Name: absorption of nasal and bronchial fluids: precision sampling of the human respiratory mucosa and laboratory processing of samples
Uploaded: 2018-01-21T14:00:00.000+00:00
Duration: 11 min 54 s
Description: Watch this Scientific Journal Video about Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples at JoVE.com

资助: 这项工作得到了皇家国立卫生研究院 (NIHR) 生物医学研究中心 (NIHR) 的资助, 该研究所在伦敦帝国理工学院的呼吸道感染中获得了健康保护研究组 (HPRU)。英国公共卫生和 NIHR 帝国患者安全转化研究中心。所表达的观点是作者的意见, 而不一定是 NHS、NIHR、卫生部或英国公共卫生部门的看法。

以下协议中使用的技术已得到伦敦西部研究伦理委员会 (参考编号 15/lO/0444) 的批准。
1. 吸鼻 (NA)
<ol><li>
NA 取样前的准备
	<ol>
<li>执行 NA 时, 先洗手, 戴上手套, 最好在病人面前。</li>
		<li>用头火炬检查鼻腔, 让临床医生用非显性拇指缩回病人的鼻子, 使鼻腔可视化。 注意:通常不需要鼻内窥镜。</li>
		<li>在取样前对鼻腔和下鼻甲进行可视化。 注意:鼻孔 (鼻孔) 在横截面上不是圆的, 它们会直接向后。一般而言, 下鼻甲可被看作是鼻孔侧壁上的凸起或凹痕, 鼻中隔形成平滑、扁平的内侧壁。我们想从这个下鼻甲标本, 因为底层上皮细胞是一个简单的纤毛上皮的呼吸道44。</li>
	</ol>
</li>
	<li>
NA 取样
	<ol>
<li>取样时, 将 NA SAM 轻轻地放在鼻孔的流明上, 将其定位为对下鼻甲平坦。</li>
		<li>让捐献者用食指把 SAM 压在鼻腔粘膜上。NA 可以引起轻微的发痒, 可能的眼睛浇水, 因为多边基金被吸收。 注意:在成人, 我们通常执行 NA 六十年代。</li>
		<li>在吸收了多边基金后, 从鼻孔中取出 NA 装置, 再放回原来的管子。</li>
		<li>在实验室中立即处理样品, 或将其冷冻, 详见本议定书的稍后部分。 注意:NA 是研究各种鼻腔黏膜挑战模型, 也在不同呼吸道疾病。然而, 对其他呼吸取样方法的验证应针对每一个研究和病人的人口。</li>
	</ol>
</li>
</ol>2. 支气管吸收 (BA)
<ol><li>
BA 取样前的准备 注意:BA 是由专门人员在一个支气管镜组。<ol>
<li>在将 BA 装置放进导管前, 检查 SAM 是否正在挤压并取出导管。</li>
		<li>在对病人进行 ba 之前, 在支气管镜模拟器上进行模拟 ba。</li>
	</ol>
</li>
	<li>
BA 取样
	<ol>
<li>通过支气管镜下气管和右主支气管到水平的支气管中间, 只是近端到右下叶和右中叶的分裂。 注意: 我们通常从支气管中间取样, 尽管呼吸道内的其他部位可以取样。观察导管和 SAM 时, 不能看到支气管镜提示。我们观察以下简单的步骤, 为 BA, 一旦支气管镜已达到所需的取样地点。</li>
		<li>导管向下: 将 BA 导管插入支气管镜的操作通道, 直到白色尖端在气道中可见, 最大为1厘米远端至支气管镜的末端。将支气管镜和导管尖端放在气道腔的中心。注意尽量减少导管尖端与支气管黏膜之间的接触, 以减少粘膜的磨蚀风险。</li>
		<li>sam: 压低 BA 装置的手柄, 使 sam 被挤压到右中或右下叶气道的腔内。在直接视觉下, 弯曲支气管镜的尖端, 以确保 SAM 正在与在气道壁上的多边基金进行接触。离开山姆在气道, 平的到黏膜壁三十年代。</li>
		<li>萨姆在: 通过支气管镜检查, 以确保潮湿的 SAM 探头是直的, 而不是弯腰在导管的末端。如果需要, 导管和支气管镜提示可以带回整顿 SAM。直视下, 将直的 SAM 轻轻缩回导管。 注: 如果有困难缩回 sam 回到导管, 撤回整个装置与 sam 挤出的气道。</li>
		<li>导尿管: 从支气管镜的操作通道中取出整个导管。</li>
		<li>萨姆: 萨姆是用无菌剪刀切掉的, 然后放在冰上的 cryotube 上。这些示例可以使用不同的方法进行处理, 详见本协议后面的详细内容。</li>
	</ol>
</li>
