Name: noninvasive sampling of mucosal lining fluid for the quantification of in vivo upper airway immune-mediator levels
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Description: Watch this Scientific Journal Video about Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels at JoVE.com

我们感谢我们向儿童和家庭的 COPSAC2010队列研究他们的支持和承诺。我们承认并感谢 COPSAC 的研究团队和技术帮助从技术员利斯贝思 Buus 罗斯霍尔姆，丹麦技术大学的细胞因子和趋化因子测量的独特努力。

根据赫尔辛基宣言 》 的指导原则进行了研究。得到了从哥本哈根 (KF 01-289/96 COPSAC2000年) 和 H-B-2008年-093 COPSAC2010年和丹麦数据保护机构伦理委员会的批准，并从父母双方各学科入学前获得了书面的知情同意。
1.实验装置
<ol>
	<li>使用的筛选器纸张 （纤维羟基化聚酯床单，见表的材料）6，7。<ol>
		<li>带 3 x 15 毫米大小为 1 个月的孩子和一个 L 形适合大龄儿童和成人 (5 × 20 × 10mm （短臂上的 L）)。</li>
	</ol>
	</li>
	<li>使用镊子婴儿每个鼻孔中插入一张过滤纸。将滤纸放在下鼻甲 （大约 1 厘米内新生儿鼻孔和 1.5 厘米，所有其他年龄段） 的前部。<ol>
		<li>将新生儿放在一张床上，给他们糖水舒适。 注: 为所有其他年龄段的儿童，进行取样最轻松地坐在椅子上的主题。</li>
		<li>大一点的孩子，将插入到每个鼻孔的 L 形过滤纸长的胳膊。</li>
	</ol>
	</li>
	<li>应用鼻夹鼻孔，尽量减少不适，避免意外损失的过滤纸周围。</li>
	<li>2 分钟的吸收后删除筛选器文件。</li>
	<li>将滤纸放在标记管和冻结他们立即在-80 ° c。</li>
	<li>记下呼吸道感染采样日被孩子显示任何症状。</li>
	<li>记录任何打喷嚏、 持久性哭泣，或在 2 分钟的采样过程中发生的鼻出血。</li>
</ol>
2.量化的气道免疫调解员
<ol>
	<li>从冰柜里拿达 10 个随机样本。记录的标识号。在整个工作过程中保持样品在冰上。</li>
	<li>解冻后在冰上，浸泡过滤纸 （每个主题） 从两个鼻孔在 300 微升的新鲜缓冲 （见材料表） 包含一个完整的蛋白酶抑制剂平板电脑 （见表的材料） 每 25 毫升的缓冲区。<ol>
		<li>调整音量的过滤纸大小的缓冲区。<ol>
			<li>用于过滤纸 3 x 15 毫米大小 300 微升的缓冲区。</li>
			<li>如果只有一个过滤纸可用，使用一半的缓冲区卷 （150 μ）。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>样品将转移到板振动筛 (400 rpm) 5 分钟使用计时器。</li>
	<li>湿润的滤纸和缓冲液转入杯醋酸纤维素管滤清器 （0.22 微米孔隙大小），放置在离心管 （见表的材料）。</li>
	<li>在 16,000 x g 5 分钟 （在 4 ° C) 的冷却离心机离心获得筛选鼻提取物管中。</li>
	<li>删除杯和保持同时 aliquoting 冰上的管鼻提取物 （见表的材料） 的低蛋白结合存储板入井。</li>
	<li>保存在-80 ° C，直到分析。</li>
	<li>确定浓度的细胞因子和趋化因子在鼻提取使用高灵敏度，基于 electrochemiluminesce 的多路复用的芯片系统 （见表的材料）。 注: 人类 10 丛 TH1/TH2 细胞因子测定，9 丛趋化因子测定，与单丛 IL-17A、 TGF-β 1 和 TSLP 在这里执行。<ol>
		<li>为测定，孵育鼻提取一夜之间在 4 ° c。进行测量，每硬件制造商的标准协议 （见表的材料）。 注: 免疫分析 （见表的材料） 通常具有高动态范围测量介于 1 &#38; 10,000 pg/mL，但对于一些检测方法，这可以是 100,000 pg/mL。这意味着，所有的样品可以运行在相同的稀释，从而限制在健康和疾病的学科，不同稀释的影响，正如在其他类似的免疫情况。所有细胞因子检测的下限是 1ml pg 或更少，和趋化因子，它介于 1 &#38; 50 pg/mL。TSLP 不是年龄的 98%的样本中检测到 1 个月。</li>
	</ol>
	</li>
</ol>
3.统计数据
<ol>
	<li>日志-变换之前获得的细胞因子和趋化因子水平通常分布式残差的分析数据。</li>
	<li>如果样品分析在多个批次，纠正批围绕对数转换数据的细胞因子和趋化因子的数据。<ol>
		<li>纳入总体均值和反对数变换，以获得原始的度量单位的数据。 注: 对于每个组所作的分析，考虑调整变量会影响细胞因子和趋化因子水平。这可能是孕产妇的影响 （例如，酒精消费和怀孕期间吸烟）、 母乳喂养、 不良事件在采样、 兄弟姐妹、 带宠物在家里，等</li>
	</ol>
	</li>
</ol>

