笔者想给Dumenil实验室的所有成员，特别是西尔克·席尔瓦感谢手稿的批判性阅读。手术部门总医院东欧人乔治 - 蓬皮杜（HEGP），大卫Maladry博士。迈克尔Hivelin和帕特里克Bruneval博士，病理学系HEGP。动物设施在PARCC，由伊丽莎白于克为首。这项工作是支持的以下授权机构：玛丽·居里奖学金IEF没有。 273223（KM），ATIP-艾文莉格兰特从INSERM，CODDIM设备补助金（法兰西岛地区），FRM（倒基金会LA RECHERCHEmédicale）设备补助金，卓越联盟的IBEID实验室，ANR（法新社国立倒拉RECHERCHE）授予“臭虫式流动“。资助者在研究设计，数据收集和分析，发布的决定，或准备稿子没有任何作用。

1。风险和权限<ol><li> 当地的伦理委员会必须批准所有动物的协议，该协议是按照法国和欧洲法规对实验动物的护理和使用（Décrets87-848，2001-464，2001-486和2001-131和既定的方针进行欧盟指令2010/63/UE），并经当地伦理委员会科米特德ETHIQUE恩MATIERE德实验安尼米尔，巴黎大学笛卡儿 ，法国巴黎。编号：CEEA34.GD.002.11。 </li><li>对人体皮肤，书面知情同意书患者按照法国国家指导方针，并获得所有程序进行，并经当地伦理委员会， 科米特D&#39;评价ETHIQUE DE L&#39;INSERM的IRB 00003888 FWA 00005881，法国巴黎意见：11-048 。 </li><li> 脑膜炎双球菌是一种潜在的有害的人类病原体和处理奥尔加当所有必要的预防措施，必须采取NISM。这些注意事项包括接种疫苗和生物安全下2级的条件下工作。 </li></ol> 2。植皮<ol><li>收集人体皮肤接近嫁接时间成为可能。大多数皮肤这项工作的来自整形手术操作涉及的腹部。其他来源，如乳房，腿，甚至面部已被成功地使用。 </li><li>通过清洁皮肤表面用70％乙醇和躺在其平放在一个良好的清洁的表面，优选地在一个流罩制备人皮肤样品。 </li><li>从用皮带预置厚度皮肤的顶片200-400微米的切片。 </li><li>检查皮肤切片的完整性和削减他们成2厘米×2厘米的正方形，然后在PBS的地方（没有二价阳离子），如果有10％的血清用于短期存放（3-12小时立即或在4℃的DMEM嫁接）。 </li><li>麻醉6-8周龄SCID.bg小鼠（约20g），氯胺酮（100毫克/千克）和甲苯噻嗪（10毫克/千克），麟蹄反恐执行局的ip </li><li>一旦老鼠被麻醉，通过执行一个脚趾捏，建立疼痛反射的一种标准方法检查疼痛反应。应用凝胶对眼睛防止角膜干燥和剃除动物的左侧的肩部，其中，接枝将放在后面。按住鼠标温暖一个加热垫的整个过程。 </li><li>准备推出小鼠手术后，清洗，用70％EtOH中的剃区域。 </li><li>局部应用局部麻醉剂的接枝位点（Tronothane）并通过小心取出小鼠皮肤有一对弯曲的剪刀的表面层制备的接枝位点。要小心，不要破坏底层的血管。 </li><li>除去皮肤的面积大于人体皮肤切片的面积稍小。 </li><li>从PBS（或DMEM）去除皮肤切片，放置在移植床，保证表皮的一面朝上。步骤2.8-2.10应尽可能快地进行，保证了移植床不干燥。 </li><li>对准一个角落或组织胶边和修复。皮肤切片的其他部分，适合移植床，放置少量的组织胶周围的边缘。总是修剪人体皮肤的大小，而不是使移植床大。 </li><li>不要拉扯皮肤过紧过移植床的小鼠皮肤是非常灵活的。 </li><li>确保移植物是固定的组织胶在每个角落。 </li><li>清洁与碘溶液的区域。 </li><li>套用一个创可贴与缓冲区域对移植物。确保创可贴是坚定的，但不紧，而动物的呼吸不受影响。 </li><li>修复创可贴包扎带磁带。 </li><li>让动物恢复上一个温暖的表面。 </li><li>一旦醒来，回到动物笼子。动物圈养2-3个笼子。 </li><li>恢复疼痛或困难的症状时定期检查小鼠。嫁接后10-14天，取出创可贴，小心不要破坏移植物。根据我们的经验术后疼痛是小正常。然而动物应每天监测特别是在以下操作中的几天。疼痛或痛苦的标准有以下几种。 <ul><li>体重下降超过10％ </li><li>物理外观，皮毛，驼背姿势，眼睛</li><li>增加呼吸或心脏速率</li><li>减少或不寻常波动。 </li><li>体温上升</li><li>万一疼痛的体征检测paracetamole口服给药（300毫克/公斤，每4小时）。人性化的端点被严格遵守，如果疼痛持续。 </li></ul></li><li>如果移植物看起来干燥适用婴儿油每隔2-3天，以保持肌肤水嫩。 </li><li>接枝准备好实验后21天移植。 </li></ol> 3。感染<ol><li>感染前一天，由裸奔到GCB琼脂平板上用适当的抗生素准备菌株。在5％的CO 2一夜之间成长在37℃。 </li><li> 活菌制剂 <ol&gt; <li> Reinoculate细菌到5ml的人类内皮细胞SFM培养基中含10％血清的OD 600 0.05。 </li><li>孵化一边摇动2小时在恒温箱（37℃，5％CO 2）与松盖子，直至约OD 600 0.1细菌密度。 </li><li>离心细菌3分钟15,000转。 </li><li>去除上清，重悬在PBS中，然后再次离心。 </li><li>两次重复洗涤步骤（2.2.4-2.2.5）。 </li><li>重悬细菌沉淀在PBS中。 </li><li>测量外径600。 </li><li>使无论是外径600 0.1（108 CFU / ml），或外径600 1.0（10 9 CFU /毫升）的文化。从这个已知浓度，稀释至10 7 CFU /毫升。试着编写细菌接近注射时间成为可能。 </li></ol></li><li> 老鼠 <ol><li>称取小鼠。 </li><li>麻醉小鼠用氯胺酮（100毫克/千克）和赛拉嗪（10毫克/千克）腹腔注射</li><li>一旦睡着了，C赫克的疼痛反应是不存在由脚趾捏，用热垫保持温暖的小鼠，并把眼药膏，以防止干燥。 </li><li>给每只小鼠8毫克人转的，悬浮在生理盐水或PBS，腹腔注射</li><li>取一小滴血从尾部蔓延到一个琼脂平板上检查血是之前感染无菌。 </li><li>经眶后或尾静脉注射静脉注射100微升10 7 CFU /毫升细菌培养（106 CFU总）。 CFU采取5分钟后注射三围是一致的，无论注射部位。 </li><li>几分钟后，剪断尾部和传播血样到琼脂平板上以适当的抗生素与一种稀释10倍来建立血液CFU紧随感染。 </li><li>封尾与组织胶。 </li><li>注射后，稀释细菌接种并传播到琼脂平板上用适当的抗生素，以建立CFU。 </li><li>让老鼠恢复上一个隔热垫，直到他们和移动</li><li>在6小时后感染吸取少量的血液从尾巴，稀释和板建立CFU 6小时。 </li><li>所有培养琼脂平板上，37℃，5％CO2过夜。 </li></ol></li></ol> 4。牺牲<ol><li>在感染选定的时间，麻醉小鼠氯胺酮（100毫克/千克）和赛拉嗪（10毫克/千克）腹腔注射</li><li>一旦睡着了，按住鼠标温暖，隔热垫。 </li><li>当反射性疼痛已经消失了（脚趾捏），剪断尾尖和抽血。 </li><li>蔓延到血液琼脂平板上用适当的抗生素; 5微升直接和10μl1:10稀释建立CFU。 </li><li>颈椎脱位牺牲鼠标。 </li><li>根据所有相关机构和道德准则牺牲鼠标后，确认死亡缺乏心脏的跳动。做一个正中切口，通过胸廓切开，使切口在听万吨。 </li><li>收集尽可能多的血尽可能从心脏/胸腔和地方在无菌试管中。 </li><li>除去内脏（ 如肝，脾，脑）进入无菌板。 </li><li>去除皮肤移植物和小鼠皮肤的部分进入无菌板。 </li><li>配置鼠标的身体。 </li><li>后采集的血液已被允许凝结15分钟，离心它15分钟，以3,000 rpm。 </li><li>从血液和存储中删除血浆在-80℃下用于以后的分析，例如用于细胞因子检测通过ELISA或流式细胞仪为基础的方法。 </li></ol> 5。器官CFU计数<ol><li>称取均质包含每个之前，采取活检标本500微升PBS管。 </li><li>通过各个组织4毫米活检标本用无菌活检穿孔。 </li><li>地方活检到含有500微升PBS预先称重均质管。均质皮肤样本为2 mm珠，使用更小的珠子其他器官。 </li><li>将剩余的吨在4％的多聚甲醛（PFA），并储存在4℃下进行显微镜问题（见下文）。 </li><li>称取均质含有活组织切片检查，以确定活检重量（为CFU / mg组织以后计算）管。 </li><li>均质样品。 <ol><li>均质肝，脾，脑，在6000转，持续30秒。 </li><li>均质皮肤样品以6,000 rpm进行30秒，并在必要时重复。 </li></ol></li><li>从各均化管扩散稀释到琼脂平板上，并在5％CO 2生长过夜，在37℃建立器官的CFU计数。 </li></ol> 6。组织学/免疫组化<ol><li> 固定术 <ol><li>修正了在4％PFA组织一夜之间在4°C。固定步骤可能会较长，如果存放时间较长的地方组织在0.25％的煤灰。 </li><li>冲洗组织在PBS中，然后在20％蔗糖溶液放置并返回到4℃过夜，或直到组织下沉在该溶液中。 </li><li>在PBS冲洗组织，切成一定尺寸和地点华侨城解决方案（无论是在组织模具或粘附于基板）。 </li><li>快速地冻结在培养皿上漂浮在液态氮的组织中。 </li><li>冻结后，将组织到干冰转移至-80℃存放前。 </li></ol></li><li> 切片 <ol><li>从-80℃取出的组织，并允许平衡至-20℃的低温恒温器。 </li><li>切片的组织的厚度为约10-15微米，并放置到涂覆的显微镜载玻片。 </li><li>店幻灯片在-20°C，直到需要。 </li></ol></li><li> 染色法 <ol><li>让幻灯片来室温。 </li><li>借鉴与疏水笔滑动片左右。等待5-10分钟。 </li><li>再水合/洗涤切片在PBS中5分钟，重复两次。 </li><li>通透切片在0.1％的Triton的PBS 10分钟。 </li><li>在PBS 2-3X 5分钟洗净。 </li><li>在PBS块0.1％的Triton 1％BSA的10〜60分钟之间取决于所用的抗体或污垢。 </li><li>除去封闭液，并添加所需的模块缓冲液稀释一抗。孵育确定的时间。 （通常1小时，在室温或过夜，4℃）。 </li><li>在PBS 2-3X 5分钟洗净。 </li><li>应用适当稀释在博克缓冲器的第二抗体; DAPI通常包含在这一步骤。孵育确定的时间，通常1-2小时，室温下，在黑暗中。 </li><li>在PBS 2-3X 5分钟洗净。 </li><li>在山褪色保护安装介质有玻璃盖和密封用指甲油。 </li><li>图片立刻染色玻片或在4°C店</li><li>用共聚焦或荧光显微镜设置了适当的用荧光滤镜/激光进行成像。 </li></ol></li><li> 组织学 <ol><li>允许使用幻灯片组织学来室温。 </li><li>使用作为染色玻片TANDARD苏木精/伊红染色协议。 <ol><li>放置在玻片架的幻灯片。 </li><li>转机架解决方案之间的幻灯片按以下顺序： </li><li> 2秒进入自来水。 </li><li> 3分钟在过滤苏木素液。 </li><li> 10秒到自来水。 </li><li> 10秒到蒸馏水中。 </li><li> 10秒为乙醇100％。 </li><li> 2秒进入过滤曙红。 </li><li> 10秒到自来水。 </li><li> 10秒为乙醇100％ </li><li> 2分钟到二甲苯×3（浴I，II，III）。 </li><li>马上转移，并添加几滴胶水固定到顶部上每个幻灯片，地方盖玻片，排除气泡，让干几个小时。 </li><li>一个标准的白色光显微镜下的图像幻灯片。 </li></ol></li></ol></li></ol>

