根据德州 A & M 大学机构动物护理和使用委员会制定的指导方针和条例, 进行了动物研究。可能需要在您的机构中审查和批准来自生物危害安全官员或委员会。
警告: 所有程序都需要 BSL3 控制。人员必须随时佩戴个人防护用品。所有的操作都是在生物安全柜 (BSC) 内进行的, 所有的锐度都在锐化容器中处理。工作表面和平衡计分卡清洗与缓冲苯酚和70% 乙醇之前开始工作和下班后。程序通常在生物安全级别3与结核分枝杆菌进行, 但为了说明和拍摄目的, 提交人在2级生物安全级别上展示了这些程序, 毒性较低的细菌。
1. 菌株和文化条件
注意: 在本研究中使用了m. 结核应变 CDC1551, 但任何结核分枝杆菌菌株都可以以同样的方式使用。
<ol><li>在 OADC 中生长细菌 (7H9 汤补充0.5% 甘油, 10% 油酸葡萄糖复合体, 不含过氧化氢酶, 0.05% Tween-80) 培养基, 其浓度为37摄氏度, 至600 0.5 (~ 0.2 x 107 CFU)。</li>
	<li>稀释 OADC 中的培养 (1:10 稀释的细菌系列)。将细菌的稀释 (105, 106, 107, 108) 在选择性7H11 板上, 以允许 CFU 的测定。</li>
	<li>孵化四周在37°c 或直到殖民地可以准确计数。</li>
	<li>将细菌接种在 8534 x g 处进行离心, 以5分钟获得颗粒。用10毫升生理盐水 (0.9% nacl) 清洗颗粒, 并在15毫升生理盐水 (0.9% 氯化钠) 中重新悬浮颗粒。</li>
</ol>2. 使用麦迪逊室的小鼠气溶胶感染
<ol><li>允许老鼠适应新环境一周。</li>
	<li>在把老鼠装进房间前先称其重量。</li>
	<li>将三根电线插入电源条: 主腔电源、真空泵和空压机, 按顺序排列。</li>
	<li>小心拧开玻璃罐。把玻璃瓶带到生物安全柜, 加入挑战接种悬浮在15毫升盐水 (0.9% NaCl), 以实现 ~ 104 -106 CFU 细菌在肺部。</li>
	<li>在生物安全柜中关闭玻璃罐的盖子。将罐子连接到雾化装置上, 调整垂直不锈钢管, 使下部 (进气) 端大约一英寸, 低于罐内流体的水平。</li>
	<li>加载实验所需的所有动物 (房间可以容纳多达90只老鼠) 进入房间, 并关闭所有闩锁在门上。</li>
	<li>检查主 (室) 空气流量计。确保浮动 (球) 的中心运行约50升/分钟, 测量的左边的刻度。</li>
	<li>在会议厅的控制面板上按 "开始" 按钮。将气流速率通过压缩机空气流量计 (左侧较小的仪表) 设置为4升/分的刻度。目视检查以确保挑战接种正在雾化。</li>
	<li>15分钟后, 当 "控制面板" 前面的红灯亮起, 并有一个发声信号指示运行结束时, 请按 "控制面板" 右下角的 "复位" 按钮重置计时器。</li>
	<li>按住房间门上的小红色按钮释放真空。</li>
	<li>打开房间门, 把动物移走。把老鼠放回笼子里去。</li>
	<li>取下玻璃雾化器罐并放置在密封的、防泄漏的运输容器中。将容器放在生物安全柜内, 将挑战悬浮液丢弃到指定的废物容器中。</li>
	<li>将所用的喷雾器罐放在生化袋内, 并密封袋, 运输到高压釜。</li>
	<li>在感染程序结束时, 用缓冲的苯酚和70% 乙醇喷洒室内, 并允许分室坐10分钟。</li>
	<li>然后, 彻底擦拭所有可访问的内部表面。处理所有受污染的纸巾,等。在生化垃圾桶里用蒸压器把垃圾、废物容器和使用的喷雾器罐。</li>
	<li>把动物放回封存室直到成像时间点。</li>
	<li>在成像当天, 将动物放在辅助容器中传送到成像室。</li>
</ol>3. 动物麻醉
<ol><li>用特制的气体麻醉系统麻醉小鼠与异氟醚。</li>
	<li>每两个木炭过滤器的重量都位于麻醉装置的顶部。</li>
	<li>替换为新的罐子, 如果重量是50克以上的初始重量。</li>
	<li>检查蒸发器单元以确保该过程有足够的异氟醚。</li>
	<li>将鼻锥架置于成像腔内。放置程序所需的鼻锥数, 并用鼻锥阻滞剂封住其余开口。</li>
	<li>打开高压气缸的氧气供应, 并将其设置为 55 psi。</li>
	<li>打开位于麻醉装置前面的疏散泵, 并将其设置为8升/分钟。</li>
	<li>打开位于麻醉装置前面的氧气开关。</li>
	<li>将气体流向麻醉感应室, 将气体流量设置为1.5 升/分. 把煤气流关了。</li>
	<li>将气体流向成像室, 将气体流量设置为0.25 升/分. 把煤气流关了。</li>
