作者希望感谢马克罗杰斯概述感染程序和 l. Eoin 卡和布赖恩 Lopresti 的指导, 建立这些成像程序。这项工作的经费由比尔和梅琳达·盖茨基金会 ( J . L . F . 、成功率 ) 、国家卫生研究院、国立反应和传染性疾病研究所 R01 AI111871 ( 成功率 ) 、国家心肺和血液研究所 R01 HL106804 ( J.l.f), R01 HL110811。

这项工作中列出的所有方法都已获得匹兹堡大学动物保育和使用委员会的批准。所有程序都遵循了机构生物安全和辐射安全性要求。CT 扫描需要穿铅围裙和喉盖。生物安全3级 (BSL3) 的外衣和与非人灵长类一起工作的程序必须按照机构的指导方针来遵循。所有扫描都是在 BSL3 设施中进行的. 
 1. 动物感染程序 <ol> <li> 安静的动物, 氯胺酮 (10 毫克/千克, 肌肉注射) 或 telazol (5-8 毫克/千克, 肌肉) 如果动物对氯胺酮有不良反应. </li>
	<li> 使用喉镜, 可视化会厌和声带。麻醉声带通过喷洒 cetacaine 喷雾 1 s (不超过2秒). </li>
	<li> 使用喉镜, 引导一个支气管镜 (2.5 毫米外径) 进入气管 通过 直接可视化进入右尾肺叶. </li>
	<li> 准备一个注射器, 由大约 5-20 (取决于研究) 的菌落形成单位的 m 结核 在2毫升的无菌生理盐水和管理的解决方案通过支气管镜通道。准备一个单独的注射器, 由2毫升无菌生理盐水和管理生理盐水通过支气管镜通道, 其次是5毫升空气, 以确保完全沉积细菌 22 . </li>
	<li> 取出支气管镜并观察猴子直到完全清醒和警觉. </li>
</ol> 2。图像采集、直方图和重建过程 <ol> <li> 准备用于成像的动物。
	<ol> <li> 镇静动物, 氯胺酮 (10 毫克/千克, 肌肉注射) 或 telazol (5-8 毫克/千克, 肌肉) 如果动物对氯胺酮有不良反应. 
		注意: 动物需要在夜间禁食, 以减少在成像过程中呕吐的风险, 并保持葡萄糖 PET 扫描的一致性. </li>
		<li> 将静脉导管插入两条腿的隐静脉, 并用布带固定. </li>
		<li> 将大约5居里剂量的葡萄糖与无菌生理盐水稀释到一个塑料注射器的总容积5毫升. </li>
		<li> 使用剂量校验仪记录注射器中的预放射性水平, 记录时间, 并将注射器放置在注射器支架上. </li>
		<li> 慢慢地通过 IV 导管注入放射性剂量, 并跟随5毫升无菌生理盐水。记录注射时间。注射时间应协调约45分钟-1 小时前 PET 成像. </li>
		<li> 使用剂量校验仪记录注射器的后放射性水平并记录时间。在适当的废物容器中处理注射器. </li>
		<li> 使用喉镜, 形象化的会厌和声带和麻醉与 cetacaine 喷雾. </li>
		<li> 引导气管插管 (3.5 毫米-4.5 毫米, 视猴大小而定) 进入气管, 并在导管的插入端充气. </li>
		<li> 使用长薄的无菌纱布条, 将试管周围的小条包裹好, 用动物的每只狗刺穿条带, 然后在鼻口的桥周围用剩余的纱布绑一个结, 最后绕在后面 o头. </li>
		<li> 用人工泪液遮住眼睛, 防止在成像过程中干燥. </li>
	</ol> </li> <li> 执行 CT 和 PET 扫描。
	<ol> <li> 将动物放在扫描床上. </li>
		<li> 将插管管连接到具有以下设置的呼吸器: 呼吸速率 = 15, 峰值压力 = 15-17, Oxygen% = 40, 窥视 (正端呼气压力) = 3, 潮汐容积 = 60, T i (吸气时间) = 0.4, i: E (吸气呼气时间) 比 = 1:3. 4, T 高原 (在失效前吸气停顿) = 0.5, 峰值流量 = 9.0 (这些值可以根据动物特定的肺顺应性或实验性需求来调整). </li>
		<li> 通过呼吸机开始吸入麻醉 (2% 异氟醚) 并继续, 直到动物对物理刺激没有反应. </li>
		<li> 将动物置于俯卧位置, 并支持其头部和腿部. </li>
		<li> 将动物置于 CT 视中, 并进行预览扫描, 以确保完整扫描中包括整个肺容积范围. </li>
		<li> 用以下参数获取 CT 扫描 (螺旋扫描, 轴向视 = 250 mm, 电压 = 140 伏, 电流 = 2.0 毫安, 切片厚度 = 1.25 mm, 锐度 = 额外锐度), 同时进行呼吸机呼吸保持. 
		注: 造影剂 CT 是可选的。如果进行对比扫描, 在注射造影剂和图像采集之间需要延迟, 因为在心脏中汇集造影剂会干扰 PET 扫描中肺部空间的正确重建, 并在肺部产生伪影。在 ct 扫描上 </li> <li> 确保在扫描过程中将异氟醚浓度降低到 0.7-0.8%. </li>
		<li> 将动物置于宠物视. 
		注: 该系统到位的这项工作是一个内嵌系统的一个单独的 CT 和 PET 扫描仪。根据 CT 坐标, 手动计算 PET 定位坐标. </li>
		<li> 获取每个床位置的 600s PET 图像. 
		注: 焦点220系统的轴向视为7.6 厘米。这项工作是使用四床的位置, 是手工缝制在后期处理. </li>
		<li> 关闭异氟醚, 逐渐关闭呼吸器, 从呼吸机管袖中取出空气, 一旦动物恢复了咳嗽反应并正常呼吸, 就将其取出。取出静脉导管, 在注射部位保持压力直到血流停止. </li>
