作者希望承认Vertex Pharmaceuticals和囊性纤维化研究创新奖，为R.Quinn和NIH / NIAID提供资金赠款1 U01 AI124316-01，一种用于治疗多重耐药性病原体的系统生物学方法。我们还要感谢UCSD本科机械工程高级设计课程的机械和航空航天工程系，以便与这项工作的工程方面进行协作。

1.股票的研制人工痰媒体<ol><li>创建一个5％粘蛋白的溶液。添加1.0克脱水猪胃粘蛋白的〜20毫升去离子水。高压釜中加入所得溶液。 注:粘蛋白消毒会破坏它的内在结构;其他方法来灭菌粘蛋白在其干燥形式包括紫外灭菌和辐射。这些方法还没有被广泛用于WinCF系统不过。 </li><li> 2.2克氯化钾添加到50毫升的去离子水，并允许溶解。 5.0克的NaCl添加至50mL的去离子水，并允许溶解。高压灭菌这两种解决方案。 </li><li> 100毫克的鲑鱼精子DNA的添加至10mL无菌去离子水。在水浴中加热该溶液至约85℃下进行几个小时，以确保溶解。 </li><li> 5.0毫克铁蛋白添加到5.0毫升无菌去离子水。 </li></ol> 2.人工痰介质的制备<ol><li>结合以下组分:16mL粘蛋白储备溶液，2.0mL KCl储备溶液，2.0mL NaCl储备溶液，200μL蛋黄乳液，5.6mL DNA储备溶液，120μL铁蛋白储备溶液，5.78mL必需碱氨基酸溶液，5.78mL非必需氨基酸溶液和2.44mL无菌水。 <ol><li>如果出现少量沉淀物，请轻轻晃动混​​匀。 </li><li>将5.0mL培养基吸入八只无菌的15mL离心管中。 注意:每个管的化学条件可以根据需要进行操作。例如，可以将缓冲溶液和pH指​​示剂添加到每个管中，目的是比较不同pH值下的微生物行为。在代表性结果部分中显示了这一点，其具有8个不同的pH水平，从5.0到8.5，0.5个pH增量。 </li></ol></li><li>一旦介质的化学条件成功操作，冻结以供以后使用。媒体将保持稳定禅在-20℃下数月。在解冻时旋涡。 </li></ol> 3.毛细管的控制运行的制备<ol><li>在无菌biohood，填充的每一媒体250μL8个无菌微量离心管中。 </li><li>获得八个无菌的15个毫升离心管中，每管对应于在步骤3.1中提到一个微量离心管中。 <ol><li>消毒用70％乙醇溶液纸巾并晾干。一旦干燥，撕毛巾成关于每个4平方英寸件，并在一个15毫升的离心管的底部揉碎每一块。对于另外的管，喷雾和干燥的附加纸巾根据需要。 </li><li>在每个离心管的底部的纸张一个丛，稍微蘸用约1.0毫升的无菌水中每个纸丛创建一个潮湿的环境。 </li></ol></li><li>获得三个玻璃毛细管在步骤3.1制得的每个微量离心管中并毛细管油灰seala的方块NT。 </li><li>对于每个微量离心管中，填充介质3个的毛细管至约5mm从靠近管的顶部的蓝色标记程。 <ol><li>通过保持在微量离心管中的管的一端并且朝向水平方向倾斜，从而允许毛细作用于介质引导到管填充（参见图1）。 </li><li>轻轻放置带手套的手指在管的开口端停止填充，然后通过按下它分解成密封剂块密封管的另一端。 </li></ol></li><li></li></ol><img alt="图1" src="/files/ftp_upload/55532/55532fig1.jpg" /> 图1:实施例的pH梯度，填充毛细管人工痰平台。该介质是通过将管插入液体的一端，并且倾斜以促进毛细管作用加入。本例中的介质的着色是由于pH指示剂加入到帮助妖STRATE培养后在酸性电位的变化。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li>将每个组三个毛细管放入盛有无菌水，腻子密封侧完全润湿向下纸巾15个毫升离心管中。盖上管和标签。这三个毛细管是用于每个控制条件复制。 </li><li>当所有15个毫升离心管中填充有其指定的毛细管，将管在保持架上。放置架，使得管将被水平地温育（捉气泡）（参见图2）。在37℃下孵育离心管（包含湿纸上团块）内的毛细管48小时。 </li></ol><img alt="图2" src="/files/ftp_upload/55532/55532fig2.jpg" /> 图2:实例pH梯度，毛细管准备孵化。