	<li>打开异氟醚蒸发器并将其设置为 2-2.5%。根据实验所用动物的数量和重量, 调整异氟醚的水平, 在异氟醚蒸发器上旋转表盘 (2-2.5% 对一只老鼠称20克; 4% 为一只体重300克的豚鼠)。</li>
	<li>把老鼠放进麻醉室, 关上盖子。打开气体流向麻醉感应室。</li>
	<li>把老鼠放在麻醉感应室里 5-10 分钟, 直到完全麻醉。</li>
	<li>一旦老鼠被麻醉, 在眼睛上涂抹一个光学软膏来保护它们。</li>
	<li>将小鼠置于腹侧或胸骨卧床, 使其鼻子放置在鼻锥内, 以方便在成像过程中对小鼠进行麻醉。</li>
</ol>4. 报告酶荧光 (REF) 成像
<ol><li>注射基质 (20 µM, 2.5 µL/克重量) 的腹腔注射入感染和控制小鼠。小鼠在成像室内麻醉下不到1分钟。</li>
	<li>启动成像系统。</li>
	<li>通过单击 "初始化" 来初始化系统。等到温度条变成绿色。 注: 成像室由加热平台组成, 用于在成像时保持动物体温。</li>
	<li>对于成像采集设置, 选择荧光 |在采集控制面板中, 对整个动物或对肺组织、结构和覆盖物进行全光照明。</li>
	<li>设置视场为 B, 单鼠标和灯级至高。</li>
	<li>设置曝光时间为自动, 中等 binning, 2-3 为 f 或停止, 并且励磁过滤器在745毫微米和放射过滤器从780毫微米到840毫微米。</li>
	<li>对于序列设置, 单击序列设置, 选择 9-12 在肺部区域的反光照点。</li>
	<li>点击获取图像获取。</li>
	<li>把鼠标放回到笼子后成像。</li>
	<li>监测小鼠直到完全恢复或牺牲, 以量化 CFU, 如果成像在一个时间点的实验。执行修改后的卡氏评分评分, 以监测小鼠成像后的福祉。</li>
</ol>5. REF 成像分析
<ol><li>打开映像软件。</li>
	<li>通过单击 "浏览" 图标加载图像文件。</li>
	<li>对于光谱分解, 单击工具调色板中的光谱分解, 然后单击 unmix 所需的光谱。点击开始分解。</li>
	<li>对于光学曲面重建, 请单击曲面地形。</li>
	<li>选择背方向, 受皮毛鼠标的左右, 单击 "生成曲面"。</li>
	<li>绘制裁剪图像并为感兴趣的区域设置阈值图像。单击 "完成"。</li>
	<li>对于器官注册, 请转到3D 光学工具下拉菜单中的 "注册" 选项卡。</li>
	<li>在器官图谱中登记感兴趣的器官。</li>
	<li>使用在面板注册工具上/关闭的变换工具调整器官大小或位置以生成的表面形貌。</li>
	<li>转到荧光层析成像 (掠过) 3D 重建, 并选择 "分析" 选项卡. 选择要包含的图像进行分析, 图像类型为规范化的传输荧光 (NTF) 效率, 请单击 "开始"。</li>
	<li>检查 "选择全部" 并在 "数据预览" 选项卡中重建。</li>
	<li>对于荧光量化, 请转到感兴趣的区域 (roi) 工具, 将 ROI 多维数据集添加到感兴趣的机构中, 并调整多维数据集大小以覆盖感兴趣的器官。点击测量 3D ROIs。</li>
	<li>在 "roi 度量" 窗口中, 转到 3D roi 度量值, 选择 "数据类型" 到 "源素和度量单位" 以 pmolM-1cm-1。</li>
	<li>将3D 重建分析的结果保存和/或导出到图形或数据文件中。</li>
</ol>6. CFU 细菌的量化
<ol><li>腹腔注射0.1 毫升戊巴比妥钠 (390 毫克/毫升) 弄死小鼠。</li>
	<li>通过挤压小鼠脚垫来检查踏板反射, 以确保没有反射反应。</li>
	<li>用无菌钳和剪刀将小鼠的肺组织外植体。融汇肺组织在1毫升的1x 磷酸盐缓冲生理盐水 (PBS)。</li>
	<li>在无菌 1x PBS 中进行肺匀浆的10倍串联稀释。</li>
	<li>为电镀, 斑点三整除数20µL 每各自稀释在琼脂板材。</li>
	<li>在37摄氏度的孵化器中孵化板块, 并监测细菌的生长。</li>
	<li>在孵化时间后, 在每一个斑点的殖民地计数, 并表示为 CFU/毫升通过纠正体积和稀释使用以下等式: CFU/毫升 = (C/V) x M 那里 C = 殖民地计数每点, V = 样品容量接种在每个板材 (mL) 和 M = 增殖因素 (被使用的稀释的倒数)。 注: 如果将11个菌落计算在一个点上, 样本体积为0.002 毫升, 样本稀释量为 10-4, 则使用该等式, 菌落计数将计算为 (11/0.02) x 104 = 5.5 x 106 CFU/毫升</li>
</ol>