	</ol> </li> <li> 执行 PET 图像直方图和重建。
	<ol> <li> 使用以下参数执行 PET 图像直方图: 3D 直方图无平滑, 跨度: 3, 环差:47, 全球平均死校正. </li>
		<li> 使用以下参数执行 PET 图像重建 = OSEM3D (有序子集期望 Maximum-3 维度) 算法与 ct 衰减, 斜坡投影滤波器, 和散射校正产生一个284层的图像. </li>
	</ol> </li> <li> 联合注册 PET 和 CT 图像. </li>
	<li> 将合作注册的 PET 和 CT DICOM 图像导出到软件 ( 例如 , OsiriX). </li>
</ol> 3。识别和分析个别病变 <ol> <li> 在轴向方向上从 OsiriX 数据库中打开 pet 和 ct DICOM 图像 (CT 图像将与 pet 图像融合, 并会有一个单独的 pet 图像窗口). </li>
	<li> 将扫描 (或串行扫描) 设置为轴向方向. </li>
	<li> 在 ct 扫描时单击 (任意位置) 并更改 和 #34; 西城/WW 和 #34; 在顶部菜单栏中 和 #34; ct 和 #8211; 肺和 #34; 。
	<li> 滚动扫描以确定肺裂片的起始和结束位置。(识别肺裂隙)
	<ol> <li> 滚动整个扫描, 聚焦于肺部空间的小区域一次. </li>
		<li> 注意正常肺部呈深色, 解剖特征显示较轻 (取决于密度)。气管出现黑色, 而血管几乎是白色的. </li>
		<li> 沿轴向切片滚动时, 跟随船只和呼吸道移动. </li>
		<li> 裂缝可以在没有船只或空中航线的地区识别。(这些是肺部的区域, 只出现在黑暗中, 没有其他解剖结构.) </li>
	</ol> </li> <li> 使用熔融 PET/CT 识别病灶。
	<ol> <li> 滚动整个扫描, 同时聚焦于肺部的小面积. 
		注意: 集中在一个肺叶的时间, 以确定和计数病变. </li>
		<li> 在肺部发现葡萄糖-狂热的病灶。它们看起来像是热球体, 与肺部背景非常不同。小的, 冷的病灶将不太明显, 更难以辨认。它们将在扫描时显示为在滚动时不移动的稠密结构 (如容器). 
		注意: 小病变和血管看起来非常相似。区分两者的一个简单方法是将光标悬停在有问题的结构上, 然后向上和向下滚动一两个切片。如果结构停留在光标下方, 则结构是一个损伤。如果在向上或向下滚动切片时结构从光标处移开, 则最有可能是容器或气道. </li>
		<li> 用于标识目的, 请使用 和 #34; 箭头和 #34; 工具指向扫描中的每个病灶. </li>
		<li> 为了便于定位, 请使用 和 #34;P 点和 #34; 工具并单击病变部位, 使 ROI (感兴趣的区域) 直接位于肉芽肿的中心。此 ROI 中包含的信息将包括可在其中找到病灶的笛卡尔坐标 (XYZ 坐标). </li>
	</ol> </li> <li> 使用 #34; 长度和 #34; 和 #34; 椭圆形和 #34; 用于测量每个病灶大小 (mm) 和葡萄糖亲和力 (SUV) 的工具。
	<ol> <li> 若要测量病变的大小, 请删除 PET 信号, 以便只有 CT 可见. </li>
		<li> 选择 和 #34; 长度和 #34; 工具 </li>
		<li> 滚动直到包含病灶的最大部分的切片被识别 (病灶表面看起来是最大的切片). </li>
		<li> 在病变最长的长度上画一条线。此 ROI 中包含的信息将表示病灶直径的长度 (毫米). </li>
		<li> 要测量病变的葡萄糖亲和力, 首先点击 pet 扫描并打开 pet。转到 #34; #38; 查找和 #34; Osirix 菜单在屏幕顶部, 选择 和 #34; 在 "西城/ww" 下拉菜单中手动和 #34 设置。在对话框中, 将 0 输入到 和 #34; 从和 #34; 和 20 进入 和 #34; #34; 以将窗口限制为0到 20 SUV. </li>
		<li> 选择 和 #34; 椭圆和 #34; 来自 和 #34 的工具; 鼠标按钮功能和 #34; 工具下拉菜单. </li>
		<li> 在病灶上方滚动以评估病灶的最热部分。在病灶周围画一个椭圆形。 #34; 椭圆和 #34; 工具 ROI 信息包括区域内所有体的 suv 的描述性统计数据。在该区域中记录 最大 SUV . </li>
		<li> 每个 和 #34; 椭圆和 #34; ROI 只代表该特定轴向平面的 SUV 值和典型病变是球形的形状, 在几个切片上绘制椭圆形, 以确保该病变的实际最大 suv 捕获. 
		注意: 如果 pet/ct 扫描被人工重建, pet 和 ct 图像可能不会完全匹配。如果是这样的话, 所有的 SUV 分析和 roi 应进行 pet 扫描, 而不是融合 pet/CT 扫描。由于许多病变都小于 PET 探测器晶体的分辨率, 所有测量的单个病灶的 suv 都被输入一个恢复系数计算器电子表格, 对每个病灶执行部分音量校正 23 . </li> </ol> </li> </ol> 4。全肺葡萄糖亲和力测定法测定全肺炎症 <ol> <li> 在轴向方向上从 OsiriX 数据库中打开 pet 和 ct DICOM 图像 (ct 图像将与 pet 图像融合, 并会有一个单独的 pet 图像窗口). </li>
	<li> 在 CT 图像上执行肺容积的分割。
	<ol> <li> 在 CT 扫描的任意位置单击以确保它是活动窗口. </li>