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L., 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=Transgenic+mice+expressing+human+transferrin+as+a+model+for+meningococcal+infection.">Transgenic mice expressing human transferrin as a model for meningococcal infection.</a> Infect. Immun. 75, 5609-5614 (2007).</li><li>Melican, K., Michea Veloso, P., Martin, T., Bruneval, P., Dumenil, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+Neisseria+meningitidis+to+dermal+vessels+leads+to+local+vascular+damage+and+purpura+in+a+humanized+mouse+model.">Adhesion of Neisseria meningitidis to dermal vessels leads to local vascular damage and purpura in a humanized mouse model.</a> PLoS Pathog. 9, (2013).</li><li>Murray, A. G., 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=Human+T-cell-mediated+destruction+of+allogeneic+dermal+microvessels+in+a+severe+combined+immunodeficient+mouse.">Human T-cell-mediated destruction of allogeneic dermal microvessels in a severe combined immunodeficient mouse.</a> Proc. Natl. Acad. Sci. U.S.A. 91, 9146-9150 (1994).</li><li>Nassif, X., 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=Antigenic+variation+of+pilin+regulates+adhesion+of+Neisseria+meningitidis+to+human+epithelial+cells.">Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells.</a> Mol. Microbiol. 8, 719-725 (1993).</li><li>Geoffroy, M. C., Floquet, S., Metais, A., Nassif, X., Pelicic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+analysis+of+the+meningococcus+genome+by+gene+disruption:+resistance+to+complement-mediated+lysis.">Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis.</a> Genome Res. 13, 391-398 (2003).</li><li>Ho, M., Hickey, M. J., Murray, A. G., Andonegui, G., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Plasmodium+falciparum-endothelium+interactions+in+human+microvasculature:+mimicry+of+leukocyte+recruitment.">Visualization of Plasmodium falciparum-endothelium interactions in human microvasculature: mimicry of leukocyte recruitment.</a> J. Exp. Med. 192, 1205-1211 (2000).</li></ol>

