这项工作是由比尔和梅林达·盖茨基金会（大挑战探索奖SBG），美国国立卫生研究院（NIHR），在微生物疾病的生物医学研究中心，以及NIHR地方综合性研究网络。我们认识到，NWDA基础设施支持。我们感谢教授J.Ň韦瑟，美国宾夕法尼亚大学的教授和P赫曼斯，荷兰奈梅亨大学的肺炎球菌菌株。感谢到的EHPC的安全委员会的支持和建议，我们所有的志愿者的参与和马修·班格特拍摄的照片。

1。志愿者选拔和筛选<ol><li>健康志愿者招募海报广告和大学的网站上，根据NHS研究伦理委员会的指导。 </li><li>参与的入选标准是： <ol><li>年龄在18-60岁的成人。 </li><li>能说一口流利的英语，并能很容易沟通，移动电话和短信。 </li></ol></li><li>参与的排除标准是： <ol><li>亲密接触风险的个人（儿童，免疫抑制的成年人，老年人，慢性疾病）。 </li><li>目前吸烟者或显着的吸烟史（&gt; 10包年）。 </li><li>哮喘或其他呼吸系统疾病。 </li><li>妊娠。 </li><li>对青霉素或阿莫西林过敏。 </li><li>另外的临床试验，除非观察或在非介入相电流参与。 </li><li>无法给予充分的知情同意。 </li></ol></li><li>初步筛选的访问，大约一个星期前接种，包括一个中心的临床病史和有针对性的临床检查。 </li><li>如果发现了先前未知的异常，志愿者将被排除在研究之外，并会安排适当的调查，通过初级保健。 </li><li>在初步筛选获得，以确保白细胞计数在正常范围内的接种访问前，访问全血计数。一个洗鼻，排除自然肺炎球菌携带者（见6.1）。 </li><li>在接种前，志愿者们参与这项研究涉及的风险教育，和提供急诊病人传单，数字温度计，紧急电话号码和3天的课程阿莫西林。 </li></ol> 2。制备接种股份的<ol><li>接种股，在一个干净的环境中，必须做的下游。用移液管只应使用d为接种物的制备。所有的玻璃和塑料制品应是无菌的。我们建议准备接种股票在一个专门的房间，使用专用通风柜和孵化器。 </li><li>肺炎球菌菌株接种到血琼脂平板上。板在37℃下温育，在5％CO 2晚 。以下所有天的细菌是从血琼脂平板上刮下，并接种到胎圈的库存中保存系统。这些珠库存用于接种备料的。测试前准备接种股票珠为污染和菌落均匀性的股票。 </li><li>要准备接种股票，接种于血琼脂平板上的两颗珠子所需的肺炎球菌血清型珠股票，裸奔，以覆盖整个板。温育平板在37℃下在5％CO 2的过夜。所有进一步的的孵育将使用这些相同的条件下。同时孵化的Vegitone的过夜，以保证无菌的媒体。 </li><li>第二天早上拭子板的一半，小心不要删除任何血琼脂，和添加12毫升预热的Vegitone肉汤。重复使用的板的另一半。保留这两个12毫升小瓶在37°C，2小时或，直到浊度的变化变得明显，以先到者为准。 </li><li>加入12毫升〜40毫升预热的Vegitone肉汤调匀。重复的其他12毫升小瓶。这是时间点= 0。取出100微升，在600nm处读取光密度（OD）。取出20微升为量化的细菌菌落形成单位（CFU）的使用的方法的变化的计数由Miles和Misra（M＆M）7。孵育两个小瓶。 <ol><li>对于M＆M，把一个血琼脂平板上分为六个部分。加入180微升无菌PBS六个孔中的96孔板（U形底）。加入20μl的培养液的顶部以及和组合。串行1:10稀释样品的6倍，从第六的好丢弃20μL。