镜下观察。 <a href="https://www.jove.com/files/ftp_upload/52862/Video3JorienClaes.mov" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions at JoVE.com

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 ...

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JoVE Journal

Immunology and Infection

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

10.5K 次观看.  KU Leuven. 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. 

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

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

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 ...

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 ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from 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 ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

人源化的小鼠模型来研究细菌感染瞄准微血管

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive 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 ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Vitronectin 在细菌黏附和血清耐药性中作用的实验研究

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...