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所有作者申报没有潜在、 感知，或实际的利益冲突关于这份手稿的内容。供资机构并没有任何作用，设计并进行研究;收集、 管理和解释数据;或制备、 审阅和审批的手稿。没有一个制药公司是参与这项研究。

这里介绍的技术，我们得以确定体内上呼吸道黏膜免疫中的配置文件从早在 1 个月的年龄，以前也没有过以前的儿童。我们观察到这种存在的特定气道细菌和核糖核酸7，11，以及其他前和围产期风险敞口，映出的气道新生儿免疫配置文件中。此外，我们用这些气道免疫配置文件数据来研究高剂量维生素 D.随机微量营养素审判的作用机制这些结果强调的正当性和黏膜衬里流体技术作为一种无创的采样技术随后体内定量的气道免疫调解的效用。
以下几点是值得考虑在方法执行之前: 为了测量细胞因子在黏膜衬里流体矩阵中的，很重要的是使用基于高灵敏度的免疫分析方法，如所提供的电致化学发光的协议。酶联免疫检测法的使用可以导致中吸光度读数的推理。此外，虽然鼻子及支气管的紧密关系，上呼吸道免疫剖面测量仍然是限制。抽样进行上部的粘膜层;因此，我们期望流体主要代表的凝胶层，不溶胶层组成。我们不度量粘膜层标记，证实了这一点，这是一种限制。在理论上鼻液分泌的个体差异可能会影响细胞因子和趋化因子的检测的水平。然而，我们已经能够确定明确和不同免疫表型在不同的曝光度7，8，，910，，1112，响应指示在鼻分泌动力学间的个体差异不可能为这种技术的主要障碍。同时演奏技巧，是必须执行所有程序在低的温度下 （冰冷却），以避免加工过程中的蛋白质降解。推荐的蛋白质蛋白酶抑制剂加入蛋白提取缓冲液可以进一步减少蛋白质的降解。
然而，这种技术的力量是它是无创性，易于执行，使气道粘膜免疫签名从新生儿期及以后的纵向评估。这种信息可能可以提供知识如何本地气道免疫系统成熟和发展整个童年和成年。此外，采样技术进行原状和在体内-相关，未稀释的方式，使免疫调解，本来无法察觉的鼻腔灌洗技术，在哪里未知的稀释因子减少结果13的有效性的检测。我们把合成、 纤维、 羟基化聚酯的媒介，是商业上可用的情况下 （见表的材料），设计用于样本采集、 存储和共轭的释放。材料是亲水性，与低生物分子结合力，并含蛋白质样品稳定贮藏于-80 ° c。这种材料是高吸水，和纤维表面已被修改，以提高水的润湿性。
在 COPSAC2010 年的研究，我们选择 21 细胞因子和趋化因子所先验来表示免疫系统在航空代理在当地的主要表型路径选择一个小组。这些调解员，TSLP，唯一一个在一个月的年龄就没有检出。
重要的未来应用程序会收集黏膜衬里流体从较低的航空公司 （例如，通过使用支气管镜珍稀技术） 来评估低气道免疫学。纤维支气管镜珍稀是纤维支气管镜诊断，能够收集当地支气管上皮衬里流体; 新程序它利用护套涤纶纤维探头14。当远端气道后，探测器被推出鞘尖端吸收支气管的上皮液支气管腔内。纤维支气管镜珍稀曾经在急性呼吸窘迫综合征14，，1516和慢性阻塞性肺疾病17中。然而，在哮喘、 存在的问题是探头可能引起出血疾病所致的上皮脆弱性和增加的血管。黏膜衬里流体技术应用支气管镜珍稀程序可能有重要的临床意义与我们的理解的病理生理学，以及监测、 治疗和预后的几种急性和慢性肺疾病的儿童和成人。
从理论上可以使用多重免疫分析平台的使用在这里的商业系统来衡量所有类型的介质 （见表的材料）。该平台能够单克隆抗体适用于免疫分析技术的发展对可以获得的任何调解员为其灵敏度高、 准确量化。这个平台也带来了高精度和较少对流体分析中的 ph 值、 温度和粘度变化的灵敏度。
将来的研究，它将会是很有价值扩大免疫调解当前面板测量中，例如: 抗菌肽;总的和具体的 IgA;急性期反应物，如 C-反应蛋白;脂质介质，如前列腺素;和神经介质，如脑源性神经营养因子 (BDNF)。这可以提供更详细的视图，在身体部位的黏膜反应系统的性质与广泛的机载曝光。建议调解人可能来衡量，通过有针对性的定量高灵敏度免疫分析，如在当前的设置，或由有针对性或不相关核磁共振 (NMR) 光谱-或液相色谱-质谱法 (LC-MS)-基于代谢组学技术。后者的方法已经在某种程度上与呼凝析方法，有一些方法的缺点，对生物流体力学18的采样并不明显的黏膜衬里流体技术。
纵向取样从新生儿期到成年，COPSAC2010队列，在执行将大大增加我们的理解的基础免疫学因素之前或与气道疾病，如哮喘和过敏症伴随地行事。这样的结果可能是重要的新型治疗方法，针对具体疾病 endotypes 的发展和微量营养素调查和药物及其作用机制的随机临床试验。