将三个从顶部以及到首节的10微升滴血琼脂平板上。其他五个井重复上述步骤。确保板是干之前，反转和培养。 </li><li>第二天可见的菌落数一数部分，并记录每个稀释度。使用的稀释部30和300之间的一个计数，由三个可见的菌落数除以得到一个平均值。 </li><li>乘以稀释因子的平均数量的菌落，然后除以10（的量的10微升降）。这使CFU /μL。 </li><li>这个数字乘以1000 CFU /毫升得到。 </li></ol></li><li> M＆M方法每隔一小时，直到进行OD 600的读数和定量的细菌月中上旬对数期，外径为0.25。 </li><li>一旦达到这个OD，加灭菌的10％甘油，并准备一小瓶1毫升等分。将等分试样储存于-80℃。 </li><li>对于更浓缩的库存中，离心处理，以使其他的小瓶在3,345 xg离心15分钟，除去上清液。重悬在22.5毫升Vegiton的球团E培养基，并添加10％的甘油。准备1毫升等分并存储在-80℃下</li><li>将股票在-80°C至少48小时前解冻和量化。 </li><li>量化冷冻细菌股票的M＆M方法。单独测试三种股票的管，以确保重现性。使用稀释股票所需的接种物在生理盐水中的浓度，（如下文所述）所获得的值。 </li><li>对于接种，肺炎双球菌的数量在接种前和接种后的计算。这种定量细菌由M＆M的方法的是，用于确定肺炎球菌的接种物占所花费的时间来完成整个过程的平均数目。 <ol><li>准备接种物，稀释到所需的浓度，并执行定量分析由M＆M为“预接种”的计数的方法。 </li><li>在我们的临床研究设施，它需要团队30分钟就完成了整个接种程序。净光合速率eumococcal悬浮在生理盐水中，通常会显示在此区间内下降计数。考虑到这一点，我们让稀释后接种在室温下放置30分钟，然后执行“量化的细菌接种后的M＆M方法。 </li><li>的前，后取平均，以确定最终的接种浓度。调整“坐”的时间根据您的临床试验方案，但保持的时间间隔尽量简短。 </li></ol></li><li>所有新的股票，应送到参考实验室进行确认股票的纯度和特性。 </li></ol> 3。接种物的制备<ol><li>制备的接种计划的志愿者接种任命开始前30分钟。 </li><li>拿一个以前的库存管在-80℃的冰箱中，解冻和旋转的微量离心管中，17,000×g离心3分钟。 </li><li>温血琼脂平板上，在37℃培养箱中培养。准备在96孔板上进行细菌定量由M＆M的方法和制备稀释管，根据所要求的剂量。 </li><li>移除肉汤从离心管的上清液，将沉淀重悬于1毫升的生理盐水。此步骤将删除从接种肉汤。 </li><li>重复离心步骤。 </li><li>移除生理盐水的上清液，将沉淀重悬于1毫升的生理盐水。 </li><li>所需的接种浓度进行稀释组和量化的M＆M方法。 </li><li>以接种的志愿任命的，接种后的志愿者，在实验室重复的量化。标记管与日期和接种剂量并存储在-80℃下</li><li>从接种到接种，以确保一致性，重要的是使用相同的移液管和专用实验室设备每次。 </li></ol> 4。接种<ol><li>志愿者应舒适地坐在一个半卧位。 </li><li使用P200移液器和无菌过滤嘴的100微升的接种和尖端插入鼻腔内。 </li><li>慢慢地驱逐接种到鼻黏膜的圆周运动。 </li><li>这是非常重要的，吸头不接触的鼻黏膜上皮细胞的完整性中断，可能会导致细菌进入血液循环。 </li><li>同样重要的是接种太靠后放置，否则将跑下来的喉咙，它应位于鼻腔内。 </li><li>重复的其他鼻孔。 </li><li>让志愿者保持半卧位10分钟无嗅或擤鼻。这让整个黏膜的细菌分散时间。 </li></ol> 5。监控<ol><li>志愿者们每天监测接种。这包括一个由志愿者每天发来的短信，到下午2点之前的调查。 </li><li>如果文本我不是下午2时收到的志愿者将进行联络，确保他/她的幸福。如果他/她不回答，所分配的近亲联系，以确保志愿者的幸福。 </li><li>志愿者被要求报告任何上呼吸道症状，在每一个预约。临床工作人员会问志愿者来形容他/她的症状和会跟进任何投诉。 </li><li>抗生素的志愿者只有在三种情况下应采取的事件，他们感到不适，并且被要求把他们的研究团队，如果他们携带肺炎球菌在研究结束时，或如果他们感到不适无法联系的研究团队。志愿者是鼓励他们在任何时候都在研究过程中保持这种应急包，并在未来7天每天被要求联系的研究团队。 </li><li>我们的团队提供24/7在工作时间内与护士接触和两名医生的豪“市区重建策略”。所有的查询都通过电话和/或直接审查处理。规定是，如果需要，可以立即，直接进入传染病病房。 </li></ol> 6。洗鼻器（NW）程序<ol><li>鼻洗液（NW）进行初步筛选的访问，以及48小时，7，和14天接种后。 NW方法是采取8从Naclerio 等。的志愿者自然殖民由S.被排除在接种肺炎和跟进。 </li><li>志愿者落座舒适和头部倾斜时，从垂直方向30°。 </li><li>要求志愿者深吸了一口气，屏住呼吸，同时把自己的舌头向上和向后反口的屋顶。虽然在这个位置上，志愿者被要求，以表示他们已经准备好。 </li><li>填充用20ml生理盐水的注射器插入到鼻前空间和5 ml生理盐水被逐出。该volunteer，则立即向前倾，并迅速通过自己的鼻子呼气，铝箔碗，排出的液体。 </li><li>此过程重复3次以上，使每个鼻孔已洗涤两次，并已被用于完整的20毫升。 </li><li>游泳池所有样品一起在离心管中，并在室温的温度进行处理，发送到实验室。 </li></ol> 7。洗鼻器处理<ol><li> 10分钟，3,345×g下离心样品。 </li><li>除去上清液并存储标记的Eppendorf管中为1毫升的等分试样在-80℃下</li><li>加入100μl的STGG的颗粒介质9调匀。确保被确定，在这一点上在管中的总体积。这的稀释将用于计算CFU马车正NW。 </li><li>板的STGG到血琼脂平板上含有庆大霉素和条纹，整个板块的再悬浮颗粒的20微升下降的。 <ol><li>如果NW接种后，取出10μL从的STGG再悬浮颗粒和使用细菌定量的M＆M方法。 </li></ol></li><li>加入800μl的STGG西北管调匀。 </li><li>板25微升到血琼脂平板和25μl一个巧克力琼脂平板上，裸奔，整个板块。 </li><li>除以剩余量细菌悬浮在STGG 2冷冻管和存储介质进入在-80℃下</li><li>孵育板在5％CO 2中在37℃培养过夜。 </li><li>检查板的第二天，肺炎球菌和其他潜在的呼吸系统病原体。 </li><li>肺炎球菌确定平板上菌落形态。阿尔法溶血的菌落草拟专员形态子培养到血液琼脂与一个奥普托欣的光盘和孵育过夜。 </li><li>推定奥普托欣敏感的肺炎球菌菌落，革兰氏染色确认革兰氏阳性球菌血清型的Statens血清研究所肺炎motest乳胶试剂盒。 </li></ol>

