The study was supported by the Swedish Research Council, the Swedish Rheumatism Asociation, Karolinska Institute Foundations, Stockholm County Council, The Wallenberg Foundation, and the GV 80 Years' Foundation for research. The authors would also like to thank Hannah Aucott for proofreading the manuscript.

所有的动物实验均根据照顾和使用由当地伦理委员会Karolinska研究所，斯德哥尔摩批准动物的指导进行。当地伦理委员会如下动物保健的欧盟指令。 注意:从原始协议的主要变化是恢复时间后手术（6小时对 1小时）和LPS的水平注射（2mg / kg的与 15毫克/千克）。否则，与手术本身不同的设置没有被改变。 1.准备材料刺激<ol><li>打开计算机和连接到刺激电极（ 图1A）中的数据采集系统上。 </li><li>进入应答程序。 </li><li>以5毫克/毫升在1×PBS中，等分它制备浓度LPS的储备溶液，并且将其存储在-20℃。在实验的当天，解冻等分并准备ADE为了根据重量至约100微升注入到动物，LPS（0.5毫克/毫升）的样品quate。 </li></ol> 2.准备麻醉动物<ol><li>使用C57BL / 6小鼠。保持它们与12小时亮/暗循环的气候受控条件下，喂它们标准啮齿动物食物，并给予他们随意饮水 。 </li><li>对周围25克打压实验当天小鼠进行手术。 注:当炎症反应引起的，它进行定期检查，并在动物临床反应的观察就显得尤为重要。如果动物条件不满足道德标准需要通过CO 2吸入过早安乐死。 </li><li>设置麻醉机。 <ol><li>确保油管连接是否正确，并以任何方式不被损坏。确保通风正常工作。关键过滤器连接到异氟醚瓶，并填补了VAPOrizer用异氟烷的足够量。 </li><li>打开气源（空气和氧气），并确保该瓶含有足够的天然气用于实验。三通连接器，然后能够将异氟醚流发送到所述吸气室或掩模。 </li></ol></li><li>打开三路连接器的感应室。匹克从家笼一个鼠标和插入动物进入室内。调节流量调节器，以1.0升/分钟的氧气和1.0升/分钟的空气。调节异氟烷浓度为4 - 5％。 </li><li>当达到麻醉的期望的水平，当达到麻醉的期望电平，剃手术区和从所述腔室到所述掩模移动的动物。打开三路连接到面罩流。调节流量调节器，以0.25升/分钟的氧气和0.25升/分钟的空气。 <ol><li>调节异氟烷浓度至1.5 - 2.5％。在开始手术再修改前检查通过神经反射控制和呼吸速率麻醉水平即</li></ol></li><li>修复鼠标使用胶带工作台的腿。确保动物的鼻子还在仔细位于掩模。 </li></ol>迷走神经的3外科和刺激<ol><li>消毒用70％乙醇手术区。 </li><li>使用手术刀，仔细在颈部（约1至1.5厘米的切口）的电平切开皮肤。 注:在该协议中，手术过程结束这里假手术的动物。事实上，这已经表明，刚刚接触到神经用金属工具已经能够刺激到一定程度。因此，使用LPS少量时获得更准确的手术控制动物，停止在这一步的手术。 </li><li>用显微镜（12.5X物镜）的帮助下，从隔离使用解剖镊颈动脉左侧迷走神经。首先通过去除皮肤和脂肪层定位胸锁乳突肌，然后收回它为了把镊子神经和动脉都落后。 注:以下步骤是非常棘手的，因为神经和动脉贴紧对方。因此，这是很容易切断血管，杀死动物。但是，通过将镊子非常仔细的神经和动脉之间，他们最终分开，并有可能对神经隔离。 </li><li>放置电极（ 图1B）的迷走神经下。针状电极是很长，所以即使神经刺激过程中轻微移动，它总是会在与电极接触。 </li><li> （关于细胞因子水平对所读出的， 即，为了测量下调效应）进行LPS的腹膜内（ip）注射（2毫克/千克）与注射器的帮助。 </li><li>在开始刺激前等待5分钟。 </li><li>通过在确认程序按下启动按钮刺激5分钟的迷走神经在5V和1Hz。 </li><li>回覆移动电极和缝合手术缝合线的动物的伤口。 </li><li>喷在伤口无刺阻隔薄膜（NSBF），以提高治疗和感染保护。 </li></ol> 4.动物的恢复<ol><li>手术后，将动物返回其家笼觉醒和恢复。在为了保持体温的红外光，确保监测动物，直到全意识已经恢复。 </li><li>让动物在笼子里收回牺牲分析前6小时。 注:迷走神经刺激的效果是非常快，也被证明是持久（可达48小时），所以恢复时间可以由实验者根据研究的需要进行设置。 </li></ol> 5.牺牲作进一步的分析<ol><li>放置动物在链接到一个CO 2管理装置的保持架。 </li><li>在设备上设置一个5CO 2吸入-min周期。 </li><li>当安乐死完成后，收集感兴趣的器官，并直接将它们冷冻用于进一步的分析干冰（ 例如，细胞因子水平的中脾提取物使用鼠标TH1测量/ TH2 9-Plex的测定）3。 </li></ol>