鼻黏膜衬里流体弥补了上呼吸道系统的液体部分。它是由复杂的矩阵的调解员来自上皮和弥补防御入侵的微生物的第一行的免疫细胞之间的相互作用。鼻黏膜是方便，还有鼻子和支气管1强大功能和免疫学关系。这种间隔是特别感兴趣就是在童年，如哮喘和变应性鼻炎，但也对一系列的以后的生活中更为普遍其他呼吸道疾病中常见的呼吸道疾病。
在这里，我们描述与样品不受干扰的黏膜衬里流体从鼻腔用基于过滤纸、 无创的技术，以及以后的提取过程，用来洗脱基于蛋白质的分析物从过滤纸之前，其量化方法的实现。这种技术为例，可以用于获取体内免疫签名的健康个体和个人的多种呼吸道疾病。此外，有可能审查暴露为一个特定的免疫签名的重要性，并评价如果它是一个预测或以后的疾病发展的调停者。
黏膜衬里液得到以前了鼻腔灌洗2，这之前通常会出现鼻内激发试验，在高水平，以刺激炎症反应3，4，过敏原介绍了。。然而，鼻腔灌洗技术在年幼的孩子是不可行和介绍了未知的稀释因子，迷惑的成果，作为水平可以低于检测限的测定5稀释调解员。此外，由于未知的稀释因子，从鼻内激发试验的实测分析物反应不可同日而语之间个人，从而限制了鼻腔灌洗技术在设置队列的有用性。最后，过敏原挑战只是适用在敏化的主题，和其他挑战，如组胺的挑战，不是生理上有关，有可能导致对介质释放的天花板效应。在提出的基于过滤纸的技术为黏膜衬里流体集合，在那里个别分泌液和分析物水平是影响个体间方差的唯一因素规避了这些问题。
在提取过程中，分析物是从洗脱过滤纸相同的卷的缓冲区在加入后的所有样品。这有利于类似体外稀释的所有样品。基于白蛋白的等渗盐溶液提取缓冲区用于提取步骤;它支持基于蛋白质的调解员的提取和稳定限制之前，量化洗脱蛋白质在随后冻结变性的蛋白质。为了避免在提取阶段的蛋白质降解，蛋白酶抑制剂的鸡尾酒被加入提取缓冲液。
允许不受干扰，体内的量化的技术执行-粘膜部位生成免疫调解员是至关重要。首先，粘膜网站构成了最大的免疫器官，在正文中。第二，鼻位置是机载暴露在主站点和紧密连接到肺1呼吸系统免疫室。第三，测量与无创技术这重要器官的可能性打开的可能性与健康和疾病的气道重要微生物免疫相互作用轴上提供大量的信息。第四，有很多其他可能的应用，这种技术，例如研究当地的免疫学改变的随机对照试验的药物和微量营养素。
我们最初在童年2000年(COPSAC2000年) 队列，在那里我们确定免疫流体分布的黏膜衬里在变应性鼻炎与健康对照组137 岁患儿在哥本哈根前瞻性研究哮喘实施技术。随后，我们成功地将此技术应用到 COPSAC2010的纵向队列和分摊气道免疫配置文件在 1 个月、 2 年和 6 年的年龄和急性呼吸道症状的实例。从 1 个月岁新生儿表明免疫签名和早期生活环境暴露7，8，9，10，，1112之间的重要关联。