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A comparison of radiography, computed tomography and magnetic resonance imaging.</a> J Bone Joint Surg Br. 75 (2), 233-239 (1993).</li><li>Soussan, 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=Patterns+of+pulmonary+tuberculosis+on+FDG-PET/CT.">Patterns of pulmonary tuberculosis on FDG-PET/CT.</a> Eur J Radiol. 81 (10), 2872-2876 (2012).</li><li>Sule, P., 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=New+directions+using+reporter+enzyme+fluorescence+(REF)+as+a+tuberculosis+diagnostic+platform.">New directions using reporter enzyme fluorescence (REF) as a tuberculosis diagnostic platform.</a> Tuberculosis. 101, 78-82 (2016).</li><li>Heuts, F., Carow, B., Wigzell, H., Rottenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+non-invasive+bioluminescent+imaging+to+assess+mycobacterial+dissemination+in+mice,+treatment+with+bactericidal+drugs+and+protective+immunity.">Use of non-invasive bioluminescent imaging to assess mycobacterial dissemination in mice, treatment with bactericidal drugs and protective immunity.</a> Microbes Infect. 11 (14-15), 1114-1121 (2009).</li><li>Kong, 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=Application+of+fluorescent+protein+expressing+strains+to+evaluation+of+anti-tuberculosis+therapeutic+efficacy+in+vitro+and+in+vivo.">Application of fluorescent protein expressing strains to evaluation of anti-tuberculosis therapeutic efficacy in vitro and in vivo.</a> PLoS ONE. 11 (3), 0149972 (2016).</li><li>Xie, H., 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+point-of-care+detection+of+the+tuberculosis+pathogen+using+a+BlaC-specific+fluorogenic+probe.">Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe.</a> Nature Chem. 4, 802-809 (2012).</li><li>Nooshabadi, F., 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=Whole-animal+imaging+of+bacterial+infection+using+endoscopic+excitation+of+&#946;-lactamase+(BlaC)-+specific+fluorogenic+probe.">Whole-animal imaging of bacterial infection using endoscopic excitation of &#946;-lactamase (BlaC)- specific fluorogenic probe.</a> Proc SPIE. 9715, 97150 (2016).</li><li>Ghiasi, M., Pande, T., Pai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+tuberculosis+diagnostics.">Advances in tuberculosis diagnostics.</a> Curr Trop Med Rep. 2 (2), 54-61 (2015).</li><li>Rao, J., Andrasi, A. D., Yao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+in+vivo:+recent+advances.">Fluorescence imaging in vivo: recent advances.</a> Curr Opin Biotech. 18 (1), 17-25 (2007).</li><li>Marshall, M. V., Draney, D., Sevick-Muraca, E. M., Olive, D. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,7-disubstituted+cephalosporin+derivatives+as+beta-lactamase+substrates+and+methods+for+their+use+for+the+diagnosis+of+tuberculosis.">2,7-disubstituted cephalosporin derivatives as beta-lactamase substrates and methods for their use for the diagnosis of tuberculosis.</a> US patent. , (2015).</li><li>Shao, Q., Zheng, Y., Dong, X., Tang, K., Yan, X., Xing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Covalent+Reporter+of+&#946;-Lactamase+Activity+for+Fluorescent+Imaging+and+Rapid+Screening+of+Antibiotic-Resistant+Bacteria.">A Covalent Reporter of &#946;-Lactamase Activity for Fluorescent Imaging and Rapid Screening of Antibiotic-Resistant Bacteria.</a> Chem Eur J. 19 (33), 10903-10910 (2013).</li><li>Li, L., Li, Z., Shi, W., Li, X., Ma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+Selective+Near-Infrared+Fluorescent+Off-On+Probe+and+Its+Application+to+Imaging+Different+Levels+of+&#946;-Lactamase+in+Staphylococcus+aureus.">Sensitive and Selective Near-Infrared Fluorescent Off-On Probe and Its Application to Imaging Different Levels of &#946;-Lactamase in Staphylococcus aureus.</a> Anal Chem. 86 (12), 6115-6120 (2014).</li><li>Ashizawa, K., 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=Coexistence+of+lung+cancer+and+tuberculoma+in+the+same+lesion:+demonstration+by+high+resolution+and+contrast-enhanced+dynamic+CT.">Coexistence of lung cancer and tuberculoma in the same lesion: demonstration by high resolution and contrast-enhanced dynamic CT.</a> BRIT J RADIOL. 77 (923), 959-962 (2004).</li><li>Figueroa, C. 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当使用像 REF 这样的成像技术时, 有一些关键策略允许生成健壮和一致的数据。光学成像在组织中产生散射光, 这会影响穿透深度, 因为很难捕捉到所有方向发出的光。利用近红外线荧光 (近红外) 基质进行 REF 成像, 在 Querange 中具有激发和发射波长的 700-900 nm, 有利于哺乳动物组织对荧光信号的最小吸收。定制设计的基底是通过将近红外荧光, IRDye 800Cw 到淬火, IRDye QC-1, 通过一个内酰胺环, 允许荧光共振能量转移为基础的淬火构造。IRDye 具有良好的组织穿透和光散射特性, 对哺乳动物没有任何明显的有害影响36, 从血液和器官清除 24 h. 荧光信号显著增加起始4小时后基板管理, 达到最高水平后6小时管理。
在开展大型、复杂的实验前, 应规范细菌感染剂量、感染剂量的管理方式、基质以及影像后感染的时间点。试验性研究可以大大减少成像大量动物的时间和成本, 因为一个标准程序可以优化之前进行的关键实验。细菌负荷应确定的器官/组织的整个身体成像后, 使用反光照和体外肺成像, 利用 epi 照明来验证信号的来源和确定相关的质量与细菌数字存在8。试点研究将提供洞察的阈值检测, 动态范围的技术, 以及确定的最佳实验条件的成像。
与其他荧光和发光策略相比, 使用 REF 成像的主要优点是它具有较高的灵敏度和对自然的m. 结核菌株的成像能力。REF 成像利用催化健壮的酶拉丁, 保存在所有的m. 结核临床分离株和结核复杂菌株。由于拉丁的快速催化速率和宿主细胞对荧光产物的保留, 导致了 REF 成像的极大灵敏度。只要基底可用, 信号就会不断增加, 从而使感染细胞和组织内的信号几乎无限地积聚。与其他成像方法相比, 此提高了 REF 成像的灵敏度, 允许对m. 结核进行特定检测体外和在体内8,23,24, 29。
REF 成像可用于细菌检测, 没有基因修改, 使其直接应用到任何感染模型, 实验动物8,37或人体临床资料29,38.REF 可用于检测和图像范围广泛的病原体39,40, 因为荧光基板可以开发为许多酶的目标以外的拉丁, 如蛋白酶, 激酶, ureases 和β galactosidases。但是, 应仔细考虑目标, 以确保其显示最佳特征的成像。拉丁代表一个良好的模型酶的特点, 将确保成功地应用这一战略。REF 成像提供了一个即时读取的细菌负荷在肺部感染期间, 这大大加快了研究结核病发病机制, 因为细菌数量的测定通常需要三周到六星期, 但即使在更快速生长的生物体这种方法将节省大量的时间。参考文献也可用于鉴别癌症的结核病, 一个关键问题诊断的结节性病变的患者41,42。REF 作为一种新的工具, 以加速翻译结核成像, 甚至可以应用于人类, 可能允许快速预测的治疗结果。