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作者什么都没有透露。

自21世纪初发现的，盖的机制已经被彻底研究。我们现在有途径的好照片，特别是，靶器官，脾，其中NE，记忆性T细胞，乙酰胆碱，和巨噬细胞作为一种非常有效的团队工作，下调炎症介质2。我们最近还发表了关于功能性前列腺素系统的小鼠中的重要性，特别是PGE 2，这显然是对乙酰胆碱的释放脾脏中盖3的活化后的强制性成分数据。 <p class="jove_conten...

先天免疫提供了针对感染和疾病在广泛的生物体防御的立即第一行。它不仅启动初次免疫应答以消除威胁，但它也起着活化和教育，其执行在病原体特异性方式二次免疫应答的适应性免疫中起关键作用。炎症细胞因子和趋化因子过多，这反过来又吸引其他免疫细胞对感染部位的能力和诱发炎症的主要体征，如红，肿，痛，功能丧失，发热策划。的持续时间和炎症的强度取决于几个因素，但解决炎症和恢复稳态是避免慢性炎症性疾病的发病的关键步骤。在神经科学和免疫学领域的最新进展没有完全揭开特定的神经机制，与巨大的治疗潜能控制INFL无论是在中枢神经系统和外周ammation。一这些机制之一是胆碱能抗炎通路（CAP），也被称为炎性反射，这是由自主神经系统4,5驱动。 这是目前认为炎症介质激活感觉神经和发送有关炎症的状态，以中枢神经系统的信号。一种反射反应，然后通过传出迷走神经活性。在CAP的解剖细节进行了广泛的研究揭示了两个神经组成的副交感神经，交感神经模型，迷走神经和神经脾，分别为6。在CAP中，活化胆碱能传出迷走神经在腹腔 - 肠系膜神经节结束，导致肾上腺素能神经脾的活化的机制尚待探索。脾神经，从而激活，已知的是内瓦泰岛接近免疫细胞在白色的纸浆，边缘区，和脾，主要和强制器官CAP 7,8的红髓。去甲肾上腺素（NE）从脾神经末梢结合于脾T淋巴细胞上表达相应的β2肾上腺素能受体。这引起胆碱乙酰转移酶（ChAT的）介导的乙酰胆碱（ACh）的释放，这反过来又激活巨噬细胞上α7烟碱型乙酰胆碱受体（α7nACh），从而限制了细胞因子的产生和炎症2。因此，现在很清楚，神经系统能够调节炎症外周组织和恢复局部免疫稳态。 由于通路顾名思义，乙酰胆碱系统是至关重要的这种神经免疫调节通路的功能。有趣的是，在所涉及的激活的机制的CAP似乎是在周边和在中枢神经系统中的不同。而烟碱受体（α7nAChR）在脾的重要性在前面已经证明9，毒蕈碱受体（的mAChR）是强制性的通路10，11的中心活化。最近，一个中枢作用M1毒蕈碱激动剂的外周施用显著抑制血清和脾脏肿瘤坏死因子α（TNFα）致死小鼠内毒素血症期间，所要求的完整迷走神经，脾神经信令12的动作。最近，我们还表明，缺乏前列腺素E 2小鼠（PGE 2）无法对迷走神经刺激作出反应，并没有下调血清和脾脏3中的细胞因子的LPS诱导释放。因此，CAP也可能比主乙酰胆碱pathw其他系统调节唉。 迷走神经已被命名为这样的，因为其在体内游荡当然，支配主要器官，包括肝，肺，脾，肾和肠13。考虑到这个大神经支配和迷走神经的非常有效的免疫效果，CAP的治疗潜力可以覆盖广泛的炎症性疾病。迷走神经可电（或机械）活化，具有过电压和频率，并与传统的治疗控制，无药物加入到所述主体。试验目前在风湿病患者进行，例如，以测试在治疗慢性炎症14 VNS的临床意义。总之，神经·免疫通信和炎症的调节目前正在研究中，这将提供一个可能的替代治疗对常规治疗。因此，迷走神经的分析stimulatio在不同的神经支配的器官n效果，同时也表征在慢性炎症的动物模型中的潜在治疗作用的，肯定会给予的见解，并希望新的潜在治疗靶点。 由特蕾西和他的同事4开发的原始方法不能移位，我们的研究领域，由于炎症反应（由LPS的致死剂量）的过度刺激和CAP激活和读出之间太短的时间范围。在本论文中，我们将提出对原始协议所做的修改，对细胞因子水平比较两个不同的方法，并突出于靶器官（脾）的新和相反观察。