		<li> 转到 ROI 下拉菜单并选择 和 #34; 增长区域 (2 维/3 维) 分割和 #8230; #34; . </li>
		<li> 为了捕获正常肺部的密度, 将下限阈值设置为-1024, 将上限阈值设为-200。这些都是 Hounsfield 单位的指示, 尽管 "分割" 框并没有这样命名. </li>
		<li> 一旦设置了下限和上限阈值, 请单击肺部内的任意位置。整个肺部应以绿色突出显示. </li>
		<li> 下一步, 单击 和 #34; 计算和 #34; 在 "分段参数" 对话框中。这将扩大生长区域从一个切片到整个肺部体积. </li>
	</ol> </li> <li> 移动和 #34; 增长区域和 #34; 从 CT 扫描到 PET 扫描的肺部。
	<ol> <li> 单击 CT 扫描名称左侧的小图标, 然后将图标拖动到 PET 扫描. </li>
		<li> 选择 和 #34; 复制 roi 和 #34; 。现在应该有一个覆盖的肺部在 PET 扫描. </li>
	</ol> </li> <li> 从 CT 扫描中删除 ROI (可选). 
	注意: 它有助于能够看到整个肺部没有 roi 的 CT 扫描, 以确保所有病理在肺部被抓获。为此, 请删除 roi。请确保 ct 窗口处于活动状态 (单击 ct 扫描) 选择 ROI 下拉菜单, 然后选择 和 #34;D elete 本系列中的所有 roi. 和 #34; </li> <li> 填充在 PET 扫描中显示为间隙的肺部的高密度区域. 
	注: 在许多情况下, PET 扫描的 ROI 有孔, 在 CT 扫描中肺组织密度大于-200 胡 (如果不发生此步骤可以跳过)。
	<ol> <li> 突出显示 ROI 下拉菜单并选择 和 #34; 画笔 roi 和 #34; #160; #8594; #34; 关闭. #34; 当出现对话框时, 将箭头滑动到 3, 以便对话框的顶部读取 和 #34; 结构元素半径: 3 和 #34; 并检查 和 #34; 应用于同名的所有 roi. #34; 注意: 当有很大一部分疾病 (如合并的密度比周围的肺部组织), 经常关闭刷子 roi将不足以填补整个肺部。如果是这种情况, 则必须手动填充间隙. </li>
		<li> 转到 #34; 鼠标按钮功能和 #34; 区域在顶部菜单上, 单击右侧的小箭头. </li>
		<li> 选择 和 #34; 画笔和 #34; 工具. </li>
		<li> 一旦选择了此工具, 请在 ROI 中手动绘制以填充孔. </li>
	</ol> </li> <li> 在 PET 扫描中隔离肺部 ROI。
	<ol> <li> 现在有一个代表整个肺部的宠物扫描, 删除肺部以外的所有像素. </li>
		<li> 突出显示 ROI 下拉菜单并选择 和 #34; 将像素值设置为和 #8230; #34; . </li>
		<li> 单击 外部 roi " 复选框, 并将 roi 以外的所有像素设置为 0. </li>
	</ol> </li> <li> 隔离和 #34; 热 #34; 病理。
	<ol> <li> 使用任何希望用作和 #34 的阈值; 热的 #34; 超过2.3 的 suv 被认为是和 #34; 热 #34; 基于文献价值的结核病损害诊断 24 . </li>
		<li> 选择 ROI 下拉菜单并选择 和 #34; 将像素值设置为和 #8230; #34; . </li>
		<li> 单击 内部 ROI 复选框。确保单击 "#34" 和 "#34"; 框, 使 0 和 2.3 之间的所有值都设置为 0. </li>
	</ol> </li> <li> 确保在 ROI 中只考虑病理学。
	<ol> <li> 请注意, 某些区域 (如肝脏) 的温度高于2.3。通过删除 roi 并创建另一个增长区域, 确保只捕获所需的区域。其他常见的组织, 在这个时候干扰包括心脏, 纵隔淋巴结, 椎骨, 肋骨. </li>
		<li> 突出显示 roi 下拉菜单并选择 和 #34;D elete 本系列中的所有 roi. #34; 下一步, 转到 roi 并选择 和 #34; 增长区域 (2 维/3 维) 分割和 #8230; #34; 。
		<li> 更改阈值以将阈值降低到 2.3, 将上限阈值转换为 100. </li>
		<li> 滚动整个 PET 窗口, 点击疾病病理学和点击 和 #34; 计算. #34; 对热疾病的每个区域重复。在 roi 菜单下, 确保使用 #34; 保存 roi 和 #34; 选项, 以节省整个肺投资回报率. </li>
	</ol> </li> <li> 将原始值导出到电子表格中。
	<ol> <li> 转到下拉菜单栏中的 2D 查看器 , 然后选择 和 #34; 还原系列. #34; </li> <li> 下一步, 转到 插件 下拉菜单栏. </li>
		<li> 选择 和 #34; ROI 工具和 #34; #160; #8594; #34; 导出 roi #34; 命名并保存导出的原始数据文件。请确保在该对话框的底部选择 #34; CSV 和 #34; . </li>
	</ol> </li> <li> 从原始数据中计算总的葡萄糖亲和力。此电子表格中的每一行表示扫描中的单个切片。感兴趣的列是 and #34; RoiTotal #34; <ol> <li> 以计算和 #34; 总葡萄糖亲和力, #34; 添加所有的 和 #34; RoiTotal 和 #34; 将切片的 组合在一起。计算列 F (RoiTotal) 的总和。这是总的葡萄糖亲和力测量. </li>