<table><tbody><tr><td>Fibrous hydroxylatedpolyester sheets</td><td>Accuwik Ultra&amp;nbsp;</td><td>SPR0730</td><td>Filter paper</td></tr><tr><td>Milliplex Assay Buffer&amp;nbsp;</td><td>Millipore</td><td>L-AB</td><td>Buffer</td></tr><tr><td>Low-protein binding storage plates&amp;nbsp;</td><td>Thermo Scientific</td><td>CLS8161</td><td>Plates</td></tr><tr><td>Protease Inhibitor</td><td>Roche</td><td>11873580001</td><td>complete EDTA-free Protease Inhibitor Cocktail</td></tr><tr><td>Reader of multi-spot plates</td><td>Mesoscale</td><td>NA</td><td>Sector imager 6000</td></tr><tr><td>Assays&amp;nbsp;</td><td>Mesoscale</td><td></td><td>Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-&amp;beta;1and TSLP. A description can be found online on www.mesoscale.com</td></tr></tbody></table>

noninvasive sampling of mucosal lining fluid for the quantification of in vivo upper airway immune-mediator levels

此协议说明原状的上呼吸道黏膜衬里流体的取样。它还详细介绍了之前在流体溶提免疫调解分析用于研究气道局部免疫签名，而不需要刺激程序 （经常使用的其他技术） 的提取工艺。黏膜衬里流体采样滤纸条上放置在前部的下鼻甲及左 2 分钟的吸收。分析物从滤纸，洗脱和提取基于蛋白质的洗脱液基于电致化学发光免疫分析法，允许高级别中的分析物同一样品的高灵敏度量化分析。我们测量 20 预选免疫调解有关的信号转导通路在上呼吸道粘膜的特异性免疫的体内含量，但技术并不局限于该特定的面板或采样点。第一次在 7 岁儿童从哥本哈根前瞻性研究哮喘在变应性鼻炎的童年2000年(COPSAC2000年) 队列中实现了技术。此后，它被用在纵向 COPSAC2010的出生世代，采样在 1 个月、 2 年和 6 年的年龄和急性呼吸道症状的实例。我们成功地获得和分析样品从 620 （89%) 的 700 名 1 个月岁儿童; 几个样品均低于检测检出限 （据中位数 (四分位数范围 (IQR))。低于检测限 （即从 0 到设定点下限为） 为每个调解员样本数目是检测的 29 (7.25-119.5)。这种技术使体内气道粘膜免疫配置文件从出生的量化，可以纵向，应用和可应用于研究遗传学和早期生活环境暴露、 病理生理学、 endotyping，和监测呼吸系统疾病，以及发展和评价的新型疗法的疗效。