作者宣称没有竞争的财务权益。

动物模型是细菌致病的研究非常重要。这是不可能完全模仿体内环境在细胞培养物和它正变得明显，宿主-病原体相互作用是由许多动态因素的影响。一些临床上重要的病原体，如N.人类特异性脑膜炎双球菌 ，艾滋病病毒，丙型肝炎病毒， 恶性疟原虫，单增李斯特菌和伤寒杆菌限制了使用体内模型对这些感染。然而，当我们开始明白这是参与特异性感染措施，正在制定人性化的模式。这里描述的协议是一个演示，这跟人介绍微血管到小鼠体内，使体内感染北路广脑膜炎 ，导致血管损伤和偶尔紫癜皮疹的发展。 利用该模型，我们已经能够确定该TFP的粘接性能都参与了体内血管定植通过使用细菌的突变体，且血管损伤减小在无粘附性18。此前，循环细菌产物有牵连的这种损害，但我们的研究结果表明局部黏附和血管定植了决定性的作用。这开辟了新颖的治疗目标发展的新的可能性。如果致病细菌的粘附可药用阻止它也可能防止皮肤的病变的发展，并导致对脑膜炎球菌幸存者在组织坏死，清创术和截肢方面更好的结果。工作也展示了感染的复杂性和免疫反应和凝血级联的参与。我们确定感染小鼠的血清中人类细胞因子信号尽管相对少量的人类内皮细胞存在18。这个指示的显著细胞因子应答，随着鼠标的免疫细胞群的渗入区。 动物模型当然可以永远不能完全复制人类疾病并且所有结果从他们身上获取了必须考虑到这一点。例如，在该模型中的血液循环和细胞是小鼠来源的，我们不能忽视它们可以有不同的行为对人类细胞。这样做的优点然而，如在我们最近的出版物18，是由从该循环的小鼠细胞的人类血管内皮细胞分化的信号始发的能力。在此模型中使用的小鼠的免疫功能低下的背景也将允许人的免疫细胞群的同种异体传递，增加了进一步的“人源化”的方面。然而小鼠的免疫功能低下的背景可能会掩盖对于NK细胞，T细胞或B细胞，它们都缺乏或缺陷在该模型中的作用。相对肖尔在这个模型中采用t时间内（24小时）主要关心的是先天的反应，但对于长期的感染和免疫等方案的开发可能需要探讨。 皮肤是一种重要的感染部位为N。脑膜炎但具有相对少量的人力船也意味着数据外推到一个涉及许多器官全身性感染是困难的。虽然这种模式可以用于皮肤病灶发展的研究中，不包括脑膜炎双球菌感染，如上皮细胞和血脑过路的重要步骤。这些人性化的模式需要进一步发展，以解决这些感染等方面。不过这款机型提供了众多人的特定的病原体，特别是那些针对血管潜力巨大。