<ol>
	<li>Ferreira, D. M., Jambo, K. C., Gordon, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+human+pneumococcal+carriage+models+for+vaccine+research.">Experimental human pneumococcal carriage models for vaccine research.</a> Trends in Microbiology. 19, 464-470 (2011).</li><li>Mera, R., Miller, L. A., Fritsche, T. R., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+and+multiple+resistance+in+Streptococcus+pneumoniae+after+the+introduction+of+the+conjugate+pneumococcal+vaccine.">Serotype replacement and multiple resistance in Streptococcus pneumoniae after the introduction of the conjugate pneumococcal vaccine.</a> Microbial Drug Resistance. 14, 101-107 (2008).</li><li>Tocheva, A. S., 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=Declining+serotype+coverage+of+new+pneumococcal+conjugate+vaccines+relating+to+the+carriage+of+Streptococcus+pneumoniae+in+young+children.">Declining serotype coverage of new pneumococcal conjugate vaccines relating to the carriage of Streptococcus pneumoniae in young children.</a> Vaccine. 29, 4400-4404 (2011).</li><li>Weinberger, D. M., Malley, R., Lipsitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+in+disease+after+pneumococcal+vaccination.">Serotype replacement in disease after pneumococcal vaccination.</a> Lancet. 378 (10), 1962-1973 (2011).</li><li>Wu, H. Y., 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=Establishment+of+a+Streptococcus+pneumoniae+nasopharyngeal+colonization+model+in+adult+mice.">Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice.</a> Microb. Pathog. 23, 127-137 (1997).</li><li>Wright, A. K. A., Ferreira, D. M., Gritzfeld, J. F., Wright, A. D., Armitage, K., Jambo, K. C., Bate, E., Batrawy, S. E. l., Collins, A., Gordon, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Nasal+Challenge+with+Streptococcus+pneumoniae+is+Immunising+in+the+Absence+of+Carriage.">Human Nasal Challenge with Streptococcus pneumoniae is Immunising in the Absence of Carriage.</a> PloS Pathogens. 8 (4), e1002622 (2012).</li><li>Miles, A. A., Misra, S. S., Irwin, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+estimation+of+the+bactericidal+power+of+the+blood.">The estimation of the bactericidal power of the blood.</a> The Journal of Hygiene. 38, 732-749 (1938).</li><li>Naclerio, R. M., 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=Mediator+release+after+nasal+airway+challenge+with+allergen.">Mediator release after nasal airway challenge with allergen.</a> Am. Rev. Respir. Dis. 128, 597-602 (1983).</li><li>O'Brien, K. L., Nohynek, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+from+a+WHO+working+group:+standard+method+for+detecting+upper+respiratory+carriage+of+Streptococcus+pneumoniae.">Report from a WHO working group: standard method for detecting upper respiratory carriage of Streptococcus pneumoniae.</a> Pediatr. Infect. Dis J. 22, 133-140 (2003).</li><li>Gritzfeld, J. F., Roberts, P., Roche, L., Batrawy, S. E. l., Gordon, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+nasopharyngeal+swab+and+nasal+wash,+using+culture+and+PCR,+in+the+detection+of+potential+respiratory+pathogens.">Comparison between nasopharyngeal swab and nasal wash, using culture and PCR, in the detection of potential respiratory pathogens.</a> BMC Research Notes. 4, 122 (2011).</li><li>McCool, T. L., Cate, T. R., Moy, G., Weiser, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+to+pneumococcal+proteins+during+experimental+human+carriage.">The immune response to pneumococcal proteins during experimental human carriage.</a> J. Exp. Med. 195, 359-365 (2002).</li></ol>