一旦三个毛细管被填充和密封，它们被放置在离心管中，在底部带有湿纸巾。然后将该管封盖并放入架子中。在孵化期间，机架必须侧向定位，如图所示，以便一旦孵化完成就可观察到气体产生。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 孵化后控制毛细管成像<ol><li>从培养箱中取出离心管架，确保管子保持水平。仔细地将毛细管从离心管中滑出，保持每组三个与其他组分离。 </li><li>将毛细管彼此排列在灯箱上，均匀排列，使管的内容物相对可见并照亮。每隔三个管子分隔不同的化学条件。 </li><li>管子对齐，灯箱开启后，直接从上方拍照。 （ 见图3 ） </li></ol><img alt="图3" src="/files/ftp_upload/55532/55532fig3.jpg" /> 图3:实施例pH梯度，对照运行，预孵育，无痰添加。人造痰培养基被添加到三套毛细管中后，pH从左到右增加。添加到培养基中的指示剂的组合导致更多的酸性管出现更多的黄色，而较少的酸性管变得更加紫色。管子水平排列，从下方照射，从上方拍摄。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="4"><li>处理控制材料的生物危害性的废物。 </li></ol> 5.痰样品接种WinCF毛细管<ol><li>在无菌biohood，填充的每一媒体225μL8个无菌微量离心管中。 </li><li>通过抽出，并用3mL注射器（塑料注射器没有针头）反复喷射痰均化痰样品。这样做，直到痰是光滑的一致性。 </li><li>添加25μL匀浆痰的在步骤5.1中制备的各微量离心管（1/10稀释在ASM介质）。然后涡管30秒以充分混合。 </li><li>媒体添加到毛细管按照相同的程序与步骤3.2至3.5。 </li></ol> 6.成像样本毛细管温育后<ol><li> 48小时的温育时间之后，按照相同的程序除去和图像毛细管与步骤4.1至4.3。 </li><li>如果存在气泡在管中，使确保拍摄的照片清楚地描绘了管中气泡和介质之间的描绘。如果存在生物膜，请确保照片可以清楚地描述其存在。 </li></ol> 7.删除下游应用程序的媒体<ol><li>为了方便下游分析，成像后从毛细管中取​​出培养基。潜在的应用包括培养和DNA / RNA测序和代谢组学分析。 注意:填充有病原体的玻璃毛细管是一个重要的生物危害因素，因此，必须使用特定的设备非常仔细地进行这些步骤。如果毛细血管破裂，则放在适当的生物危害尖锐的容器中。 </li><li>使用25号长度为0.5英寸的平头针，以移除介质。将钝头针插入管的堵塞端，以破坏密封。 </li><li>打破密封后，将毛细管上下颠倒，介质将从顶部滴出。一世f介质不会轻易滴落，使用200μL吸头的移液器，并将其插入毛细管末端时，通过向下移动移液器柱塞，将介质从管中排出。将排出的管介质收集在合适的容器（1.5mL离心管）中。 注意:对于转录组学或其他RNA分析，培养基可以直接排出到RNA稳定缓冲液中。 </li></ol> WinCF FLUD系统注意:WinCF流体加载实用程序（FLUD）系统是一套可选的补充设备，旨在优化WinCF系统的吞吐量。 WinCF FLUD系统主要由3D可打印材料组成。 3D印刷制造允许快速和容易地更换材料，以确保研究人员的最短停机时间以及最低的制造要求。设计，stl文件，3D打印说明和WinCF FLUD手册可在线补充NT。 <ol><li> 对于毛细管装载准备媒体 <ol><li>在无菌biohood，填充与每个介质的900μL8个无菌2个毫升微量离心管中。 </li><li>通过抽出，并用3mL注射器（塑料注射器没有针头）反复喷射痰均化任何痰样品。这样做，直到痰是光滑的一致性。 </li><li>加入100μL匀浆痰的在步骤8.1.1制备的每个微量离心管（1/10稀释在培养基中）。然后涡管30秒以充分混合。 </li><li>时隙中的填充和开放的微量离心管到定向成使得管是垂直旋转体管保持架。 </li></ol></li><li> 毛细管的位置 <ol><li>检索用于每个微量离心管3个的毛细管在步骤8.1。 </li><li>槽三个管入橡胶支架，使得它们与在该装置的另一端的离心管对准。