作者承认温家宝仔细审阅的手稿发疯。

结核护理国际标准，所有怀疑有肺结核患者应提交至少有两个痰标本细菌学检查标准2。如果可能的话，至少有一个清晨的标本应该得到，此时痰收集具有最高产量11 Xpert山地车/ RIF检测可应用于痰标本或浓缩痰沉积物准备从诱导或咳出的痰是要么抗酸杆菌（AFB）涂片阳性或阴性6，12，13拟从患者标本，对他们来说，是临床怀疑肺结核的人使用该法是：1）不接受抗结核治疗，2 ）曾&lt;7天的治疗，或3）没有收到在过去60天的治疗。要注意，它不应该被用于监测药物治疗7的影响，因为细菌的DNA可能坚持抗生素治疗后。 <p类=“jove_content”的治疗临床标本，包括使用的墨盒，仿佛发射传染性。使用适当的预防措施，如穿着一次性防护手套，实验室外套和保护眼睛，并按照您的机构的安全程序和指引。 1。咳出痰液样本 注意：进程只作为许多标本在同一时间，因为有GeneXpert DX系统模块运行测试！ <ol><li>每个Xpert山地车/ RIF墨盒上标明相应的标本ID。 </li><li>转让1.0毫升咳出痰一个圆锥形，螺丝帽的管，用无菌移液管。此外，整个标本可在原有的防漏痰收集容器处理。 </li><li>咳出痰，用无菌移液管加入2.0毫升Xpert山地车/ RIF样品试剂（2:1 V / V）。 </li><li>更换的盖子，并大力摇晃管10-20倍。另外，混合物可涡旋（30秒）。 </li><li>允许管站直立在室温下5分钟。 </li><li>大力摇动管10-20倍。另外，使用一个旋涡（30秒）。 </li><li>允许管直立站在另一个在室温10分钟。 </li><li>检查标本：样品应与液化痰不可见的团块。 </li></ol> 2。集中沉积物 注：过程中只有一次是GeneXpert DX系统的许多标本模块运行测试！ <ol><li>每个Xpert山地车/ RIF墨盒上标明相应的标本ID。 </li><li> 1.5毫升Xpert山地车/ RIF样品试剂转移到一个圆锥形，螺丝帽的管，用无菌移液管。 </li><li>新增至少0.5毫升浓缩沉积物样品试剂含有使用无菌移液管的管。 </li><li>更换盖子，摇管至五igorously 10-20倍。另外，使用一个旋涡（30秒）。 </li><li>允许管站直立在室温下5分钟。 </li><li>大力摇动管10-20倍。另外，使用一个旋涡（30秒）。 </li><li>允许管直立站在另一个在室温10分钟。 </li><li>检查标本：样品应与液化痰不可见的团块。 </li></ol> 3。载入Xpert山地车/ RIF墨盒 注意：样品墨盒30分钟内开始测试！ <ol><li>检查的Xpert山地车/ RIF盒和标本编号的标签。 </li><li>打开墨盒盖。 </li><li>使用无菌移液管，移液管吸进液化标本，直到半月板以上的最低标志是，转移到开放端口的Xpert山地车/ RIF墨盒样品。 </li></ol> 注意：免除慢慢气溶胶的形成，以尽量减少风险！ <ol start="4"><li>合上墨盒盖。 </li></ol> 4。开始测试 注：在开始测试之前，确保的GeneXpert DX系统配备与GX2.1软件，软件进口的Xpert山地车/ RIF检测到9！ <ol><li>打开计算机，然后打开了GeneXpert DX仪器。 </li><li>在Windows桌面上，双击的GeneXpert DX快捷图标。 </li><li>登录到的GeneXpert DX系统软件，使用您的用户名和密码。 </li><li>在GeneXpert DX系统“窗口，单击” 创建测试 。 扫描盒条码对话框。 </li><li>在样品编号 “框中，扫描或输入样品编号。 </li><li>扫描Xpert山地车/ RIF墨盒上的条形码。 创建测试窗口出现。 </li><li>点击开始测试 </strong&gt;，然后在出现的对话框中输入密码。 </li><li>绿色指示灯闪烁的仪器模块门打开，装入墨盒。 </li><li>关闭的大门。在测试开始和指示灯停止闪烁，并保持不断的绿色。当测试完成后，指示灯熄灭。 </li><li>等到系统在运行结束后释放的门锁，然后打开模块的门并取出硒鼓。 </li><li>弃置在适当的废物容器使用的墨盒，根据你的机构的标准做法。 </li></ol> 5。代表结果每个Xpert山地车/ RIF墨盒包括试剂MTB的复杂和RIF耐药的检测以及样本处理控制（SPC）控制目标细菌有足够的处理和监测PCR反应抑制剂的存在（S） 。探头检查控制（PCC）验证试剂补液，PCR管充填墨盒，探针的完整性，和染料的稳定性。9的Xpert山地车/ RIF检测的引物扩增的 rpoB基因含有81个碱基对的“核心”地区的一部分。五个不同颜色的荧光核酸杂交探针，称为分子信标，询问整个81 bp的核心。14每个分子信标被设计成具体的，它并没有绑定到它的目标，如果目标序列从野生型的rpoB基因不同如单核苷酸替代序列。因为只有当它们绑定到自己的目标，即野生型rpoB基因序列的分子信标荧光，没有任何一个在检测的五种颜色区分核心区域与野生型之间的保守序列和突变RIF的阻力。 SPC应该负样本的积极和消极或积极的，可以在一个积极的范例。测试结果将是“无效”，如果最高人民法院不以一种消极的测试检测。 PCR反应开始前，GeneXpert DX系统的措施，从探针的荧光信号的监测，反应管内充珠补液，探针的完整性和染料的稳定性。该委员会通过，如果从探针的荧光信号满足指定的验收标准。结果解释的GeneXpert DX系统测量荧光信号和嵌入式计算算法，并在9 查看结果窗口中显示，如下所示： 结核菌检测：山地车目标的DNA检测，这两个控制，SPC和PCC，满足分配的验收标准。较低的Ct值代表的DNA模板起始浓度较高;更高的Ct值代表低浓度的DNA模板。在结核菌检测结果没有检出“RIF的电阻检测”（ 图1A），“RIF的抵抗”（ <strONG&gt;图1B），或“RIF的电阻不确定”上会显示一个单独的行。 山地车未检出：山地车的目标DNA检测不到，这两个控件，SPC和PCC，满足指定的验收标准（ 图2）。 无效：不能确定存在或没有山地车的SPC不符合验收标准，即该样品不妥善处理，或PCR的抑制（ 图3）。注：重复与多余的试样测试！ 错误：一个或多个失败（FAIL）政协结果。山地车和SPC都显示没有结果。注：重复与多余的试样测试！如果该委员会通过（PASS），错误是由系统组件故障引起的。 图1：山地车目标DNA检测，这两个控制，SPC和PCC，满足分配的验收标准。较低的Ct值代表在更高的起点DNA模板的Ct值较高的浓度，低浓度的DNA模板。 “RIF的阻力在山地车检测结果没有检出”（ 图1A），“RIF的电阻检测”（ 图1B），或“RIF的电阻不确定”上会显示一个单独的行。 <img alt="图1A" src="/files/ftp_upload/3547/3547fig1A.jpg" /> 是目前的6。 <a href="http://www.jove.com/files/ftp_upload/3547/3547fig1A.jpg" target="_blank">点击这里查看大图</a> 。图1A山地车检测RIF的电阻没有检测到，即只有野生型的81-BP RIF的耐药决定区（RRDR）rpoB基因核酸序列。 <img alt="图1B" src="/files/ftp_upload/3547/3547fig1B.jpg" /> 图1B山地车检测; RIF的电阻检测。完成下降探测器B（箭头），其中包括职位513至517的R<html>共和的rpoB基因。在这个例子中，部分测序结果显示，检测rpoB基因突变D516V。8 <a href="http://www.jove.com/files/ftp_upload/3547/3547fig1Blarge.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3547/3547fig2.jpg" /> 图2山地车目标DNA检测;。这两个控件，SPC和PCC，满足分配的验收标准， <a href="http://www.jove.com/files/ftp_upload/3547/3547fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/3547/3547fig3.jpg" /> 图3的测试结果无效，即存在或山地车的情况下，不能确定：最高人民法院不符合验收标准，因为样本不妥善处理，或因为PCR的抑制。3547fig3large.jpg“目标=”_blank“&gt;点击这里查看大图。