		<li> 如果 OsiriX 没有导出 ROI 插件, 请转到 插件 在下拉菜单中。选择 #34;P lugins 管理器和 #8230; #34; 单击对话框顶部的 & #34;D ownload 和 #8230; #34; 选项卡。从 和 #34; 可用插件和 #34; 下拉菜单中选择 #34; ExportROIs 和 #34; 。选择 #34;D ownload 和 #38; 安装. #34; </li> </ol> </li> </ol> 5。测定 #34 中葡萄糖摄取量的分析程序; 热 #34; 淋巴结肿大 <ol> <li> 在轴向方向 OsiriX 数据库中打开 pet 和 ct DICOM 图像 (CT 图像将与 pet 图像融合, 并会有一个单独的 pet 图像窗口). </li>
	<li> 确保在对 PET 图像执行手动 ROI 分析时, 图像强度窗口是一致的。
	<ol> <li> 单击 PET 图像窗口以确保它是活动窗口. </li>
		<li> 在 "OsiriX" 菜单中, 单击 " 和 #34; 妇女协会/ww 和 #34; 下拉菜单, 然后单击 和 #34; 手动和 #34; 。
		<li> 当下拉框出现在活动 PET 窗口中时, 请在 和 #34 中填充所需的最小强度值; 从和 #34; 字段和在 和 #34 中所需的最大强度值; to 和 #34; 字段 (如 到窗口PET 图像从0到 20 SUV, 类型0进入和 #34; 从和 #34; 字段和20进入和 #34; #34; 字段). </li>
		<li> 或者, 如果希望始终加载具有相同亮度值的图像, 请在顶部菜单突出显示 Osirix -#62; PET 和 #62; 然后根据 和 #34; 窗口级别和 #38; 宽度和 #34; 部分, 单击 使用固定级别 气泡并在 & #34 中插入所需的值; 从和 #34; 和 和 #34; #34; 字段. </li>
	</ol> </li> <li> 一旦确定了所需的淋巴结, 请在淋巴结边缘周围手工绘制一个 ROI。
	<ol> <li> 突出显示 PET/CT 融合图像, 以确保它是活动窗口. </li>
		<li> 由于对此分析使用彩色查找表非常有用, 要更改此设置, 请单击 和 #34; 查找和 #34; 主 OsiriX 工具栏中的下拉框, 并选择所需的查找表设置 (UCLA 首选). </li>
		<li> 若要在淋巴结周围绘制手动 ROI, 请单击 和 #34 的右侧的下拉菜单; 鼠标按钮功能和 #34; 在主工具栏中选择 和 #34; 关闭多边形和 #34; 。点击边缘的淋巴结根据宠物视窗查找表建立第一个点的 ROI. </li>
		<li> 单击淋巴结外边缘的另一点, 继续追踪, 直到淋巴结几乎封闭. </li>
		<li> 若要确定 roi 的最终点, 请双击以在 roi 中关闭. </li>
		<li> 在多个切片上重复此过程以确保确定淋巴结内的最大 SUV. </li>
		<li> 在单独的电子表格中记录所需的 SUV 数据. </li>
	</ol> </li> </ol> 6。葡萄糖浓度的测定标准值规范化的背景摄取量 注意: 为了保持多个成像时间点在葡萄糖摄取和动物体内代谢活性变化的一致性不同的时间, 所有的 PET 分析应归为肌肉, 并提出这样的。在这项工作中提出的所有定量 PET 数据表示为一个 SUVCMR (标准摄取值缸肌比). 
<ol> <li> 在轴向方向上从 OsiriX 数据库中打开 pet 和 ct DICOM 图像 (ct 图像将与 pet 图像融合, 并会有一个单独的 pet 图像窗口). </li>
	<li> 单击 Co 注册的 PET 和 CT 图像以确保它是活动窗口. </li>
	<li> 滚动图像, 直到到达包含主支气管 (底座) 的汇点的切片. </li>
	<li> 在背部肌肉上绘制 roi 以获取背景 SUV 值。
	<ol> <li> 在 OsiriX 主菜单中选择 & #34; 鼠标按钮选项和 #34; 的 "ROI" 下拉工具. </li>
		<li> 突出显示 和 #34; 椭圆和 #34; 作为 ROI 工具. </li>
		<li> 在脊柱后部和侧面的肌肉上绘制近似相同大小的 roi. </li>
		<li> 单击 Co 注册 pet/CT 扫描左侧的图标, 然后将图标拖动到 pet 窗口. </li>
		<li> 选择 和 #34; 复制 roi 和 #34; 。现在应该在 PET 扫描窗口中看到 roi. </li>
		<li> 在主菜单上, 选择 和 #34; 模式和 #34; 复选框并确保 和 #34; MIP 和 #8211; 最大强度投射和 #34; 在右侧的下拉菜单中立即选中. </li>
		<li> 确保 和 #34; 厚板和 #34; 滑动比例设置为 10 。这表明, 10 切片是结合在 PET 图像作为最大强度投影, 使 #34; 气缸和 #34; 兴趣量 (气缸肌肉比的来源). </li>
	</ol> </li> <li> 在电子表格中记录两个 roi 的平均 SUV 值. </li>
	<li> 平均两个值以获取背景葡萄糖摄取值。这是用于获得目标部位摄取量与基础代谢率之间的比值值的值. </li>
</ol>