没有利益冲突的声明。

的EHPC平台有许多潜在的用途，包括用作粘膜疫苗模型，和作为替代的保护，用于测试新的蛋白质疫苗。该方法依赖于一个统一的接种技术和高品质的NW。 重现性是一个问题的接种。由于可变性质的冷冻的细菌等分，也可以是难以复制所需的剂量。所需的剂量减半或加倍的考虑范围之内。至关重要的是，细菌定量立即完成之前，和下面的接种，为了精确地确定由CFU计数量化细菌。出于这个原因，它可能是有益的，如果志愿者约会发生同步发展，只有一个的接种需要准备和完成前和接种后发生的量化电镀相距不超过30分钟。 NW方法需要coope定量的志愿者和在儿童中是一种理想的方法。如果产率是小于5毫升，该过程可重复最多使用一个额外的20毫升。戴假牙可能会降低的NW产量。如果志愿者有的封锁/鼽和生理盐水运行前方插入后，吹鼻，插入注射器一点点进一步，如果需要更多地倾斜头部。 NW是一个技术，获得更好的实践。一些体积通常会被丢失，所以其目的是为至少10毫升回报。如果志愿者可以品尝盐水在西北，后鼻咽和口咽之间的联系还没有得到充分的封闭的志愿者的舌头。如果志愿者燕子的盐水，解释的过程，强调对嘴的屋顶上推的舌头。然后重复同样的程序，以增加量。 如果NW包含一个substant的，IAL的黏液，这是最好的离心前将其删除。涡流样品放松任何可能被绑定的粘液，然后删除尽可能多的大块尽可能生理盐水尽可能小，同时消除。 先前的研究在美国完成EHPC安全，无不良事件11。为确保持续安全的EHPC平台，我们已经成立了一个紧急协议，该协议是基于24小时访问的研究团队。志愿者接触的细节，从而使他们的研究团队每天24小时。任何医疗需要的是在皇家利物浦大学医院。作为进一步的保护措施，安全委员会，临床医生和科学家组成的研究小组从外面接收每周一次的安全报告。该报告包含细菌的剂量，每名志愿者收到，是否有任何症状或疾病的报道。可以使用此报告的安全日工程研究，在外部不带偏见地评论。安全委员会也可以每天24小时在不太可能发生的紧急情况。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 猫＃ </td></tr><tr><td>甘油</td><td>费舍尔</td><td> G/0650/08 </td></tr><tr><td> 0.9％氯化钠</td><td>贝朗</td><td> 3627667 </td></tr><tr><td>脱脂奶粉</td><td> Fluka公司</td><td> 70166 </td></tr><tr><td>胰蛋白胨大豆肉汤</td><td> Becton Dickinson公司</td><td> 211768 </td></tr><tr><td>葡萄糖</td><td> BDH </td><td> 284504S </td></tr><tr><td>哥伦比亚chocolated马血琼脂</td><td> Oxoid公司</td><td> PB0124 </td></tr><tr><td> Pneumotest乳胶试剂盒</td><td>国立血清研究所</td><td> 51823 </td></tr></tbody></table>

experimental human pneumococcal carriage

在科学上是非常重要的，因为鼻咽部肺炎链球菌是两个主要的传染源和侵入性疾病的前提下，人体实验肺炎球菌马车（EHPC）。运输的模型能够准确地确定运输的免疫保护相关的免疫效果和对病原体的影响，主压在鼻咽部的利基。此外，运输检测有用的流行病学研究，包括疫苗研究的方法，可以进行比较。 目的我们的目标是到发展的EHPC的平台，这是一个安全有效的重复性，可用于下选择候选人的新型肺炎球菌疫苗运输作为替代疫苗诱导的免疫预防。这将致力于测试的候选疫苗从carria EHPC和疫苗的保护机制和说明GE 1。目前的对抗肺炎球菌结合疫苗保护儿童免受侵入性疾病，虽然新疫苗的迫切需要目前的疫苗并没有赋予最佳的保护，对非菌血性肺炎，并有证据显示非疫苗血清型，血清型替换2-4。 方法我们接种与S.肺炎悬浮于100μl的生理盐水。安全是发展的一个重要因素的EHPC模型，是通过密集的志愿者进行筛查和监测。临床医生和科学家的研究是独立的一个安全委员会，由每周一次的基础上提供客观的反馈。 细菌接种是标准化的要求，没有动物产品被接种到志愿者（蔬菜为主的媒体和生理盐水）。殖民（10 4〜10 5）所需的剂量要低得多THAŇ在动物模型中所使用的（10 7）5。检测肺炎球菌托架被增强高的体积（理想情况下&gt; 10毫升）洗鼻相对粘液无。该协议将涉及回合协议中最重要的部分。 （一）志愿者选拔，肺炎球菌接种物的制备（二），（三）接种，（四）跟进及（e）运输检测。 结果我们目前的协议已经在超过100名志愿者的安全，在使用两种不同的细菌血清型6的剂量范围。剂量范围研究使用S.肺炎 6B和23F，目前正进行，以确定最佳的接种剂量为50％的滑架。一个预测的50％的税率运输允许的EHPC模型具有很高的敏感度疫苗的有效性，研究数字小。