<ol>
	<li>Sudre, P., Dam, t. e. n., Chan, G., C, A., Kochi, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis+in+the+present+time:+a+global+overview+of+the+tuberculosis+situation.">Tuberculosis in the present time: a global overview of the tuberculosis situation.</a> World Health Organisation Document WHO/TUB/91. 158, 1-47 (1991).</li><li>World Health Organisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+tuberculosis+control:+epidemiology,+strategy,+financing.">Global tuberculosis control: epidemiology, strategy, financing.</a> WHO report 2010. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The global plan to stop TB, 2006-2015. , (2006).</li><li>Morris, 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=Molecular+mechanisms+of+multiple+drug+resistance+in+clinical+isolates+of+Mycobacterium+tuberculosis.">Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis.</a> J. Infect. Dis. 171, 954-960 (1995).</li><li>Traore, H., Fissette, K., Bastian, I., Devleeschouwer, M., Portaels, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampicin+resistance+in+Mycobacterium+tuberculosis+isolates+from+diverse+countries+by+a+commercial+line+probe+assay+as+aninitial+indicator+of+multidrug+resistance.">Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as aninitial indicator of multidrug resistance.</a> Int. J. Tuberc. Lung Dis. 4, 481-484 (2000).</li><li>Helb, D., 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=Rapid+Detection+of+Mycobacterium+tuberculosis+and+Rifampin+Resistance+by+Use+of+On-Demand+Near-Patient+Technology.">Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand Near-Patient Technology.</a> J. Clin. Microbiol. 48, 229-237 (2010).</li><li>Blakemore, R., 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=Evaluation+of+the+Analytical+Performance+of+the+Xpert+MTB/RIF.">Evaluation of the Analytical Performance of the Xpert MTB/RIF.</a> Assay. J. Clin. Microbiol. 48, 2495-2501 (2010).</li><li>Telenti, 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=Detection+of+rifampicin-resistance+in+Mycobacterium+tuberculosis.">Detection of rifampicin-resistance in Mycobacterium tuberculosis.</a> Lancet. 341, 647-650 (1993).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GeneXpert Dx System Operator Manual. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Standards for Tuberculosis Care (ISTC). , (2009).</li><li>Boehme, C. C., 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=Rapid+Molecular+Detection+of+Tuberculosis+and+Rifampin+Resistance.">Rapid Molecular Detection of Tuberculosis and Rifampin Resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li><li>Marlowe, E. 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=Evaluation+of+the+Cepheid+Xpert+MTB/RIF+assay+for+the+Direct+Detection+of+Mycobacterium+tuberculosis+Complex+from+Respiratory+Specimens.">Evaluation of the Cepheid Xpert MTB/RIF assay for the Direct Detection of Mycobacterium tuberculosis Complex from Respiratory Specimens.</a> J. Clin. Microbiol. 49, 1621-1623 (2011).</li><li>El-Hajj, H. H., Marras, S. A. M., Tyagi, S., Kramer, F. R., Alland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampin+resistance+in+Mycobacterium+tuberculosis+in+a+single+tube+with+molecular+beacons.">Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons.</a> J. Clin. Microbiol. 39, 4131-4137 (2001).</li><li>Hillemann, D., Ruesch-Gerdes, S., Boehme, C., Richter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+extrapulmonary+tuberculosis+by+automated+GeneXpert+MTB/RIF+system.">Rapid molecular detection of extrapulmonary tuberculosis by automated GeneXpert MTB/RIF system.</a> J. Clin. Microbiol. 49, 1202-1205 (2011).</li></ol>

