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?...

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 border="0" cellpadding="0" cellspacing="0" width="1688">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Fibrous hydroxylatedpolyester sheets</td>
			<td style="width:103px;">Accuwik Ultra&#160;</td>
			<td style="width:119px;">SPR0730</td>
			<td style="width:1084px;">Filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milliplex Assay Buffer&#160;</td>
			<td>Millipore</td>
			<td>L-AB</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-protein binding storage plates&#160;</td>
			<td>Thermo Scientific</td>
			<td>CLS8161</td>
			<td>Plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor</td>
			<td style="width:103px;">Roche</td>
			<td align="right" style="width:119px;">11873580001</td>
			<td>complete EDTA-free Protease Inhibitor Cocktail</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Reader of multi-spot plates</td>
			<td style="width:103px;">Mesoscale</td>
			<td style="width:119px;">NA</td>
			<td>Sector imager 6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Assays&#160;</td>
			<td style="width:103px;">Mesoscale</td>
			<td style="width:119px;"></td>
			<td>Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-&#946;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 sample...

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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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

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

科研

免疫与感染

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

单核细胞化学策略活性在Vivo的定量和血液单核细胞衍生巨噬细胞的特征

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

小鼠动脉粥样硬化的定量

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...