脑膜炎双球菌败血症是一种常见的致命性血液生感染引起的细菌病原体脑膜炎奈瑟菌 。脑膜炎双球菌败血症患者往往表现为对自己的皮肤瘀点或紫癜样皮疹先前已用所造成的循环细菌和细菌产物1血管破坏有关。从临床患者的皮肤活检显示与微血管相关的细菌，经常加注容器2。除了 ​​细菌，广泛血栓形成，凝血，充血和血管渗漏是出现在紫癜地区3-5。此血管损伤可以导致皮肤和周围组织的广泛坏死，导致清创，甚至截肢中脑膜炎球菌幸存者。了解如何感染导致该血管损伤重要的是要优化的预防和治疗策略。多数对脑膜炎双球菌败血症研究已经在体外被执行上由于N的人类特异性的人细胞系中脑膜炎双球菌 。感染的许多方面进行了研究， 在体外包括细菌粘附，宿主细胞应答以及细胞因子反应6-9。 IV型菌毛（TFP）有牵连的主要黏附细胞器为N。脑膜炎奈瑟氏菌的上皮和内皮细胞10，这也被证明N的那粘附脑膜炎奈瑟氏菌的宿主细胞是剪切应力依赖性，因此被认为与血液流速在微血管11。这表明，细菌在体内遇到的动态应力是非常重要的发病机制。然而它是非常困难的小血管微环境模拟体外 。 的粘附受体的TFP 奈瑟菌仍是未知的，因此敲入战略，以实现细菌附着在动物模型不能在这个时候可以设想。 CD46被认为是TFP受体和转基因动物生产作为小鼠模型。然而，在感染这些动物不会导致大量的感染或皮疹发展12,13。已经被描述为奈瑟感染的菌血症方面的其他动物模型中考虑的细菌倾向于人转铁作为铁源14,15。无论是补充人体转铁蛋白或转基因导致增加细菌负荷在血液流在延长的时间内表达，但是，这个模型显示没有细菌粘附或皮疹发展16,17。 在这个协议中，我们描述了在其中人体皮肤，包括真皮的微血管，被移植到免疫缺陷小鼠18,19的人源化小鼠模型。这将导致在功能性人类血管，灌注用鼠标循环。结合人转铁蛋白补充剂，此米ODEL占至少两个N的人类特定方面脑膜炎奈瑟氏菌，即人类内皮细胞和人转铁蛋白， 在体内环境。N.脑膜炎静脉引入到这个模型特别坚持以人血管内皮细胞，产生病理类似于什么是报道在临床患者中，包括血管损伤和紫癜样皮疹发展18。