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作者没有什么可透露的。

从 PET/CT 获得的数据可以作为替代测量的许多方面的m 结核感染, 将观测没有这样的技术。PET/CT 比 x 射线技术更灵敏, 它通常用于猕猴的研究。PET/CT 提供结构, 空间和功能的信息。上述分析有许多实际应用, 如监测疾病进展, 评估药物治疗的有效性, 并为重新激活6,10,11提供风险因素, 13。
跟踪肉芽肿的扩散和葡萄糖摄取的个体病变可以比较控制和实验组不仅提供具体的感染部位, 而且还要遵循传播疾病的25。例如, 在科尔曼和 al. 描述蟹猕猴早期感染的工作中, 继感染进展后, 可以确定动物体内的感染是否会继续活跃、恶化或被免疫系统所控制 (即LTBI)11。这只是一个例子的力量, PET/CT 成像有研究进展的疾病与肉芽肿。同样的方法可以用来研究各种实验参数随着时间的推移。例如, 由4周感染建立的肉芽肿的计数可以为疫苗提供一个强有力的结果措施, 因为最好的疫苗将防止或限制肉芽肿的建立, 在挑战之后。疫苗的另一个结果措施可能是限制传播。这些可量化的结果措施提供重要的数据, 而无需对动物进行早期尸检。评价单个肉芽肿的局限性是 CT 扫描仪的灵敏度;可视化肉芽肿和 #60; 1 毫米的大小往往是不可能的。
评估纵隔淋巴结 (MLNs) 是重要的, 在研究m 结核感染以及。MLNs 是重要的 T 细胞启动和贩运的免疫细胞在感染期间。然而, 在几乎所有的猕猴中, 至少有一个, 有时几个 MLNs 会感染。因此, MLNs 是活性结核和 LTBI 中细菌持久性的一个额外的部位, 可以作为细菌的宿主, 可能有助于重新激活26,27。在严重的 MLN 介入的情况下, 气管可以被压缩。大的坏死 MLNs 会侵蚀到呼吸道, 导致传播感染。对淋巴结的 PET/ct 数据进行分析比肉芽肿更复杂, 因为 ct 扫描不容易看到结节的结构成分。此外, 只有新陈代谢活性的淋巴结, 可以分析由于葡萄糖摄取。因此, 在分析热淋巴结时, 必须确保将 PET 图像缩放到相同的最大和最小强度比例, 以确保图像分析的一致性。由于它们可以是大的结构, 坏死中心可以是阴性的葡萄糖亲和力, 因此, 葡萄糖亲和力可以似乎减少随着时间的推移, 甚至当疾病增加在 MLNs。
全肺葡萄糖亲和力代表肺部内的总炎症。肺炎症的数量是疾病严重程度的征兆并且与细菌负担相关6,10,28因此, 这种定量和客观的评估有许多应用。要测量总的葡萄糖亲和力, 所有体内的 PET 图像显示一辆 suv 的大于2.3 的组合在一个单一的 volume-of-interest (VOI) 和总 SUV 值的整个 VOI 是最终亲和力值。该值是从文献中选择的, 比较了在肺部肿瘤中摄取葡萄糖的 SUV 值与人类的各种传染性病症24。重要的是要注意, 这总的葡萄糖亲和力值仅限于肺部疾病和所有葡萄糖摄取病区相关或位于靠近肺部的地方, 不得考虑。此外, 全肺葡萄糖亲和力不包括 MLNs。虽然个别肉芽肿和淋巴结的亲和力使我们能够看到宿主内结核结局的变异性, 但总肺的葡萄糖亲和力是评价寄主整体的组成部分。这些方法也作为分析工具来测量结核病的药物反应。以前的研究表明, 结核病药物治疗可以减少单个肉芽肿的大小和葡萄糖亲和力, 时间为12 , 这些变化与减少细菌负担有关。在整个疗程中, 炎症的变化也可以用来评估药物的功效或失败。
由于这些程序的高度详细的性质, 为了在研究期间获得最一致的数据, 可能需要进行相当数量的故障排除。本文的目标是概述使全世界的个人都能使用这些技术的程序, 同时牢记对细节的精确注意是必不可少的。评价图像的人应该非常熟悉的解剖和生理学, 以识别异常扫描。图像阅读器需要识别非正常探头摄取整个身体, 因为结核病可以蔓延超出胸腔。此外, PET 和 CT 在成像中的配准不是一个完美的过程, 偶尔会出现图像配准的偏差;在评估非常小的疾病特征 (即1-2 mm 肉芽肿) 时, 认识到这一点非常重要。原始扫描可以是特别有用的比较器, 以确定正常的肺 (和其他器官) 结构和 PET 模式, 并承认那些是新的或改变感染。这项分析的另一个重要组成部分是背景测量。所有的 PET 数据被规范化为肌肉摄取作为一个生理基线, 因为葡萄糖摄取是基于新陈代谢。背部的菱形和锯肌肉的结合用于背景测量, 因为靠近胸腔和禁食动物的葡萄糖摄取相对一致。在m. 结核-感染的猕猴的情况下, 这比使用其他器官, 如肝脏更可取, 为背景测量作为m. 肺结核能传染肝脏, 并且肝脏新陈代谢在对待动物时可能影响与各种抗结核药物。考虑上述因素, 并确保所有的图像区保存在分析结束时, 应产生高度重现性的结果。
类 = "jove_content" >> 总之, PET/CT 提供了一个独特的和强大的方法, 调查m 结核感染非人灵长类动物, 提供定量的结果措施, 与初始感染, 传播和细菌的负担。这样就可以在不同的时间点上跟踪感染的可变结果, 而不需要尸检, 从而节省资源和减少动物的使用。这项技术是直接可翻译的人, 因为 PET/CT 已被用于一些研究, 以评估药物治疗结核, 以及 LTBI 的艾滋病毒和艾滋病毒 + 主题10,29,30,31。最后, 这一技术和定量的工具, 用于分析 PET/CT 数据, 可能是有用的, 在未来的疫苗效能研究, 并可能被用作一个模板, 分析其他传染病的动物模型和人类的主题。