我们有经验，接种159人，其中35人已携带肺炎球菌。我们达到的最低剂量使用血清型6B运输为11,100 CFU/100微升每鼻孔，最高剂量为313,000 CFU/100微升每鼻孔。我们取得使用血清型23F运输的最低剂量为每鼻孔9,000 CFU/100微升，，每鼻孔的最高剂量为84,500 CFU/100微升。 我们的马车评估的方法是选择一个洗鼻，因为最近的一项研究显示，91％的志愿者发现，洗鼻咽拭子更舒适，比洗鼻，也更容易使用微生物培养检测病原体。恢复与NW接种于血琼脂平板上的微生物菌群，可以是可变的，难以阅读。庆大霉素的第一个血琼脂平板上使用，，选择肺炎球菌。巧克力琼脂板和第二血琼脂平板上，被用来检测其他潜在的呼吸系统路径的ogens这可能有助于或妨碍肺炎球菌定植。血液琼脂平板接种洒水，很容易阅读的一个例子示出在图1a中，含有许多种植物群的一个困难的板是图1b中所示。 在每一个预约志愿者被要求描述任何上呼吸道症状，如果存在，运营商和非携带者之间的比较突出。最近接种肺炎球菌145人，28主诉症状（ 表1）。八，这些携带者中，20人没有。最常见的症状，非特异性的鼻症状（ 表1）。 ，其中有全身症状，包括发烧及/或身体不适的情况下，进行的调查显示，没有证据病毒感染并发肺炎球菌疾病和症状是一致的，除了在一种情况下扁桃体炎。两种肺炎球菌阳性将Ë志愿者用抗生素治疗。一个抱怨喉咙痛和耳朵疼痛，在满足研究的医生，建议服用抗生素作为预防措施。其他志愿者扁桃体炎临床诊断。所有的志愿者迅速缓解症状。 <img alt="图1" src="/files/ftp_upload/50115/50115fig1.jpg" /> 图1。血琼脂平板上接种洒水血琼脂平板上接种洒水可以是难以阅读。1a是的板的一个例子，很容易读取与肺炎球菌清晰可见。1b是板有很多合作定植菌群，使它更难以检测，如果肺炎球菌存在。 <table border="1"><tbody><tr><td> 症状 </td><td></td><td> 运输组（n = 32） </td><td> 无运输组（n = 112） </td></tr><tr><td>所有症状</td><td></td><td> 24％ </td><td> 18％ </td></tr><tr><td>系统的</td><td>热</td><td> 3％ </td><td> 4％ </td></tr><tr><td></td><td>马氏* </td><td> 9％ </td><td> 4％ </td></tr><tr><td>当地</td><td>耳朵</td><td> 9％ </td><td> 3％ </td></tr><tr><td></td><td>鼻子</td><td> 0 </td><td> 10％ </td></tr><tr><td></td><td>喉咙</td><td> 12％ </td><td> 3％ </td></tr></tbody></table> 表1中。症状表征志愿者S.肺炎 6B接种。所有投诉的外部安全委员会进行了审查，经过充分调查，没有任何症状是由于肺炎球菌疾病。最常见的症状是喉咙痛，塞入鼻孔和类似流感的疾病。

Watch this Scientific Journal Video about Experimental Human Pneumococcal Carriage at JoVE.com

Experimental Human Pneumococcal Carriage

人体实验肺炎球菌马车运输和潜在的模型中使用的疫苗的开发提供了一种自然的模型。这种技术是有价值的，但复杂的，涉及临床风险，为人类的病原体。我们已经制定了详细的方案。

人体实验肺炎球菌运输

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Research

JoVE Journal

Medicine

15.4K 次观看.  Liverpool School of Tropical Medicine. 人体实验肺炎球菌马车运输和潜在的模型中使用的疫苗的开发提供了一种自然的模型。这种技术是有价值的，但复杂的，涉及临床风险，为人类的病原体。我们已经制定了详细的方案。

视频: Experimental Human Pneumococcal Carriage

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

实验转移的含量

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

科研

医学

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

步骤人类隐静脉<em&gt;前体内</em&gt;灌注和外部强化

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

后皮肤微移植生成一个新的方法解聚表征的组织

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

大鼠实验性生殖研究中的超声检查

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.