Baseline Characteristics of the Airway Immune Profiles: 
Complete data on upper airway mucosal immune mediator levels at 1 month of age was obtained in 620 (89%) of the 700 children enrolled in the COPSAC2010 cohort. Ten neonates were enrolled before the technique was established, and 19 did not attend the 1-month visit. Additionally, 47 samples were excluded because they were extracted and measured in another laboratory used in a pilot study, and 4 samples were lost in transportation (See the study flowchart, Figure 1). Baseline characteristics for the cohort are shown in Table 1.
Most of the mediators had a low level of detection &#60;10 pg/mL (IL-1&#946;, IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, IL-17A, TGF-&#946;1, IFN-&#947;, TNF-&#945;, CXCL8, CCL13, CCL4, and CCL17), whereas 5 had a detection level &#62;10 pg/mL (CXCL10, CCL2, CCL11, CCL26, and CCL22) (Table 2). The median (IQR) number of samples below the detection limit (i.e.&#160;from 0 to the lower limit of detection) for each mediator was 29 (7.25 - 119.5) (Table 2). IFN-&#947; levels were below the lower detection limit in almost half (46%) of the samples, whereas CXCL8 and IL-1&#946; levels were detectable in all samples, and TGF-&#946;1, CCL4, and TNF-&#945; levels were below the lower detection limit in &#60;1% of the samples. One of the mediators, TSLP, was only detected in 2% of samples, and TSLP data was therefore not used for consecutive data analyses. A strong multicollinearity was evident for the 20 detected immune mediator levels, which is visualized in a heatmap (Figure 2).
The Influence of Airway Bacteria and Viruses in Neonates: 
We previously found an association between the airway immune profile and colonization in specific airway bacteria sampled concomitantly with mucosal lining fluid at 1 month of age. An upregulated Type 1- and Type 17-based airway immune profile was observed in neonates colonized with intracellular bacteria, whereas the presence of extracellular bacteria was associated with a Type 17-based profile7. We have also shown previously that the presence of picornavirus was associated with an upregulated profile comprised mainly of Type 1 immune mediators (Figure 3)11. These findings were obtained in children without respiratory symptoms, (i.e.&#160;asymptomatic) on the day of sampling, suggesting an immune-triggering role of colonizing bacteria and viruses in the earliest part of life.
The Influence of Pre- and Perinatal Exposure: 
We have further observed an association between the level of immune mediators at 1 month of age and a maternal history of atopy8, where children born to atopic mothers displayed an overall lower level of immune mediators than children of non-atopic mothers. We subsequently identified an association between the presence of siblings in the household at birth and a specific Type 1/Type 17-directed mucosal immune response, with evidence of this being related to an in utero immune priming effect, as the immune response was inversely correlated to the time since last childbirth (Figure 4)12. Further evidence for in utero immune priming effects was determined from an association between maternal H1N1 influenza vaccination status and the upregulation of TGF-&#946;1 levels in the neonatal airway10.
Evidence for Micronutrient Mechanisms of Action: 
In COPSAC2010, we performed a high-dose versus standard-dose vitamin D intervention trial during pregnancy, which showed an almost one-fourth reduction in the risk of asthma/recurrent wheeze in the offspring at 3 years of age9. Utilizing the measured neonatal airway immune profile, we observed that children born to mothers receiving high-dose vitamin D, compared to standard-dose, were characterized by an upregulation of specific mediators (IL-12p70, IL-13, CCL17, TGF-&#946;1, and IL-2), suggesting that vitamin D promotes an anti-bacterial defense system different from that induced by bacteria, virus, siblings, and maternal atopy9 (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55800/55800fig1.jpg" /> 