确保管的标记末端远离微量离心管。将托架底部的三个孔适当地安装在支架支架上的三个螺柱上。 </li><li>将放置的毛细管放置在设备的引导通道中，将橡胶夯实牢固地固定在其中心部分以防止移动。 （ 见图4 ） </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/55532/55532fig4.jpg" /> 图4:完全装有毛细管的FLUD系统由橡胶垫片固定在其中心部分。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="3"><li> 将介质装入毛细管 <ol><li>仔细使用一只手抓住毛细管的设备末端并用另一只手握住装有微量离心管的旋转架。 </li><li>精心旋转微量离心机架，使微量离心管几乎处于水平位置，继续将毛细管慢慢推向毛细管。 （ 见图5 ） </li><li>当毛细管的端部与微量离心管中的介质接触时，确保毛细作用立即开始填充毛细管。要调整填充率和填充量，请将设备整体轻轻旋转。在执行此操作时，请注意不要将介质从微量离心管中溢出。 （ 见图6 ） </li><li>当毛细管被填充到所需的水平时，将装置水平放置在表面上，并小心地将微量离心管的架子从毛细管的端部拉出以停止填充。微量离心管现在可以一直回到垂直位置并关闭。 </li><li>通过将密封剂块压在三份一套上密封毛细管的突出端，一次密封一套，直到所有组密封。为了降低污染的风险，请将密封块的不同部分按压到每一套三份装置上（ 见图7 ）。 </li></ol></li></ol><img alt="图5" src="/files/ftp_upload/55532/55532fig5.jpg" /> 图5:具有中等管的FLUD系统部署到水平方向，准备与毛细管接触。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/55532/55532fig6.jpg" /> 图6:带有毛细管的毛细管的FLUD系统通过毛细作用加载媒质。 </str翁&gt; <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/55532/55532fig7.jpg" /> 图7:密封充上FLUD系统1个重复的同时使用密封胶座毛细管管。此密封剂块沿着被切断，以防止在密封期间与相邻一式三份组接触边缘有塑料。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="4"><li> 孵化 <ol><li>取下橡胶夯实在管midsections并提起橡胶托架关闭主设备。这应该采取一切毛细管它。现在设置的摇篮，被关押在成像架管。该RAck有三个短杆，适合摇篮，还有小导轨，可以将管子放入。 </li><li>将成像架整体设置在透明塑料保护盒中。在无菌水中浸泡少量无菌纸巾，并沿着箱子的两个短边放置，以在孵育期间提供湿度。 （ 见图8 ） </li><li>完全关闭盒子，并置于37°C的孵化器内，确保管子保持水平。孵育48小时。 </li></ol></li></ol><img alt="图8" src="/files/ftp_upload/55532/55532fig8.jpg" /> 图8:从FLUD系统转移到成像架上的橡胶支架中的毛细管，已经放置在清洁温室中，潮湿纸巾旁边提供湿气。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig8large.jpg" target="_blank">请点击这里查看更大的版本</a>是数字<ol start="5"><li> 成像和提取 <ol><li>从保护箱中取出保持管的成像架，并将其放在灯箱上，从下方照亮。使用管已经在成像架上，一式三份的组将被适当地间隔开并准备立即成像。 </li><li>将相机聚焦在管上，使得所有在视场中可见，并且来自灯箱的光提供了管染料中的颜色的足够的对比度和可视化。照片从上方。 </li><li>要提取管的内容物，请从橡胶支架中一次一组取出毛细管。轻轻地将管子提起并从支架中取出，或将其滑出。 </li><li>对于每一组三次，请按照步骤7.1至7.3中详述的提取程序。 </li></ol></li></ol>