没有利益冲突的声明。

集成的Xpert山地车/ RIF检测山地车和RIF耐药的同时检测样品净化，免提操作，板来样加工，超灵敏的半巢式PCR，无论是在咳出痰或集中沉积物，在一个单一的易于使用的系统。该法的检测分析限制被认为是131个/毫升痰液。6 Ct值与的山地车细胞的数量之间的对数线性关系，目前拥有来自10 2 10 7 CFU /毫升痰液真实。作为临床有关的山地车浓度检测的线性范围内所有的秋天，该法提供了半定量估计的细菌负荷。该法只需要温和的实验室基础设施和培训。在于检测限至少有两个幅度较低的订单，比传统的显微镜。6特异性，是优秀的。此外，同时检测对RIF-resistanţ山地车3是在耐药结核病的时代设置的医疗点测试的显着效益。 最新的只有有限公布的检测的性能数据诊断在临床上肺结核12;。13的Xpert的山地车/ RIF检测的整体检测疑似肺结核病患者的痰结核菌的临床灵敏度为97.6％，比文化作为参考。这是一项前瞻性多中心研究，涉及五个研究基地（利马，秘鲁，阿塞拜疆的巴库，南非开普敦和德班;孟买，印度）的调查结果，共1462例。预测试整体文化研究人群中证实结核病的概率为50.7％。涂片和培养阳性的情况下，灵敏度为99.8％和90.2％，涂片阴性，培养阳性的情况下。在艾滋病毒阳性肺结核患者中，山地车/ RIF的测试灵敏度为93.9％，98.4％相比，整体检测山地车的协议。艾滋病毒阴性的患者12在低结核病患病率的设置，为89％相比，文化;涂片阴性的标本涂片阳性和72％的敏感性为98％，分别与特异性为13。灵敏度测试和未经处理的痰集中沉积物之间没有显着性差异。在同一研究中12，山地车/ RIF的测试正确检测RIF的阻力，在209 211例（敏感性99.1％）和所有RIF的易感性506例（特异性100％），测序结果时，考虑到12。研究人员发现，两个案件侦破，对RIF耐药性的歧视，表现在不同地点的类似，暗示的结果有可能广泛适用12。此外，集中沉积物比其他呼吸道痰标本，如支气管肺泡灌洗和支气管升的分泌物，具有可比的灵敏度和特异性进行了测试，13日检测的性能测试数据时，肺外结核病是新兴15。 全球结核病控制的迫切需要，允许新的结核病例的快速诊断和检测耐RIF的普遍访问山地车保健点的设置。3电流的核酸扩增方法用于检测山地车是复杂的，劳动密集型的有效工具，和技术要求。 Xpert山地车/ RIF检测有可能使结核病和耐药性，他们将能够负担得起这些设置，标准化，敏感和非常具体的诊断测试。为了方便访问，基金会（FIND）创新诊断Xpert山地车/ RIF测试谈判明显的降价。 10当前查找议价，随着折扣资格的国家名单，在互联网上提供。 </suP&gt;