Figure 1: Study Group Flowchart. Complete data on upper airway mucosal immune mediator levels at 1 month of age was obtained in 620 (89%) of the 700 children enrolled in the COPSAC2010 cohort. Ten neonates were enrolled before the technique was established, and 19 did not attend the 1-month visit. Additionally, 47 samples were excluded because they were extracted and measured in another laboratory used in a pilot study, and 4 samples were lost in transportation. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55800/55800fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55800/55800fig2.jpg" /> 
Figure 2: Heatmap of Pairwise Correlations between the 20 Cytokines and Chemokines. The red color represents a positive correlation and the blue color represents a negative correlation. The number inside every square denotes the correlation between two mediators. The figure shows that all mediators are positively correlated. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55800/55800fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55800/55800fig3.jpg" /> 
Figure 3: The Presence of Picornavirus is Associated with an Upregulated Profile Comprised Mainly of Type 1 Immune-mediators. This figures shows the Geometric Mean Ratios (GMR), with 95% Confidence Intervals (CI), of airway mediators in neonates with/without the concomitant presence of picornavirus, measured at 1 month of age. This figure has been modified from Wolsk et al.11, with permission. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55800/55800fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55800/55800fig4.jpg" /> 
Figure 4: Effects of the Presence of Siblings in the Household at Birth. A) Airway immune signature (PC1) in neonates with and without siblings at birth. B) Scatterplot showing an inverse association between the airway immune signature (PC1) and time since previous birth for children with siblings. This figure has been modified from Wolsk et al.12, with permission. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55800/55800fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55800/55800fig5.jpg" /> 
Figure 5: Principal Component Analysis Biplot of the Effect of Vitamin D Intervention in the COPSAC2010 Cohort on the Airway Immune Mediator Level at 1 Month of Age. Each point represents a sample, colored according to the intervention allocation of the child. The loadings (i.e.&#160;impact) of the original variables on the principal components are shown as arrows. The intervention resulted in a significantly upregulated immune profile in PC1, which is positively correlated with all immune mediators. This figure has been modified from Chawes et al.9, with permission. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55800/55800fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>N = 620 (%)</td>
		</tr>
		<tr>
			<td>Maternal atopy</td>
			<td>334 (54)</td>
		</tr>
		<tr>
			<td>Non Caucasian</td>
			<td>29 (5)</td>
		</tr>
		<tr>
			<td>Cesarean section</td>
			<td>134 (22)</td>
		</tr>
		<tr>
			<td>Antibiotics in 3rd trimester&#160;</td>
			<td>126 (20)</td>
		</tr>
		<tr>
			<td>Siblings in the home at birth</td>
			<td>358 (58)</td>
		</tr>
		<tr>
			<td>Pets (cat and/or dog) in the home</td>
			<td>216 (35)</td>
		</tr>
		<tr>
			<td>Fully-breastfed at 1 month</td>
			<td>570 (92)</td>
		</tr>
		<tr>
			<td>No smoking in 3rd trimester</td>
			<td>597 (96)</td>
		</tr>
		<tr>
			<td>No alcohol consumption in 3rd trimester</td>
			<td>591 (95)</td>
		</tr>
		<tr>
			<td>Family income &#62;100.000 euro/year</td>
			<td>89 (14)</td>
		</tr>
		<tr>
			<td>Gestational age &#62;37 weeks</td>
			<td>601 (97)</td>
		</tr>
		<tr>
			<td>Apgar score &#62;7 at 5 min</td>
			<td>591 (95)</td>
		</tr>
	</tbody>
</table>
Table 1: Baseline Characteristics of the COPSAC2010 Cohort.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Mediator</td>
			<td>Detection limit pg/ml</td>
			<td>&#60; Detection limit N (%)</td>
			<td>Median pg/ml</td>
			<td>IQR&#160;pg/ml</td>
		</tr>
		<tr>
			<td>IL-12p70</td>
			<td>1.4</td>
			<td>102 (16%)</td>
			<td>10.80</td>
			<td>4.99 - 22.22</td>
		</tr>
		<tr>
			<td>CXCL10</td>
			<td>31</td>
			<td>8 (1%)</td>
			<td>1787.78</td>
			<td>722.8 - 5423</td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>4.1</td>