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DMEM</td>
			<td style="width:161px;">Gibco Invitrogen</td>
			<td style="width:119px;">31885-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Phosphate buffered saline</td>
			<td style="width:161px;">Gibco Invitrogen</td>
			<td style="width:119px;">10010-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ketamine 500</td>
			<td style="width:161px;">Virbac France&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">LOT N&#176;VAL4243</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Xylazine</td>
			<td rowspan="2" style="width:161px;">Bayer Healthcare</td>
			<td rowspan="2" style="width:119px;">AMM N&#176; FR/8146715 2/1980</td>
			<td rowspan="2" style="width:167px;">LOT N&#176; KPO809S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">(Rompun 2%)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:247px;">Optigel&#160;&#160;&#160;</td>
			<td rowspan="2" style="width:161px;">Europhta</td>
			<td rowspan="2" style="width:119px;">Medicament autoris&#233; N&#176;3400933521134</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Lacrigel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tronothane</td>
			<td style="width:161px;">Lisa Pharma</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="40" rowspan="2" style="height:40px;width:247px;">GC agar Base</td>
			<td rowspan="2" style="width:161px;">Conda</td>
			<td rowspan="2" style="width:119px;">1106</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Human endothelium SFM media</td>
			<td style="width:161px;">Gibco Invitrogen</td>
			<td style="width:119px;">11111</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal bovine serum</td>
			<td style="width:161px;">P A A</td>
			<td style="width:119px;">A15-101</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Human transferrin</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3309</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">UEA lectin - rhodamine</td>
			<td style="width:161px;">Vector Labs</td>
			<td style="width:119px;">RL-1062</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Hematoxylin</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Eosin</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4009&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Xylene</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">534056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;"></td>
			<td style="width:161px;">Humeca BV, Holland</td>
			<td style="width:119px;">4.SB01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;"></td>
			<td style="width:161px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;"></td>
			<td style="width:161px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Equiptment Name</td>
			<td style="width:161px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sober Hand Dermatome</td>
			<td style="width:161px;">Humeca BV, Holland</td>
			<td style="width:119px;">4.SB01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Animal housing</td>
			<td style="width:161px;">Innovive</td>
			<td style="width:119px;">M-BTM-C8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Biopsy punch (4 mm)</td>
			<td style="width:161px;">Dominic Dutscher</td>
			<td style="width:119px;">30737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fast-Prep lysing matrix M tubes</td>
			<td style="width:161px;">MP Bio</td>
			<td style="width:119px;">116923050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MagNA Lyzer Green Beads</td>
			<td style="width:161px;">Roche</td>
			<td style="width:119px;">3358941001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MagNA Lyzer</td>
			<td style="width:161px;">Roche</td>
			<td style="width:119px;">3358976001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vectashield mounting media</td>
			<td style="width:161px;">Vector Labs</td>
			<td style="width:119px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetbond</td>
			<td style="width:161px;">3M</td>
			<td style="width:119px;">1469SB</td>
			<td>Tissue Glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">OCT tissue tek</td>
			<td style="width:161px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
		</tr>
	</tbody>
</table>

humanized mouse model to study bacterial infections targeting the microvasculature

脑膜炎奈瑟菌引起严重的，常常是致命的败血症，当它进入人体血液流。感染导致的导致血管渗漏，紫癜皮疹和最终组织坏死的发展血管大面积损坏。研究这种感染的发病机制先前被人类特异性的细菌，这使得在体内模型难以限制。在这个协议中，我们描述了一种人源化模型用于这种感染在其中人体皮肤，含真皮微血管，接枝到免疫缺陷小鼠。这些血管吻合用鼠标血液循环，同时保持自己的人性化特点。一旦引入这种模式，N。脑膜炎奈瑟氏菌粘附只对人血管，导致广泛的血管损伤，炎症和紫癜皮疹的在某些情况下的开发。该协议描述了小说的嫁接，感染和评估步骤此模型N的XT 脑膜炎双球菌感染。该技术可以被应用到感染的血液流众多人的具体病原体。