</ol>3. NA 和 BA 样品的处理
注意:有许多选择的实验室处理的样品所产生的 NA 和 BA。这些协议试图存储样本以备以后使用, 并洗从 SAM 中提取多边基金的样本。
<ol><li>
NA 和 BA 样品的直接处理方法
	<ol>
<li>选择 1: 在进一步加工之前, 在冰上储存潮湿的 SAM 几个小时。</li>
		<li>选择 2: 立即深冷冻 NA 在收集管。同样地, 用剪刀从取样装置中取出后, 在低温小瓶中冷冻 BA。</li>
		<li>选择 3: 将分离的 SAM 放在洗脱缓冲器 (300 µL) 中, 然后再直接或深冷冻处理。</li>
	</ol>
</li>
	<li>
钠和 BA 样品洗脱缓冲液的选择 注意:洗脱缓冲液的选择取决于将在多边基金样本中分析的内容, 我们建议四种主要选择:<ol>
<li>使用预准备的化验缓冲液, 适用于免疫分析程序。此类缓冲器应包含少量洗涤剂 (0.05%) 以及蛋白质,例如,牛血清白蛋白 (BSA) 在1%。</li>
		<li>或者, 使用含有大量洗涤剂的缓冲液, 使细胞裂解发生。 注意:我们使用包含海卫 x 或 NP40 1% 浓度的缓冲器。细胞裂解缓冲能使胞内和胞外细胞因子从 SAM 洗, 通常导致细胞因子和趋化因子水平增高。这些缓冲也应该含有蛋白质, 并由 BSA 组成到1%。</li>
		<li>对于 rna 测量, 如病毒 rna 的定量 PCR 或测量寄主 rna, 增加 rna 提取缓冲直接地对潮湿的 SAM。 注意:Chaotropic RNA 提取缓冲包含胍那使蛋白质。另一种方法是将 RNA 提取缓冲区添加到免疫分析或细胞裂解缓冲液中的洗多边基金中。</li>
		<li>使用有机溶剂, 如乙酸酸, 用于提取脂质和代谢物, 进行质量分光光度法评价。 注意:所有这些试剂的细节都包括在材料部分。</li>
	</ol>
</li>
	<li>
洗脱技术
	<ol>
<li>对于上述任何洗脱技术, 将 SAM 插入2毫升微离心管, 连同所需的萃取缓冲器。</li>
		<li>漩涡混合三十年代的样品来洗涤松散附着的液体和生物分子的 SAM。</li>
		<li>为了确保完整的样品回收, 执行离心洗脱, 加入湿 SAM 的旋转过滤器迷你柱插入到相同的2毫升微离心管用于洗涤。 注意:可以使用两种类型的自旋滤波器迷你柱。第一个只包含一个塑料网格, 它把 SAM 放在地方, 允许液体完全洗脱。或者, 如果使用传染性物质, 用0.22 µm 孔大小的自旋滤波器。这些过滤器将消毒样品, 适用于疑似杆菌感染的样品。然而, 这些过滤器应该是预孵化与缓冲, 以最大限度地减少对过滤器的约束力, 通过非特定的互动。</li>
		<li>使用灭菌钳转移湿 SAM 到旋转过滤器。更换样品间的镊子以防止污染。</li>
		<li>离心机样品为20分钟在 1.6万 x g 在一个微型离心机冷却了到4° c。</li>
	</ol>
</li>
	<li>
摘要示例协议
	<ol>
<li>在实验室中, 标签2毫升微离心管样本收集和添加所需的洗脱缓冲液 (通常为 NA 300 µL, 100 µL 为 BA)。密封盖和冰上的地方。</li>
		<li>在取样后 (立即或在实验室中), SAM 用镊子将其从手柄中取出, 放入含有收集管的缓冲液中 (前一步产生)。确保离心管的瓶盖安全关闭, 并将样品在冰上转移到实验室进行进一步处理。</li>
		<li>从他们的转移容器和漩涡混合物去除包含 SAM 和洗脱缓冲的管子三十年代。</li>
		<li>使用无菌钳, 删除 SAM 和放置到一个旋转柱 (有或没有0.22 µm 纤维素醋酸过滤器)。</li>
		<li>收集从收集管洗脱缓冲和保留。</li>
		<li>将自旋列与 SAM 一起放在原始集合管中 (如果是新的, 则为无菌过滤样本), 并将收集缓冲区添加到自旋列中。以这种方式, 洗涤液将通过自旋柱回收集管, 帮助洗样品从山姆。</li>
		<li>离心样品与盖子密封 (1.6万 x g, 20 min, 4 ° c)。</li>
		<li>将样品从离心机中取出, 放入机架中。</li>
		<li>打开管的密封盖, 并移除包含 SAM 的自旋列。在适当的废物容器中处理 SAM 和自旋柱。</li>
		<li>小心地将管子内的洗分到标签的低温管中。记录洗的总体积和每个分的体积。</li>
		<li>将密封的低温管转移至80° c 冷冻库, 并将其直立存放直到使用。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