			<td>285 (46%)</td>
			<td>20.14</td>
			<td>7.63 - 48.51</td>
		</tr>
		<tr>
			<td>TNF-&#945;</td>
			<td>0.6</td>
			<td>5 (&#60;1%)</td>
			<td>28.33</td>
			<td>10.06 - 88.55</td>
		</tr>
		<tr>
			<td>CCL4</td>
			<td>4.3</td>
			<td>4 (&#60;1%)</td>
			<td>194.90</td>
			<td>71.22 - 655.8</td>
		</tr>
		<tr>
			<td>CCL2</td>
			<td>16.0</td>
			<td>9 (1%)</td>
			<td>256.11</td>
			<td>134.4 - 468.5</td>
		</tr>
		<tr>
			<td>CCL13</td>
			<td>2.8</td>
			<td>13 (2%)</td>
			<td>15.17</td>
			<td>10.35 - 22.80</td>
		</tr>
		<tr>
			<td>IL-4</td>
			<td>1.1</td>
			<td>196 (32%)</td>
			<td>1.37</td>
			<td>0.42 - 2.86</td>
		</tr>
		<tr>
			<td>IL-5</td>
			<td>1.2</td>
			<td>172 (28%)</td>
			<td>3.72</td>
			<td>1.42 - 8.10</td>
		</tr>
		<tr>
			<td>IL-13</td>
			<td>2.7</td>
			<td>48 (8%)</td>
			<td>14.49</td>
			<td>6.84 - 27.51</td>
		</tr>
		<tr>
			<td>CCL11</td>
			<td>22.0</td>
			<td>39 (6%)</td>
			<td>139.54</td>
			<td>85.00 &#8211; 236.0</td>
		</tr>
		<tr>
			<td>CCL26</td>
			<td>45.0</td>
			<td>252 (41%)</td>
			<td>164.93</td>
			<td>56.33 - 350.02</td>
		</tr>
		<tr>
			<td>CCL17</td>
			<td>7.2</td>
			<td>90 (15%)</td>
			<td>41.30</td>
			<td>26.83 - 60.68</td>
		</tr>
		<tr>
			<td>CCL22</td>
			<td>25.0</td>
			<td>78 (13%)</td>
			<td>398.31</td>
			<td>243.4 - 575.7</td>
		</tr>
		<tr>
			<td>IL-1&#946;</td>
			<td>0.4</td>
			<td>0 (0%)</td>
			<td>225.49</td>
			<td>50.07 - 1169</td>
		</tr>
		<tr>
			<td>IL-17A</td>
			<td>1.5</td>
			<td>285 (46%)</td>
			<td>1,480</td>
			<td>0.43 - 4.79</td>
		</tr>
		<tr>
			<td>CXCL8</td>
			<td>3</td>
			<td>0 (0%)</td>
			<td>19465</td>
			<td>6321 - 38274</td>
		</tr>
		<tr>
			<td>TGF-&#946;1</td>
			<td>5.2</td>
			<td>1 (&#60;1%)</td>
			<td>28.66</td>
			<td>20.76 - 39.50</td>
		</tr>
		<tr>
			<td>IL-10</td>
			<td>1</td>
			<td>13 (2%)</td>
			<td>22.32</td>
			<td>9.28 - 49.16</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>1.5</td>
			<td>19 (3%)</td>
			<td>18.72</td>
			<td>8.29 - 37.33</td>
		</tr>
	</tbody>
</table>
Table 2: Mucosal Lining Fluid Samples from 1-month-old Neonates from the COPSAC2010 Cohort. Most of the mediators had a low level of detection &#60;10 pg/mL (IL-1&#946;, IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, IL-17A, TGF-&#946;1, IFN-&#947;, TNF-&#945;, CXCL8, CCL13, CCL4, and CCL17), whereas 5 had a detection level &#62;10 pg/mL (CXCL10, CCL2, CCL11, CCL26, and CCL22).

Watch this Scientific Journal Video about Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels at JoVE.com

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

此协议说明采样的原状黏膜衬里流体从上呼吸道非侵入性方法。它可以用于执行量化的体内水平的蛋白质介质，如细胞因子和趋化因子，在各个年龄段的主题。

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 ...

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Universidad San Sebastian

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Immunology and Infection

Unit 1

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

Upper Respiratory Tract Infections

SCIENTISTS IN ACTION

10.2K 次观看.  University of Copenhagen. 此协议说明采样的原状黏膜衬里流体从上呼吸道非侵入性方法。它可以用于执行量化的体内水平的蛋白质介质，如细胞因子和趋化因子，在各个年龄段的主题。

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

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

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

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

舌下免疫治疗作为一种替代起到保护作用对急性呼吸道感染

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

医学

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 ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

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 ...

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

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

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 ...

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 ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

评估对流感嗜血杆菌的呼吸道免疫应答

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

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

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

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