m. 结核的缓慢增长率是结核病快速诊断的一个主要障碍1,2,3。虽然基于区域性的诊断需要几周的时间才能产生结果, 但酸性快速涂片在儿童5中有诊断限制4 , 患者与人体免疫机能丧失病毒6,7有共同感染。光学成像技术最近被认为是替代传统的结核病诊断方法8,9。荧光和生物发光可用于实时10,11,12,13,14的活体动物中的光学图像m. 结核, 15,16,17,18,19。光学成像有助于快速而具体地评估感染与m. 结核20,21,22。
我们概述了使用 REF 对活鼠中的m. 结核进行光学成像的细节。此方法非常特定且敏感23、24 , 与其他光学方法类似, 比其他结核病 (TB) 成像25的方法成本低, 包括计算机断层扫描 (CT)26, 磁性磁共振成像 (MRI)27和 F 氟脱氧葡萄糖正电子发射断层扫描/ct (f-葡萄糖 PET/ct)28。REF 利用自定义的荧光或生物发光基底, 经细菌酶解裂后, 产生荧光产物8,29。因此, 它的优点是不需要重组结核杆菌报告器应变30,31。所述的烦恼基板由由拉丁 (β酶) 水解的β-内酰胺环连接的 fluorochrome 和淬火组成, 自然组成性由结核复合分枝杆菌 8,32. 细菌直接产生的信号, 由于 REF 催化活动, 允许放大的数量级和敏感检测的m. 结核。
本研究所用的 REF 基底在活体动物中具有良好的组织穿透力, 并且由于波长长而减少了背景。有了这个长波长的基底, 就有可能在小鼠 (整个动物)8中, 为近100个菌落形成单位 (CFU)体外和 < 1000 CFU 的m. 结核检测阈值. 33。REF 可作为一个诊断工具的痰, 临床材料, 甚至直接在微内窥镜系统的患者16,32,33,34由于其高灵敏度和特异性。REF 可以应用于任何结核病临床菌株, 因为它使用天然产生的细菌酶, 拉丁, 用于检测目前在所有菌株。这些特点使 REF 成像在临床前结核病研究中具有重要的应用价值, 有助于治疗和疫苗的评估以及发病机制的分析, 但最终也可应用于结核病的诊断。患者.