现有气道取样技术的结果被认为是高度可变的;需要采用替代抽样技术, 以便在这一领域内标准化研究5。NA 和 BA 允许以非侵入性的方式取样多边基金, 并具有令人兴奋的潜力来测量健康和患病呼吸道的免疫应答。这些技术提供了许多潜在的优势超过现有的技术, 包括更高的耐受性, 取样速度, 经常重复取样, 降低的用户间变异性, 和减少稀释免疫介质的能力5.每项研究都应优化吸收的持续时间和处理技术, 并在取样事件之间严格保持。此外, 在 BA 的情况下, 在气道内取样的地点应仔细地在个人之间复制。
NA, 特别是 BA, 仍然是相对地新颖的技术为临床研究。然而, 这些技术的好处导致了它们在许多研究中的使用, 包括对替代技术的仔细验证5。这些设备现在可以作为 CE 标记的设备, 可以广泛应用于呼吸道研究。虽然 NA 和 BA 的结果比替代取样技术的样本量要小得多, 但获得的浓度越高, 对低丰度免疫介质的敏感性就越大。
根据所需的下游应用, NA 和 BA 样品可以直接冷冻, 以备以后处理, 在临床研究环境中提高研究的可行性。该协议的样品处理也可以适应特定的下游应用。建议的处理技术可用于收集蛋白质或脂质免疫介质或核酸, 但应针对每项研究进行优化。特别是, 多边基金可以洗不同的缓冲。首先, 免疫分析缓冲可以用来测量黏膜细胞因子、趋化因子和抗体6,45。具有更高洗涤剂水平的缓冲器也可以用来保证细胞裂解发生, 从而允许包括细胞内细胞因子。Chaotropic RNA 提取缓冲液应用于病毒感染、病毒负荷、寄主 mRNA 和微生物的测定。另外, 有机溶剂可用于学和质谱。
总之, 从黏膜表面直接吸收多边基金是一种令人兴奋的技术, 在呼吸道、胃肠、泌尿生殖和其他粘膜疾病中有潜在的应用。然而, 这些有希望的吸收技术将需要对每个疾病设置的个体化验 (生物标志物) 的取样和处理技术进行精确验证。此外, 这些新的精确的黏膜取样技术将需要对常规样本进行验证, 例如从血液、呼吸和痰中提取。利用这些技术, 多边基金可以用来测量微生物、细胞因子、趋化因子、前列腺和抗体。