<ol>
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D., L&#252;nsdorf, H., Lam, J. S., R&#246;mling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcolony+formation:+a+novel+biofilm+model+of+Pseudomonas+aeruginosa+for+the+cystic+fibrosis+lung.">Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.</a> J. Med. Microbiol. 54 (Pt 7), 667-676 (2005).</li><li>Quinn, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogeochemical+forces+shape+the+composition+and+physiology+of+polymicrobial+communities+in+the+cystic+fibrosis+lung.">Biogeochemical forces shape the composition and physiology of polymicrobial communities in the cystic fibrosis lung.</a> mBio. 5 (2), (2014).</li></ol>

作者没有什么可以披露的。

与CF肺的微生物构成中含有种类繁多的生物，但在肺中的条件可能对什么样的微生物可以生存和发展13，15显著的影响。具体机制，通过这些条件的变化和它们对肺微生物的确切影响是目前通常不清楚。在该实验方法中，我们提出在模拟肺细支气管基于操纵化学条件变化微生物分析。 有协议的一些关键步骤。考虑到被分析的环境的微...

本手册中描述的方法称为WinCF系统1 。 WinCF的总体目标是提供一种能够模拟粘液填充肺支气管环境的实验装置。这将允许易感系统用粘液分泌过多的表型研究肺部疾病的微生物病原体，包括囊性纤维化（CF），慢性阻塞性肺病（COPD），哮喘等。该方法专门用于研究CF，其特征在于引起肺分泌物变稠且难以清除的突变，最终填充细支气管和其他具有粘液的小通道2 。肺中的这种阻塞抑制气体交换，因为吸入的空气不再能够到达许多肺泡，并且也为细菌定居3提供了栖息地。无法预防微生物生长肺过度粘液最终导致气道复杂的慢性感染的发展。这些社区含有多种生物，包括病毒，真菌，和类似的铜绿假单胞菌的细菌，都彼此5，6，7，8相互作用。在CF肺微生物的活性被认为是参与的症状称为肺部症状加重1，9，10，11耀斑。 WinCF能够围绕这些病情加重的微生物群落行为的研究，目前正扩大到作为一个基础的实验系统研究肺微生物生态学。传统上，加重已通过从肺部取出的样品的直接分析研究。许多混杂因素使微生物B的直接分析利用WinCF系统对肺部的行为进行挑战，除去许多这些因素，更直接地研究肺微生物组织的行为，从而更好地分析粘液塞细菌中的细菌活性。 WinCF系统提供了一种以有效模拟肺部环境的方式生长和分析细菌的方法。传统的肺细菌生长方法常常涉及在传统琼脂平板上培养样品。这些方法使样品对大气氧气开放，忽略了在粘液12,13接种的肺细支气管中发现的缺氧和经常缺氧的病症。在有氧条件下在琼脂上培养不像CF肺的环境，并且可能误导临床医生和研究人员关于他们试图治疗的病原体的行为。此外，琼脂平板上的细菌可用的营养物质与实际痰中可用的不相似，这通过使用人造痰介质（ASM）在WinCF中解决。如Sriramulu 等人的假单胞菌培养物所示。 14 ，ASM包括一组模拟可用于痰微生物的资源的组件，并且还可以复制痰的物理一致性。因为病肺具有特定的微生物组织，所以这种微生物的研究也应理想地发生在肺的特定条件下。 WinCF系统能够快速分析和轻松操作实验条件，以观察微生物的变化，类似于它们在实际肺细支气管中的发生。这种技术允许接种无数种相关样品，包括痰液，唾液，其他身体分泌物和纯的或混合的细菌培养物。实验设置的性质允许立即视觉解读微生物群落行为，旨在实现许多微生物和流行程序的下游应用。这些研究是重要的，因为细菌群落组成根据其环境的生理化学条件而变化。使用WinCF可以操纵介质的化学条件来分析对细菌活性的影响。例如，可以在接种样品之前改变培养基的酸度。孵育后，可以直接比较这些条件中的每一种的细菌活性，并且可以得出关于这些痰样品中的细菌如何响应不同pH值的行为的结论。在这里，我们概述了应用WinCF系统的程序和如何操作媒体化学来研究对肺微生物的影响的例子。