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="664">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:163px;">VETONE</td>
			<td align="right" style="width:119px;">501027</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:216px;">CNIR800</td>
			<td style="width:163px;">Custom synthesized</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Fatal Plus solution</td>
			<td style="width:163px;">Vortech Pharmaceutical Ls, Ltd</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7H9 Middlebrook broth</td>
			<td style="width:163px;">BD</td>
			<td align="right" style="width:119px;">271310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OADC Middlebrook enrichment</td>
			<td style="width:163px;">BD</td>
			<td align="right" style="width:119px;">212351</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sporcidin</td>
			<td style="width:163px;"></td>
			<td style="width:119px;">RE-1284F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7H11 Middlebrook Agar</td>
			<td style="width:163px;">BD</td>
			<td align="right" style="width:119px;">212203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Madison Chamber</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS Spectrum</td>
			<td style="width:163px;">Perkin Elmer</td>
			<td align="right">124262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">XGI-8-gas Anesthesia System</td>
			<td style="width:163px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Living Imaging software</td>
			<td style="width:163px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transparent nose cones</td>
			<td style="width:163px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">M. tuberculosis strain CDC1551</td>
			<td style="width:163px;">ATCC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Female BALB/C mice, 5-7 weeks</td>
			<td style="width:163px;">Jackson Laboratory</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging mycobacterium tuberculosis in mice with reporter enzyme fluorescence