大多数呼吸道疾病引起炎症反应, 并迫切需要从呼吸道黏膜表面取样, 在变应性鼻炎、病毒和真菌感染、肺结核、哮喘、慢性阻塞肺疾病、肺纤维化和肺癌1。鼻咽吸液、鼻腔灌洗、支气管肺泡灌洗是上、下呼吸道取样的常用技术。然而, 这些技术存在着相当大的问题, 包括耐受性差, 炎症介质的稀释, 以及无法经常重复采样2。一个替代品的抽样是使用棉签, 无论是尼龙植绒, 棉花, 或人造丝3,4, 但这些也有限制, 因为它们造成不适和损害的鼻腔上皮, 并在某些情况下不可逆转的约束力炎症介质5。这些技术通常不能连续一小时重复进行, 而替代技术可能更有效地检测低丰度细胞因子和趋化因子5,6。此外, 与这些技术相关的用户可变性可能会产生数据不一致, 从而导致更大的患者队列的需求。
另外, 使用天然和合成海绵的吸收技术也被用于从黏膜表面收集多边基金。由天然纤维素 (如、Weck) 组成的眼部海绵已用于唾液、宫颈和阴道分泌物的取样,7。此外, 由聚乙烯醇 (PVA) 和羟基聚醋酸乙烯酯 (HPVA) 制成的合成海绵已被利用8。在测量抗体9之前, 对七种不同的吸收材料进行了取样, 而聚氨酯迷你海绵则被用来收集人类的眼泪,10。
由天然纤维素组成的滤纸从棉花植株中被广泛用于吸收鼻腔分泌物自从阿拉姆的先驱纸和同事在 1992年11、12、13、14, 15、16、17。过滤纸光碟已产生的过滤卡 (如山东), 并已被用于测量胺和细胞因子后, 控制鼻过敏原的挑战和与自然过敏原暴露18,19 ,20,21。然而, 不同批次的滤纸在它们的蛋白质结合程度上不尽相同, 有些则无法释放细胞因子。使用合成吸收矩阵 (SAM) 的方法因此被开发了2,22,23。现在一般都是用钠来获取鼻多边基金。这些吸水材料是舒适的使用, 可以获得多边基金, 甚至从发炎的鼻子在很长一段时间内频繁。
鼻吸收是一种精确的黏膜取样的形式, 使用 SAM 进行上呼吸道多边基金的取样。NA 设备是制造的 CE 标志医疗设备从医疗级材料使用洁净室和无灰尘和过敏原。NA 取样器由手柄和山姆在无菌 cryotube。SAM 由聚合物, 典型地是纤维, 但它也是可利用的作为泡沫, 并且这些被选择是软的和吸收的, 以快速的吸汗为样品汇集。sam 具有最小的蛋白质结合, 使吸收的分泌物的有效洗脱。NA 是一种非常温和的, 非侵入性的技术, 可以在各个年龄段的捐赠者身上进行。此外, 连续抽样, 甚至每隔几分钟, 是可能的。NA 已经验证了现有的上气道取样技术5和重复采样允许生成的动能数据后, 挑战的气道与过敏原23,24,25,细菌内毒素26和病毒型 TLR 激动剂 (贾, A. et al., 手稿准备)。NA 也已用于婴儿调查的自然历史过敏27,28,29和病毒性毛细支气管炎30。
支气管 microsampling (BMS) 是由奥林巴斯31,32,33开发的下气道中的多边基金的收集程序。不幸的是, 这个 BMS 系统只在日本获得许可。奥林巴斯供应两个 BMS 系统: 一个带有纤维羟基聚酯 (FHPE) 探头34,35,36,37, 一个带有棉花探针33,38,39,40,41,42,43. 一个主要的绊脚石是, BMS 探头用于哮喘患者引起的粘膜接触性出血, 其中一半的样本受血液污染。作者的结论是, 在哮喘患者的外周气道上使用这种 BMS 系统来取样是不可行的43。
作为替代, 我们已经开发 BA 使用软 SAM, 可以执行在支气管调查的下呼吸道, 包括以下实验感染的哮喘患者与鼻6。BA 设备包括: 外部空心导管, 激活拉伸的手部, 以及一个中央塑料导丝, 它的末端有 sam。至于 NA, BA 套件是由医用级材料制造, 使用干净的房间, 没有灰尘和过敏原。此外, 设备是 CE 标记和提供伽玛辐照。山姆条是软的, 吸收, 并且有快速的吸汗为样品汇集。它还具有最小的蛋白质结合, 使吸收分泌物的有效洗脱。该装置可以通过支气管镜的工作通道, 可用于快速准确地在呼吸道内的特定部位对多边基金进行取样。不像球或 BMS, BA 不会导致接触出血或额外的病人不适后程序。
对 NA 和 BA 样品的处理应慎重考虑。样品可以直接冷冻和处理分批, 或可以立即处理。该类型的加工可以针对某些下游应用, 包括免疫细胞因子, 趋化因子和免疫球蛋白, 或 elutions 病毒, 细菌, 和宿主细胞相关的 RNA。我们介绍了与 NA 和 BA 相关的临床收集和实验室处理技术, 作为临床研究人员的指南。