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Color-Coded Capillary Tubes</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">22-260943</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cha-seal Tube Sealing Compound</td>
			<td style="width:93px;">Kimble-Chase</td>
			<td align="right" style="width:119px;">43510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mucin from porcine stomach</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">M1778</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ferritin, cationized from horse spleen</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">F7879</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Salmon sperm DNA Sodium salt (sonified)</td>
			<td style="width:93px;">AppliChem Panreac</td>
			<td>A2159</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM Nonessential Amino Acids</td>
			<td style="width:93px;">Corning cellgro</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MEM Amino Acids</td>
			<td style="width:93px;">Cellgro</td>
			<td style="width:119px;">25-030-CI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Egg Yolk Emulsion, 50%</td>
			<td style="width:93px;">Dalynn Biologicals</td>
			<td style="width:119px;">VE30-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Chloride</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">P2157500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Chloride</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">S271500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">15mL centriguge tubes with Printed Graduations and Flat Caps</td>
			<td style="width:93px;">VWR</td>
			<td style="width:119px;">89039-666</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50mL centrifuge tubes with Printed Graduations and Flat Caps</td>
			<td style="width:93px;">VWR</td>
			<td style="width:119px;">89039-656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5mL microcentrifuge tubes</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">MCT-150-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2.0mL microcentrifuge tubes</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">MCT-200-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

the wincf model - an inexpensive and tractable microcosm of a mucus plugged bronchiole to study the microbiology of lung infections

许多慢性气道疾病导致气道粘液堵塞。具有囊性纤维化的个体的肺是其粘液性细支气管产生微生物定植的有利栖息地的示例性情况。各种病原体在这种环境中相互茁壮成长，并驱使许多与CF疾病相关的症状。像任何微生物群落一样，其栖息地的化学条件对社区结构和动力学有重大影响。例如，不同的微生物在不同水平的氧气或其他溶质浓度下繁殖。在CF肺中也是如此，其中氧浓度被认为是驱动社区生理和结构。这里描述的方法被设计为模仿肺环境并以与它们引起疾病的方式更相似的方式生长病原体。然后使用这些微生物的化学环境的操作来研究化学物质肺部感染的疾病控制其微生物生态学。称为WinCF系统的方法是基于人造痰中和细毛细血管，旨在提供与存在于粘液细支气管中的氧梯度相似的氧梯度。操作化学条件，例如痰液的介质pH或抗生素压力，允许使用有色指示剂观察这些样品的微生物差异，观察气体或生物膜产生，或提取和测序每个样品的核酸含量。

在整个样本中引起的各种化学条件下微生物生长显着在某些情况下，更巧妙地在别人改变。在活动许多变化是视觉性的，只要潜伏期结束是显而易见的。在pH值操作的例子中，在整个pH值范围的试样，如图由温育后变得很明显多种因素变化很大。当无痰样品加入到培养基，温育48个小时后在整个pH值谱所表现出的唯一的变化是在培养基体积轻微蒸发（ 图9）产生?...

Watch this Scientific Journal Video about The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections at JoVE.com

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

粘液堵塞囊性纤维化的气道（CF）患者是微生物病原体茁壮成长的理想环境。手稿描述了用于研究CF肺微生物的环境中模仿其中它们引起的化学条件和疾病如何改变可驱动微生物动力学的新方法。

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

the-wincf-model-an-inexpensive-tractable-microcosm-mucus-plugged

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Pharmacology

Immunology and Infection

Unit 1

Lower Respiratory Disorders

SCIENTISTS IN ACTION

9.1K 次观看.  University of California, San Diego. 粘液堵塞囊性纤维化的气道（CF）患者是微生物病原体茁壮成长的理想环境。手稿描述了用于研究CF肺微生物的环境中模仿其中它们引起的化学条件和疾病如何改变可驱动微生物动力学的新方法。 

视频: The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

科研

免疫与感染

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

听话的哺乳动物细胞原生动物引发的细菌感染

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

方法基因水平转移的研究<em&gt;金黄色葡萄球菌</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

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方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

方法基因水平转移的研究

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响