结核分枝杆菌已经折磨了人类几千年, 造成的死亡率比当今世界上任何其他单一的传染性药物都要多。在 2015年, 有1050万报告的新的肺结核 (TB) 病例全球1 , 大多数病例来自印度、印度尼西亚、中国、尼日利亚、巴基斯坦和南非。估计将全球死亡人数从结核病的140万人在同一时期。这个值比100年前的死亡率低近25%。虽然药物敏感的结核病是可治疗的, 但疗程冗长需要多种药物, 依从性是一个关注的问题。多药耐药性 (MDR) 菌株的出现在2015年占新结核病病例的58万。m 结核耐多药菌株的成功治疗率估计为50% 左右。更令人担忧的是, 出现了广泛耐药 (XDR) 的m 结核菌株, 它对几乎所有可用的药物都有抗药性。因此, 在结核病研究领域需要新的技术, 提高诊断结核的能力, 提高对疾病过程的免疫学认识, 并允许对新的治疗方法和预防策略进行筛选, 包括抗生素方案和疫苗功效研究。
m. 结核是一种有氧的 acid-fast 芽孢杆菌, 其物理特征是其非常复杂的外细胞壁和缓慢的生长动力学。感染通常是通过吸入雾化液滴中的单个细菌而引起的, 它们是由有症状的、受感染的个体在咳嗽、打喷嚏或唱歌时排出的。在暴露的感染个体中, 只有 5-10% 的人发展为积极的临床结核病。其余90% 有不同程度的无症状感染, 范围从亚临床感染到无疾病, 所有这些都被归类为潜伏性结核病感染 (LTBI)2,3。在有这种无症状感染的人群中, 大约10% 将通过在其生命周期中重新激活所含的感染来发展活跃的结核病。如果有无症状感染的人染上 HIV 或接受免疫抑制剂治疗, 如 TNF 抑制剂4,5,6, 则重新激活的风险会显著增加。活动性结核病也表现为频谱, 大部分人患有肺结核, 影响肺部和胸淋巴结。然而, m. 结核可以感染任何器官, 因此感染也可以出现在外的参与部位。
m 结核感染的病理特征是宿主细胞的组织球形结构, 称为肉芽肿。巨噬细胞、T 细胞和 B 淋巴细胞是肉芽肿的主要成分, 有可变数量的中性粒细胞7。肉芽肿的中心经常坏死。因此, 肉芽肿功能作为一个免疫微环境杀死或控制杆菌, 防止扩散到其他部分的肺部。然而, m. 结核可以颠覆肉芽肿的致死, 并在这些结构中持续数十年。在新感染或重新激活 LTBI 后, 对积极结核病的发展进行持续和定期的监测是不切实际的, 具有科学挑战性和耗时。在人类和类似人类的动物模型中纵向研究这些过程的技术对于科学界进一步了解m. 结核感染和疾病的许多复杂性非常有用。
PET/CT 是一种非常有用的成像技术, 已被用来研究人类和动物模型的广泛疾病状态8。PET 是一种利用正电子发射的放射性化合物作为一个记者的功能技术。这些放射性同位素通常是功能化的代谢化合物, 如葡萄糖, 或目标组, 旨在绑定到一个受体的利益。由于 PET 同位素发出的辐射强度足以穿透组织, 因此极低的浓度可以用于研究受体靶向化合物中的饱和度, 且浓度低, 对新陈代谢没有影响。使用诸如 2-脱氧-2-(18F) 氟-d-葡萄糖 (葡萄糖) 等药剂的过程。CT 是一种三维 x 射线成像技术, 它使用不同程度的 x 射线衰减来识别人体内器官的物理特性9。当与宠物配对时, CT 被用作地图来确定特定的位置和结构, 以显示宠物示的摄取量。PET/CT 是一种功能强大的工具,在体内成像的人和动物模型感染的m 结核感染, 导致了许多重要的洞察力发病机制, 反应药物治疗, 疾病频谱等6 ,10,11,12。这项工作描述了特定的 PET/CT 分析方法, 在非人类灵长类动物模型中纵向使用参数, 如肉芽肿大小、个体病变中的葡萄糖摄取量、全肺和淋巴结葡萄糖亲和力, 以及检测外疾病6,10,11,12。
这篇手稿描述了非人类灵长类动物 (NHPs) 的成像分析方法, 特别是蟹猕猴, 用于纵向评估疾病进展和药物治疗感染后的m 结核.NHPs 是一个有价值的动物模型, 因为当接种了低剂量的m 结核艾德曼菌株, 动物显示了各种疾病的结果与〜50% 开发活动结核和其余的动物有无症状感染 (即控制感染, LTBI), 为人类所见的临床疾病谱提供最接近的模型3,13,14,15,16。LTBI 在猕猴中的重新激活是由引起人类重新激活的同一种药物引起的, 其中的例子包括人类免疫机能丧失病毒 (hiv、使用猿猴免疫缺陷病毒 (SIV) 作为猕猴版的艾滋病毒)、CD4 衰竭或肿瘤坏死因子 (TNF) 中和13,16。此外, 猕猴的病理学与人类所见的非常相似, 包括在肺部或其他器官中形成的有组织的肉芽肿17。因此, 该模型提供了重要的洞察力的基本主机-病原体相互作用的m 结核感染, 以及关于药物治疗方案和结核病疫苗的宝贵知识14,18,19,20,21。
PET/CT 成像提供了跟踪单个肉芽肿的外观、分布和进展的能力.这项工作主要是作为一个探针, 作为葡萄糖模拟, 合并到新陈代谢活性宿主细胞, 如巨噬细胞, 嗜中性细胞和淋巴细胞8, 所有这些都在肉芽肿。因此, 葡萄糖是宿主炎症的代表。本文详细的分析程序使用 OsiriX, 一个广泛使用的 DICOM 查看器可供购买和使用。图象分析方法描述跟踪个体肉芽肿的形状、大小和新陈代谢活动 (通过葡萄糖吸收) 随着时间的推移, 并利用成像作为一个地图, 以确定特定的病变后, 动物尸检。此外, 还制定了一个单独的方法来量化在一个特定的阈值 (SUV ≥ 2.3) 的肺部葡萄糖摄取总和, 并使用这个值来评估控制和实验组之间的差异, 从疫苗的研究范围感染模型的试验。这些数据支持, 这一全面的措施, 在肺部摄取葡萄糖与细菌的负担, 从而提供有关疾病状况的信息。类似的分析可以进行的葡萄糖摄取胸淋巴结的研究进展的疾病, 以及。下面的协议描述了从动物感染通过图像分析的实验过程。