<table><tbody><tr><td>Nasosorption (adult, 7mm width)</td><td>Hunt Developments</td><td>NSFL-FXI-11</td><td>Different sizes are available for different patient groups/ages.</td></tr><tr><td>Bronchosorption</td><td>Hunt Developments</td><td>BSFL-FXI-11</td><td>Minimum bronchoscope channel size 2mm; Max working length 815mm</td></tr><tr><td>Corning&amp;nbsp;Costar&amp;nbsp;Spin-X&amp;nbsp;centrifuge tube filters (without membrane)</td><td>Sigma Aldrich</td><td>CLS9301-1000EA</td><td></td></tr><tr><td>Corning&amp;nbsp;Costar&amp;nbsp;Spin-X&amp;nbsp;centrifuge tube filters (0.22um membrane)</td><td>Sigma Aldrich</td><td>CLS8160-24EA</td><td>For sterilisation of samples with infection risk.</td></tr><tr><td>Assay (elution) buffer</td><td>Millipore</td><td>AB-33k</td><td>Not listed on the Millipore website but available through enquiry or general lab supply companies, such as Cedarlane. Contains 0.05% Tween-20 and 1% BSA.</td></tr><tr><td>NP-40 Cell lysis buffer</td><td>Life Technologies</td><td>FNN0021</td><td>Add bovine serum albumin to 1% (w/v). Can recover higher absolute mediator levels.</td></tr><tr><td>Buffer RLT (RNA extraction)</td><td>Qiagen</td><td>79216</td><td>Allows recovery of RNA from nasosorption and bronchosorption samples.</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma Aldrich</td><td>302031-100ML-M</td><td>For elution of samples to be used in HPLC applications</td></tr><tr><td>2.0ml micro-centrifuge tubes</td><td>Costar</td><td>3213</td><td>2ml tubes are required for the Spin-X tube filters, traditional 1.5ml tubes will not fit these.</td></tr></tbody></table>

absorption of nasal and bronchial fluids: precision sampling of the human respiratory mucosa and laboratory processing of samples