报告酶荧光 (REF) 利用的基底, 是特定的酶存在于目标有机体的成像或检测的荧光或生物发光。我们利用拉丁, 一种由所有结核组成性菌株表达的酶。REF 允许快速定量的细菌在肺部感染的小鼠。同一组小鼠可以在许多时间点成像, 大大降低成本, 更快地列举细菌, 允许在寄主-病原体相互作用方面的新的观察, 增加统计能力, 因为每组更多的动物易于维护.由于自定义荧光共振能量转移 (拉丁) 或荧光基板使用, REF 是非常敏感的由于催化性质的酶记者和具体的。REF 不需要重组菌株, 以确保正常宿主病原体相互作用。我们描述了m. 结核感染的影像, 使用的是最大排放量为 800 nm 的烦恼基底。基底的波长允许哺乳动物敏感的深部组织成像。我们将概述小鼠的气溶胶感染的m. 结核, 麻醉小鼠, 参考基质的管理和光学成像。这种方法已成功地应用于评估宿主-病原体相互作用和抗生素对靶向结核治疗的效果。

图 1A中显示了感染m. 结核和未感染控制鼠标的小鼠的 REF 成像。受感染的小鼠在感染后6周内产生了显著的 (P = 0.0057) 的荧光信号, 与控制基质的管理相对应。在1、2、4、6、15和24后的感染后, 一个典型的时间课程, 研究使用 REF 治疗m. 结核的疗效。荧光强度是通过对肺组织的免疫照射来量化的 (图 1B)。从2周到6周的感染小鼠肺部持续增加的信号表明, REF 能够成功地检测到体内的 m. 结核菌。在以后的时间点 (图 1B) 中, 控制鼠标的背景信号下降可能归因于在6周时间内身体质量和小鼠体积的增加, 从而减少了激发波长的穿透。3D. 在图 2A中, 对感染结核的小鼠的荧光源进行重构。使用相同的激发和串联的发射滤波器, 在鼠标的多个反光照点上获取图像序列。这些图像序列, 然后用于3D 掠过重建的荧光源分布在动物的主题。图 2-d说明了小鼠层析成像与肺器官注册的不同方向 (冠状、矢状和 transaxial)。用于检查重建质量的 NTF 效率图在图 2E中表示。NTF 允许通过使用中性密度过滤器捕获的额外图像, 从反照图像中减去背景光泄漏。使用特定励磁滤镜 (图 2E 测量) 的图像被规范化为用相同的发射过滤器和开放励磁过滤器 (图 2E 模拟) 测量的传输图像, 以给出由基板单独产生的信号。在水平 (图 2F) 和垂直 (图 2G) 配置文件中, 类似的测量和模拟的 NTF 效率配置文件具有近0% 的百分比错误 (图 2E-% 错误)提供了一个良好的质量3D 重建与减少工件和改进的信号定位和灵敏度的证据。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56801/56801fig1.jpg"> 图 1.使用 REF 诊断m. 结核(A) 在2、4和6周的感染后,在体内对未感染和感染m. 结核的小鼠进行成像。颜色条表示荧光信号的强度, 每秒每个 cm2从低 (黄色) 到高 (红色)。(B) 从感染了m. 结核的小鼠身上荧光强度的量化。两个控件 (黑色) 和受感染 (红色) 的荧光值都与每个时间点的标准错误一起表示。结果的重要性由学生t测试确定, p值 < 0.05 被认为是重要的。<a href="//cloudflare.jove.com/files/ftp_upload/56801/56801fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56801/56801fig2.jpg"> 图 2. 3D. 在感染结核的小鼠中, 荧光源的重构重建.用不同方向表示的鼠标层析成像;A) 冠状, B) 矢状, C) transaxial 与 D) 肺器官注册。3D 感兴趣的区域 (ROI) 在肺中表示为源测量的红色立方体。(E) 测量和模拟的 NTF 效率图以检查重建质量。比较了实测和模拟 NTF 效率剖面, 提供了良好的3D 重建质量 (类似的测量和模拟 NTF 效率)。F) 水平和 G) 垂直信号剖面, 代表测量 (蓝色) 和模拟 (红色) NTF 效率曲线。水平和垂直红色条表示源位置。颜色条表示荧光信号的强度, 每秒每个 cm2从低 (蓝色) 到高 (红色)。<a href="//cloudflare.jove.com/files/ftp_upload/56801/56801fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence at JoVE.com

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

我们描述了使用报告酶荧光 (REF) 感染结核分枝杆菌(m 结核) 的小鼠的光学成像。该协议有助于在临床前动物模型中对m. 结核进行敏感和特定的检测, 以进行发病机制、治疗和疫苗研究。

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 ...

imaging-mycobacterium-tuberculosis-mice-with-reporter-enzyme

Research

JoVE Journal

Immunology and Infection

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

7.3K 次观看.  Texas A&M Health Science Center. 我们描述了使用报告酶荧光 (REF) 感染结核分枝杆菌(m 结核) 的小鼠的光学成像。该协议有助于在临床前动物模型中对m. 结核进行敏感和特定的检测, 以进行发病机制、治疗和疫苗研究。

视频: Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

科研

免疫与感染

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 ...

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 ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

报告酶荧光法检测小鼠结核分枝杆菌的影像学

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样品制备

增长结核分枝杆菌生物膜

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

展现着多药耐药

关于功能研究的三维人体肺组织模型

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

系统抑制剂靶向细胞内的疗效和毒性筛选

高通量兼容检测，以评估对巨噬细胞传代药物疗效

分析¹⁸葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

marinum 分枝杆菌感染成人斑马鱼结核的建模