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Computer</td>
			<td style="width:91px;">Toshiba</td>
			<td style="width:136px;">-</td>
			<td style="width:191px;">Any computer is actually compatible</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">MP-150 data acquisition system</td>
			<td style="width:91px;">Biopac Systems</td>
			<td rowspan="2" style="width:136px;">MP150WSW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Acknowledge software</td>
			<td style="width:91px;">Biopac Systems</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Mice C57Bl/6</td>
			<td style="width:91px;">Charles River</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Anesthetic machine</td>
			<td style="width:91px;">Simtec Engineering</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical oxygen bottle</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">107563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical air bottle&#160;</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">108639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetflurane (1000mg/g)</td>
			<td style="width:91px;">Virbac</td>
			<td style="width:136px;">137317</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">LPS</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">L2630</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Saline</td>
			<td style="width:91px;">Merck Millipore</td>
			<td style="width:136px;">1024060080</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PBS 10X</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5493</td>
			<td>Diluted 10 times for used concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe (1 ml)</td>
			<td style="width:91px;">BD Plastipak</td>
			<td style="width:136px;">303172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Needles 23G</td>
			<td style="width:91px;">KD-FINE</td>
			<td style="width:136px;">900284</td>
			<td>0.6 x 30 mm (blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Microdissecting forceps (curved)</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">F4142</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dissecting scissors</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z265969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical suture 4-0</td>
			<td style="width:91px;">Ethicon</td>
			<td style="width:136px;">G667G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Euthanasia unit</td>
			<td style="width:91px;">Euthanex Smartbox</td>
			<td style="width:136px;">EA-32000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Cavilon No Sting Barrier Film</td>
			<td style="width:91px;">3M Health Care</td>
			<td style="width:136px;">3346N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">TH1/TH2 9-Plex assay, ultrasensitive kit</td>
			<td style="width:91px;">MesoScale Discovery</td>
			<td style="width:136px;">K15013C-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

increased recovery time and decreased lps administration to study the vagus nerve stimulation mechanisms in limited inflammatory responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

手术后增加时间流逝和减少LPS的剂量后TNFα和白介素1β（IL-1β）的水平如前所示，使用原来的协议，VNS降低TNFα的水平（169.3±24.9皮克/在SHAM mg相比在VNS 39.7±10.8皮克/毫克，P &lt;0.001）和IL-1β（360.0±40.21微克/毫克SHAM对191.7±27.2皮克/在VNS腹膜内注射LPS（15毫克/千克）（ 图2A）以后毫克，p &lt;0.01）在脾。改变用于分析的时...

Watch this Scientific Journal Video about Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses at JoVE.com

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

increased-recovery-time-decreased-lps-administration-to-study-vagus

Research

JoVE Journal

Immunology and Infection

9.8K 次观看.  Karolinska University Hospital. Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

视频: Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

科研

免疫与感染

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 ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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 ...

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 ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

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