鼻吸收 (NA) 和支气管吸收 (BA) 的方法使用合成吸收基质 (SAM) 吸收的黏膜衬里液体 (多边基金) 的人呼吸道。NA 是一种非侵入性的技术, 它从下鼻甲中吸收液体, 造成极小的不适。NA 已经取得了重复性的结果与能力, 经常反复取样的上呼吸道。
相比之下, 呼吸黏膜的替代方法, 如鼻咽吸入和常规抽吸, 更具侵入性, 可能导致更大的数据变异性。其他方法有局限性, 例如, 活检和支气管程序是侵入性的, 痰中含有许多死亡和垂死的细胞, 需要液化, 呼出的呼吸冷凝液 (EBC) 含有水和唾液, 洗灌样本稀释变量.
BA 可通过支气管镜的工作通道进行临床。取样是很好的耐受, 可以进行在多个地点的气道。BA 的结果是, 多边基金样品的稀释比支气管肺泡灌洗 (球) 样品少。
本文阐述了钠和 BA 的技术, 以及所得到的样品的实验室处理方法, 可以根据所需的下游生物标志物进行测量。这些吸收技术是用于临床呼吸研究的常规取样技术的有用替代品。

NA 已被用于许多研究, 以方便和非创测量黏膜炎症。根据过敏原管理的鼻子, prostaglandin-D2 (PGD2) 的水平可以观察到上升和下降在几分钟内 (图 1), 符合肥大细胞脱 (茨et al., 手稿准备)。此外, 第二类炎症的调解人, 如 IL-5 (图 2, 从24转载的许可), 可以在鼻腔过敏原挑战23,24中的小时内测量。在实验性感染的过敏性哮喘患者和健康的控制, NA 被用来测量一个小组的调解人, 包括干扰素伽玛 (干扰素), 在7天 (图 3, 转载的权限, 从6) 的过程中。此外, 在自然呼吸道合胞病毒感染的婴儿, NA 表明, 与高浓度的炎症细胞因子, 如干扰素, 相对于非呼吸道病毒的婴儿毛细支气管炎和健康控制 (图 4, 从30的权限中重印)。有趣的是, 与呼吸道合胞病毒和非呼吸道合胞毛细支气管炎之间的区别在时间匹配的挪威文样本中并不显著 (图 4)。
BA 也用于过敏性哮喘的实验感染与鼻。在鼻感染的4天, 干扰素、CXCL11、IL-10 和 IL-5 的水平从基线升高 (图 5;分别为 a、B、C 和 D。此外, 这项技术显示了高 IL-5 水平的过敏性哮喘患者在鼻感染期间, 相对健康的控制 (图 5D) (图 5转载的权限从6)。
这些代表性的结果是从样品洗, 使用含有 0.05% Tween-20 和 1% BSA 的试验缓冲液 (参见材料表) 生成的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56413/56413fig1.jpg"> 图 1: 快速生成和清除 prostaglandin-D2 继鼻过敏原的挑战.prostaglandin-D2 (PGD2) 的水平从鼻腔吸收溶测定的序列样本后, 鼻过敏原挑战与蒂莫西草花粉 (n=5)。每一行代表一个个体的数据。<a href="//cloudflare.jove.com/files/ftp_upload/56413/56413fig1large.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/56413/56413fig2.jpg"> 图 2: 运动测量 IL-5 后鼻过敏原的挑战.IL-5 在鼻腔过敏原的连续鼻腔吸收标本中的生产挑战。三重复过敏原挑战研究进行, 参加者 (n=19) 接受安慰剂 (蓝色), 低剂量 (10 毫克) 口服泼尼松 (橙色), 或高剂量 (25 毫克) 口服泼尼松 (红色) 前一小时过敏原管理。线表示几何平均值和误差线是所有参与者的95% 置信区间。(图重印了以允许从泄密et al., 黏膜免疫学, 2017)。<a href="//cloudflare.jove.com/files/ftp_upload/56413/56413fig2large.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/56413/56413fig3.jpg"> 图 3: 在鼻感染期间诱导干扰素-γ.在健康成人 (n = 11, 蓝色) 和过敏性哮喘 (n = 28, 红色) 与 rhinovirus-16 的感染挑战, 鼻吸收取样被用来测量干扰素γ (干扰素) 的水平。数据代表作为一个) 的个人和 B 的原始意大利面条情节的中间水平与误差线表示分范围。(图转载的许可从汉斯et al.6).<a href="//cloudflare.jove.com/files/ftp_upload/56413/56413fig3large.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/56413/56413fig4.jpg"> 图 4: Nasosorption 将高干扰素与婴儿呼吸道合胞病毒感染联系起来.用鼻吸收和鼻咽吸入法测定婴幼儿毛细支气管炎与呼吸道合胞病毒感染 (红、n=12)、非呼吸道呼吸病原体 (绿色、n=12) 和健康控件 (蓝色、n=9)。数据的分析使用 Kruskall-瓦利斯测试与邓斯校正多个比较 (***p< 0.001)。线条表示中值和误差线是分范围。(使用茨et al.的权限修改的图30).<a href="//cloudflare.jove.com/files/ftp_upload/56413/56413fig4large.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/56413/56413fig5.jpg"> 图 5: 在鼻感染期间支气管吸收样本中的炎症介质. 继 rhinovirus-16 感染健康控制 (n=10, 蓝色) 和过敏性哮喘志愿者 (n=23, 红), 支气管吸收被用来测量水平的 A) 干扰素, B) CXCL11, C) IL-10, 和 D) IL-5 在基线和4天感染。数据通过魏氏签名的秩测试 (匹配样本) 和曼-惠特尼测试 (无与伦比的样本) 进行分析。图转载的许可从汉斯et al.6<a href="//cloudflare.jove.com/files/ftp_upload/56413/56413fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples at JoVE.com