<table>
	<tbody>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>23061</td>
			<td>Henry Schein</td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td>4866</td>
			<td>Henry Schein</td>
		</tr>
		<tr>
			<td>Cetacaine</td>
			<td>Patterson Vet Generics</td>
			<td>07-892-6862</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Hospira</td>
			<td>07-800-9721</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>7H11 agar</td>
			<td>BD</td>
			<td>283810</td>
			<td>BD Biosciences</td>
		</tr>
		<tr>
			<td>IV catheter</td>
			<td>Surflash</td>
			<td>07-806-7659</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>18F-FDG</td>
			<td>Zevacor</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>Jorgensen Labs Inc</td>
			<td>07-887-0284</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Artificial tears</td>
			<td>Patterson Vet Generics</td>
			<td>07-888-1663</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>07-806-3204</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Neurologica Ceretom CT</td>
			<td>Samsung Neurologica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Siemens Focus 220 microPET</td>
			<td>Siemens Molecular Imaging Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inveon Research Software</td>
			<td>Siemens Molecular Imaging Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>OsiriX</td>
			<td>Pixmeo</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of 18fdg pet/ct imaging as a tool for studying mycobacterium tuberculosis infection and treatment in non-human primates

结核分枝杆菌仍然是当今世界头号传染性药物。随着抗生素耐药性菌株的出现, 需要新的临床相关方法来评估疾病的过程和筛选潜在的抗生素和疫苗治疗。正电子发射断层扫描/计算机断层扫描 (PET/CT) 已经建立, 作为一个有价值的工具, 研究许多疾病, 如癌症, 阿尔茨海默氏症, 炎症/感染。这里概述了一些策略, 已被用于评估蟹猕猴的 PET/CT 图像的感染 intrabronchially 低剂量的m 结核。通过对 pet 图像中病灶和淋巴结的 ct 和摄取量的葡萄糖 (葡萄糖) 的损伤大小进行评价, 这些方法表明 pet/ct 成像能预测活性与潜伏性疾病的未来发展, 并从潜在的感染状态重新激活的倾向。此外, 通过分析肺部炎症的总体水平, 这些方法确定了药物对m 结核的抗生素疗效, 这是目前临床上最相关的动物模型。这些图像分析方法是在阿森纳对抗这种疾病的一些最强大的工具, 他们不仅可以评估一些感染和药物治疗的特点, 但它们也可直接翻译成临床设置, 用于人类研究.

个体病变的鉴别与分析
个别肉芽肿可以可视化的数量, 大小和葡萄糖摄取定性地了解感染过程的一般范围 (图 1)。使用这些图像, 计数肉芽肿随着时间的推移是一个定量的疾病传播的尺度。图 2描述了一组10只动物在一段时间内的个别肉芽肿计数。在这10动物中, 有三个被开发的活性疾病和六种潜伏感染。一只动物没有表现出积极的疾病的迹象, 但偶尔也有培养阳性 (在胃抽吸和/或支气管肺泡灌洗样品) 为m 结核, 把它置于疾病的频谱之间的活动和潜伏, 从而被删除从这个特殊实验的分析。在三动物与活跃疾病, 一动物开发了粟疾病由12星期感染并且被安乐死 (这在图 2被辨认作为 TNTC [太多计数])。从感染后的6周后, 后来发展为活动性疾病的动物, 其肉芽肿的数量比那些将潜伏感染的动物高出统计学意义。
为了更好地描述和区分活动和潜伏动物之间的肉芽肿, 对 PET 扫描的个别病变进行分析, 以确定是否有不同的葡萄糖摄取模式之间的两组。在所有的活动感染动物中, 每种肉芽肿中的葡萄糖摄取量都增加了三至六周后感染 (图 3A)。相反, 动物中的肉芽肿, 潜伏感染显示了葡萄糖摄取量的变化, 一些病变增加, 减少, 或显示相同的摄取量从三到六周 (图 3B)。这些结果被比较的组显示在三周与六周 (图 3C) 和三周vs. 24 周 (图 3D) 之间的差异, 潜在和活跃的动物之间的变化。在这两种情况下, 活动动物的肉芽肿表现出一个积极的和显着不同的变化, SUV (在每个单独的动物 (图 3A 和 3B) 和比较的动物组 (数字 3C 和 3D)。
"热" 淋巴结中葡萄糖摄取量的分析
由于纵隔淋巴结在 CT 扫描中不易被形象化, 除非大面积放大, 否则必须使用 PET 图像来识别这些病变组织。在分析淋巴结时, 关键是要使图像始终缩放到相同的最大和最小的 PET 比例, 以保持整个过程的一致性。在比较 MLNs 的动物的活动或潜伏性疾病时, 林 et al. 通过对 MLNs 的 ROI 分析表明, 在3周的活动动物和潜伏动物中, 在淋巴结中的葡萄糖摄取量是相似的, 而 MLNs 从活畜中发现明显更高的摄取率在6周11。差异在8和12周被视为更大的程度 (图 4)。因此, 除了研究感染动物的肉芽肿外, 还可以利用 PET/CT 数据来评估淋巴结的显著差异。
全肺葡萄糖亲和力
作为评价全肺葡萄糖亲和力的一个例子, Lin et al. 显示, 临床上被归类为 LTBI 的动物的肺部炎症升高与重新激活6的风险有关。在这项研究中, LTBI 蟹猕猴 (感染低剂量的m 结核) 是 PET/CT 成像 (6 月感染) 前肿瘤坏死因子 (TNF) 中和评估的频谱病变看到的临床定义 LTBI 和确定重新激活的风险。具有较高总肺葡萄糖亲和力的动物更有可能重新激活 (图 5)。超过90% 的动物与超过 103肺葡萄糖亲和力或与可见 (通过扫描) 至少一个外站点的感染后重新激活 TNF 中和。只有一只没有激活的动物超过了这个总的肺葡萄糖亲和力阈值。因此, PET/CT 参数可以是一个强有力的工具, 以预测临床结果, 虽然具体参数必须科学确定。
作为一个例子, 显示效用在药物治疗的情况下, 科尔曼 et al. 进行了一项研究测试噁在人类和蟹猕猴的地方, 总肺葡萄糖亲和力被测量 pre-drug 治疗, 和一两个月后处理10。折叠更改被计算为显示药物响应一个月 (图 6A) 和两个月后处理 (图 6B)。在两个时间点, 控制动物显示显着高的葡萄糖亲和力在整个肺部空间比药物治疗动物。在所有药物治疗的动物中, 全肺炎症在两个月的治疗方案中减少, 而在大多数的控制动物中, 总的肺部炎症随着时间的推移而增加或不变。