<table border="1"><tbody><tr><td> 试剂/设备 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Xpert山地车/ RIF的测试</td><td>造父变星</td><td> GXMTB/RIF-10 </td></tr><tr><td>转移移液器</td><td>萨尔斯塔特</td><td> 86.1172.001 </td></tr><tr><td>锥形管，15毫升，120×17毫米，PS </td><td>萨尔斯塔特</td><td> 62.553.542 </td></tr></tbody></table>

diagnosing pulmonary tuberculosis with the xpert mtb/rif test

由于结核分枝杆菌 （MTB）的结核病（TB）仍然是一个重要的公共卫生问题：感染会影响到三分之一的世界人口1，近两百万人每年结核病死亡2普及高品质。世卫组织控制结核战略，是强调以病人为中心，对所有结核病患者治疗3山地车在快速检测呼吸道标本和可靠的抗药性测试结果为基础的药物治疗是这一战略的成功实施的先决条件。然而，在世界许多地区，结核病的诊断仍依赖于敏感，不善标准化痰显微镜方法。无效的结核病检测和耐药性的结核菌株的出现和传播日益危及全球结核病控制活动。2 有效的诊断肺结核需要的可用性 - 在全球范围内的立场 -ardized，易于使用和强大的诊断工具，将使双方的山地车复杂和关键的抗生素，如利福平（RIF），阻力的直接检测。后者的结果可以作为标记为耐多药结核菌（MDR TB）的，并已在迅速提供可靠的测试结果&gt; 95％的MDR-TB菌株。4，5报道很可能直接转化成无害的病人管理决定，最终会治愈，个别病人，并打破了结核病的传播链在社区。 造父变星（桑尼维尔，加利福尼亚，美国）的Xpert山地车/ RIF检测6，7，符合上述要求的一个显着的方式。这是1 1核酸，氨基酸放大试验）山地车痰或集中痰沉积物中复杂的DNA检测;和2）的RIF的耐药相关的 rpoB基因突变检测8它是利用造父变星的GeneXpert霉素设计。系统日在集成和自动化样品处理，核酸扩增，目标序列，采用实时PCR和逆转录聚合酶链反应检测。系统由仪器，个人电脑，条码扫描器，预装软件的运行测试和查看结果。9它采用单使用一次性Xpert山地车/ RIF墨盒持有PCR试 ​​剂和主办PCR过程。由于墨盒是自足的，被淘汰样品之间的交叉污染。6当前核酸扩增方法用于检测山地车复杂，劳动密集，技术要求高。 Xpert山地车/ RIF检测有可能使结核病和耐药普遍接入点保健设置3标准化，敏感和非常具体的诊断测试，他们将能够负担得起。为了便于访问，创新诊断基金会（FIND）谈判的意义不能价格下降。 10当前查找议价，随着折扣资格的国家名单，在互联网上提供。

Watch this Scientific Journal Video about Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test at JoVE.com

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Xpert山地车/ RIF的测试样品去污，免提操作，上板来样加工，超灵敏的半巢式PCR同时检测集成<em&gt;结核分枝杆菌</em&gt;和利福平耐药，无论是在咳出痰或浓缩痰沉积物，在约两个小时。测试规范和要求只有温和的实验室基础设施和培训。

Xpert山地车/ RIF测试与诊断肺结核

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

diagnosing-pulmonary-tuberculosis-with-the-xpert-mtbrif-test

Research

JoVE Journal

Immunology and Infection

79.8K 次观看.  University of Bern. Xpert山地车/ RIF的测试样品去污，免提操作，上板来样加工，超灵敏的半巢式PCR同时检测集成结核分枝杆菌和利福平耐药，无论是在咳出痰或浓缩痰沉积物，在约两个小时。测试规范和要求只有温和的实验室基础设施和培训。

视频: Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

科研

免疫与感染

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

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 ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

一个<em&gt;体外</em&gt; Caseum结合测定，预测药物在渗透结核病变

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

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 ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

免疫沉淀法研究受体与酶的功能相互作用

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...