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

这篇手稿描述了使用鼻腔和支气管吸收技术, 特别是使用合成吸收基质 (SAM) 来取样的黏膜衬里液体 (多边基金) 的上和下呼吸道。这些方法比现有的呼吸取样技术提供更好的标准化和耐受。

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

absorption-nasal-bronchial-fluids-precision-sampling-human

Universidad de Cartagena UDEC

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Unit 1

Upper Respiratory Tract Infections

SCIENTISTS IN ACTION

29.8K 次观看.  St Mary's Hospital. 这篇手稿描述了使用鼻腔和支气管吸收技术, 特别是使用合成吸收基质 (SAM) 来取样的黏膜衬里液体 (多边基金) 的上和下呼吸道。这些方法比现有的呼吸取样技术提供更好的标准化和耐受。

视频: Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

科研

免疫与感染

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

痰液诱导和实验室处理方法

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

医学

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed due to challenges with different diagnostic modalities. Ciliary videomicroscopy, using Digital High-Speed Videomicroscopy (DHSV), one of the diagnostic tools for PCD, is considered the optimal method to perform ciliary functional analysis (CFA), comprising of ciliary beat frequency (CBF) and beat pattern (CBP) analysis. However, DHSV lacks standardized, published operating procedure for processing and analyzing samples. It also uses living respiratory epithelium, a significant infection control issue during the COVID-19 pandemic. To continue providing a diagnostic service during this health crisis, the ciliary videomicroscopy protocol has been adapted to include adequate infection control measures.
Here, we describe a revised protocol for sampling and laboratory processing of ciliated respiratory samples, highlighting adaptations made to comply with COVID-19 infection control measures. Representative results of CFA from nasal brushing samples obtained from 16 healthy subjects, processed and analyzed according to this protocol, are described. We also illustrate the importance of obtaining and processing optimal quality epithelial ciliated strips, as samples not meeting quality selection criteria do now allow for CFA, potentially decreasing the diagnostic reliability and the efficiency of this technique.

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

To guarantee a successful and high-quality ciliary functional analysis for PCD diagnosis, a precise and careful method for respiratory epithelium sampling and processing is essential. To continue providing PCD diagnostic service during the COVID-19 pandemic, the ciliary videomicroscopy protocol has been updated to include appropriate infection control measures.

使用数字高速睫状视频显微镜进行鼻刷采样和处理 – 适应 COVID-19 大流行

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

使用等温扩增进行人类呼吸道细菌感染诊断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

作者聚焦:使用微流控芯片推进呼吸道病原体的快速检测 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

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通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体