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56375/56375fig1.jpg"> 
图1。串行葡萄糖 PET/CT 图像显示在早期感染过程中的肉芽肿演变的传播和稳定模式。 (顶行)原发性肉芽肿 (白色箭头) 首先建立在3周感染, 而新的肉芽肿毗邻现有的病变 (绿色箭头) 或新的地点 (黄色箭头)。在感染过程中, 后来发展为活动性结核病的动物会产生更多的病变。(下一行)潜在动物的原发性肉芽肿 (白色箭头) 通常保持稳定, 几乎没有新的肉芽肿在感染过程中发育。wks PI, 周感染。图取自科尔曼et al.11 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56375/56375fig1large.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/56375/56375fig2v2.jpg"> 
图2。从 CT 扫描中描述肉芽肿计数中位数和范围的代表性数据比较主动感染 (红色符号) 与潜伏感染的动物(绿色符号)。 早在六周感染, 主动感染动物的肉芽肿比潜在感染动物多。P & #60; 0.05 (*) 由曼-惠特尼测试。周 PI, 周感染。TNTC, 太多计数。图改编自科尔曼et al.11 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56375/56375fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56375/56375fig3.jpg"> 
图3。利用 ROI 分析 PET 图像, 表明在早期感染时, 主动和潜在感染动物的肺肉芽肿的代谢活性不同。 活动动物的单个肉芽肿 [a] 有显著增加的代谢活动 (测量为标准摄取值的肌肉摄取 [SUVCMR]) 3 和6周后感染, 而潜在感染的动物 [B] 不。在3与6周 (C) 和 3 vs. 24 周时比较了潜伏和活动动物的代谢活性变化, 结果表明, 积极感染的动物 (红方) 在摄取上的变化明显大于潜在动物 (绿色圈子) 在两个时间点。实心黑线代表中位数。魏氏秩和检验用于分析面板 A 和 B 中的数据。对于 C 和 D 面板, 价值是由曼-惠特尼测试分析。所有面板的 P 和 #60; 0.0001 (***)。图改编自科尔曼et al.11 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56375/56375fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56375/56375fig4.jpg"> 
图4。PET 图像的 ROI 分析显示有活性疾病 (红方) 和潜伏性疾病 (绿色圆圈) 的动物在纵隔淋巴结中摄取葡萄糖的差异。 在6、8和12周感染后, 积极感染的动物摄取量较高。每个点代表一个单独的淋巴结。实心黑线代表中位数。p & #60; 0.05 (*), p 和 #60; 0.01 (**), p 和 #60; 0.001 (***) 由曼-惠特尼测试。图改编自科尔曼et al.11 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56375/56375fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56375/56375fig5.jpg"> 
图5。全肺葡萄糖亲和力测量的 ROI 分析六月后感染突出显示在动物中葡萄糖摄取的差异, 从潜伏感染 (粉红色的方块) 与仍然潜伏的动物 (绿色圆圈) 的比较中重新激活。 三四 "重新激活" 的动物, 位于虚线下有外疾病前 TNF (肿瘤坏死因子) 中和。每个点代表一种动物。虚线表示可能重新激活风险的阈值。实心黑线代表中位数。P & #60; 0.01 (**) 从曼-惠特尼测试. 从 Lin et al.6 中改编的数据<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56375/56375fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56375/56375fig6.jpg"> 
图6。有代表性的全肺葡萄糖亲和力 ROI 测量, 突出显示肺部炎症的整体变化比较未经治疗 (红圈) 和唑治疗 (蓝圈) 猴。 葡萄糖亲和力在唑治疗前被测量 (每日30毫克/千克), 在1月 [a] 和2月 (B) 治疗后, 总葡萄糖摄取量的折叠变化。每只猴子都由一个单独的圆圈来表示。实心黑线代表中位数。P & #60; 0.01 (**) 每曼-惠特尼测试.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56375/56375fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates at JoVE.com

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

在这里, 我们提出了一个协议来描述的分析, 18f-葡萄糖 PET/CT 成像的非人灵长类已经感染了m 结核研究疾病的过程, 药物治疗, 和疾病的重新激活。

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 ...

analysis-18fdg-petct-imaging-as-tool-for-studying-mycobacterium

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

18.7K 次观看.  University of Pittsburgh School of Medicine. 在这里, 我们提出了一个协议来描述的分析, 18f-葡萄糖 PET/CT 成像的非人灵长类已经感染了m 结核研究疾病的过程, 药物治疗, 和疾病的重新激活。

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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

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) ...

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 ...

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 ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

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 ...

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 ...

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 ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

利用无脊椎杆菌作为感染模型研究结核分枝杆菌复合物

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...