CFU计数该N.在这些代表性成果用于脑膜炎菌株为N。 8013 脑膜炎克隆12，C群的临床分离株，表达了一个类I SB菌毛，OPA - ，OPC - ，PilC1 + / PilC2 + 20。该菌株已被工程化以从染色体插入18表达绿色荧光蛋白（GFP）。细菌菌落形成单位计数通过计数琼脂平板上的菌落数并计算任一CFU /毫升的血液或从已知体积镀组织的菌落形成单位/毫克建立。血球计数显示，5分钟静脉注射10 6 CFU的细菌后平均1.5×10 5 CFU /毫升在血液中循环（ 图1A）有。 6小时后计数平均为4.8×10 4 CFU /毫升。通过24小时的平均计数为2.4×10 4 CFU /毫升，但在本组10只小鼠中，5例没有检测到循环的细菌，而其他5有比较高的计数（ 图1A）。从皮肤样品之CFU计数表明大部分具有相当大的数，在人体皮肤的小鼠，平均为2.1×10 4 CFU的组织在6小时/毫克和组织在24小时4.4×10 2 CFU /毫克（ 图1B）的。对侧小鼠皮肤样本显示没有细菌数为24小时，6小时只有一个非常低的数字，显示出强烈的偏好N。脑膜炎在移植皮肤（ 图1B）的人类血管。在一般的细菌计数非常低到无法检测的采样中，虽然2动物那样，可能是由于受污染的血液，因为它们具有高循环细菌数量（ 图1C）相关节目的计数的其他器官。该模型也可以用于确定致病因子在体内的作用，例如，IV型PI李。细菌菌株与在pilD基因 21限定的突变，从而导致菌株缺乏TFP，以及pilC1基因8，缺乏建议TFP“粘附素”被引入到模型中。这些突变导致没有细菌黏附在人体皮肤移植，证实TFP是打在黏附在体内（ 图1D）的关键作用。 免疫组织化学/组织学在这些实验中，我们使用N。脑膜炎奈瑟氏菌菌株中表达的绿色荧光蛋白（GFP），以使荧光检测，而不需要二次染色。人类血管是用标记物对人类内皮细胞染色，或者CD31（PECAM-1）或外源凝集素荆豆凝集素（UEA）18,22。 的UEA偶联到罗丹明允许一步法染色（ 图2A-C）。细胞核可以被染色编辑用DAPI，以帮助确定组织结构（ 图2A-B）。 使用标准的苏木精/伊红染色进行组织学检查。皮肤的表皮/真皮边界是清晰可辨。炎症，血栓形成和血管渗漏浓缩至船只靠近该边界24小时感染（ 图2D）之后。血栓形成是可见几个小真皮血管，常伴有充血和炎症。泄漏的红血细胞出来进入组织可以看出，显示的血管损伤的广泛程度。在未感染小鼠移植皮肤的组织病理表现正常，没有明显区分炎症18。在大约30％的感染的细菌粘附于皮肤导致肉眼检测紫癜（ 图2E和2F）的发展。 <img alt="图1" fo:content-width=<html> “5英寸”FO：SRC =“/ files/ftp_upload/51134/51134fig1highres.jpg”SRC =“/ files/ftp_upload/51134/51134fig1.jpg”/&gt; 图1。 CFU计数（A）每毫升血液中5分钟，6小时，24小时后感染细菌CFU计数。 （B）细菌的CFU计数从在两个6小时和24小时后的感染人类的皮肤（HS）和小鼠皮肤（MS）进行比较的皮肤样品。 （三）细菌菌落形成单位计数从其他器官采取24小时后感染。 （ 四）从24小时后感染从感染了野生型小鼠全取人类和小鼠的皮肤样本比较的细菌CFU计数脑膜炎 2C43污点（WT），与在pilD基因（pilD）或在pilC1基因（pilC1）一个突变菌株的突变菌株。所有的图表都显示为原始数据与中位数。图是从Melican 等 18修改“_blank”&gt;点击这里查看大图。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51134/51134fig2highres.jpg" src="/files/ftp_upload/51134/51134fig2.jpg" /> 图2。显微镜（A）投影共焦堆栈显示一个人的微血管沾上UEA凝集素（红色）接近真皮（D）表皮（五）边界。该容器是感染N.脑膜炎双球菌 （绿色）2小时后感染。 （B）一种光学片和切片投影（C）示出N。脑膜炎双球菌菌落（绿色）感染的人类容器（UEA -红色）内植皮2小时后感染。人类皮肤移植（四）苏木素/伊红染色感染24小时与N。脑膜炎双球菌 。表皮（E），真皮（D）边界是清晰可见和广泛血栓形成和microvess拥堵ELS（箭头）被认为是在真皮。炎症和某些血管渗漏（箭头）也可被识别。 （ 五）人体皮肤移植前感染。 （F）相同的皮肤移植物（E）24小时后感染N.脑膜炎显示紫癜区域（箭头）。 图2B，2D，2E，2F和从Melican 等 18改装<a href="https://www.jove.com/files/ftp_upload/51134/51134fig2highres.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature at JoVE.com

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

脑膜炎奈瑟氏菌是感染血管人类特定的病原体。在这个协议中人类微血管被移植人体皮肤上免疫功能低下小鼠引入鼠标。菌粘附到广泛的人类血管，导致紫癜皮疹通常在人类病例观察到的血管损伤和发育。

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 ...

humanized-mouse-model-to-study-bacterial-infections-targeting

Research

JoVE Journal

Immunology and Infection

14.7K 次观看.  Paris Cardiovascular Research Centre.  脑膜炎奈瑟氏菌是感染血管人类特定的病原体。在这个协议中人类微血管被移植人体皮肤上免疫功能低下小鼠引入鼠标。菌粘附到广泛的人类血管，导致紫癜皮疹通常在人类病例观察到的血管损伤和发育。 

视频: Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

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 ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

昆虫<em&gt;大蜡螟</em作为一个强大的的感染模型探讨细菌致病

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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

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 shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

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 ...

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 ...

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

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

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 ...