野生型（ 小牛+ / +）和基因敲除（ 小牛- / - ）小鼠，野生型（ 小牛+ / +）和基因敲除（ 小牛- / - ）的MEF由李志坚陈军大学博士（友情提供得克萨斯大学西南医学中心）13。本出版物是基于由美国国立卫生研究院（R01 CA134241和R01 DE021445）和美国癌症协会（RSG-11-162-01-MPC）赠款资助的工作。

在严格按照指南中的美国国立卫生研究院实验动物的护理和使用的建议进行的所有动物工作：操守准则 。该方案经南加州大学的机构动物护理和使用委员会（IACUC）。 1。通过实时定量PCR和分泌的ELISA方法γHV68感染小鼠细胞因子基因表达<ol><li>通过腹腔注射1.5毫克ketamine/0.12毫克甲苯噻嗪麻醉性别匹配，6-8周龄的C57BL / 6（B6）窝（8-12只/组），并与40 PFUγHV68的滴鼻接种在40微升（感染及东丰11详细说明）。在不同时间点感染后，用CO 2吸入颈椎脱位安乐死的小鼠。 </li><li>打开胸腔，沿STER左外侧切Num和通过与锋利的剪刀的排骨斩。收集肺组织。 </li><li>收集一半的肺组织（左叶）到含有500微升1.0毫米氧化锆/硅珠的无菌1.5毫升螺旋盖管。加入1毫升冷的无血清DMEM入管和均化肺组织通过珠打浆30秒。冰浴管2分钟，然后重复此过程一次。 </li><li>离心管（15,000× 克，10分钟），在4℃，收集上清液并测定如以下所述的细胞因子产生。 </li><li>根据制造商的说明书使用可商购的细胞因子ELISA试剂盒测量细胞因子。 </li><li>收集肺组织（右叶）的另一半到另一个含珠管，并加入1 ml TRIzol试剂。匀化，并根据制造商的指示，在步骤1.3和提取的RNA作为所述收集上清液。通过利用吸光度读数在260和280nm处测定RNA浓度。 </li&gt; <li>制备cDNA用1μg总RNA，并根据制造商的指令（总终体积为20μl）逆转录酶。稀释的cDNA的50倍与无DNA酶和RNA酶的水并进行定量实时PCR。 </li><li>定量实时PCR机器上执行程序。每个反应含有0.5微升对GAPDH（引物的工作浓度为10μM，CE 500nM的每种引物）的基因特异性引物或引物控制的，取5μlSYBR主混合物和4微升的稀释的cDNA。计算△△Ct法确定抗病毒细胞因子mRNA转录的相对量。 </li></ol> 2。 MEF细胞的感染和细胞因子定量ELISA法和定量RT-PCR检测<ol><li> 小牛成长+ / +和小牛- / - MEF细胞亚汇合（约80％），电镀前的细胞密度。 </li><li>拆分MEF细胞成12我们LL板以100,000个细胞/孔（细胞密度将是70％左右的第二天）。进行γHV68感染一式三份，以感染复数为5-10（MOI）进行同步并最大化细胞因子诱导。 </li><li>准备γHV68悬浮在含有病毒适量（1毫升每口井）完整的DMEM培养基。 </li><li>去除培养基，加入含γHV68悬浮MEF细胞。 </li><li>将板在组织培养箱，摇滚，每30分钟，并允许共有2小时培养的。 </li><li>删除含有γHV68培养基，用PBS洗一次，并更换新鲜的完全DMEM。 </li><li>在不同时间点感染后，收获培养基进1.5ml离心管中。用冷PBS洗涤细胞，并用胰蛋白酶消化收集被感染的细胞。 </li><li>按照步骤1.5中所述测量抗病毒细胞因子在培养基中。 </li><li>提取的RNA，制备cDNA并进行定量RT-PCR检测，以确定抗病毒细胞因子基因表达按照步骤1.5-1.7中所述。 </li></ol> 3。慢病毒制作，病毒感染和产生稳定细胞系<ol><li>分裂293T细胞转染前一天，并允许细胞达到约40％汇合在转染时。 </li><li>转染的293T细胞的10cm皿与包装质粒（1.2微克的pVSV-G和6微克pDR8.9的）和7.2 PCDH-FLAG-MAVS或PCDH控制由磷酸钙沉淀的微克。 </li><li>在6至8小时转染后更换培养基用新鲜完全DMEM。 </li><li>在72小时转染后，收集含介质慢病毒，离心机在4000×g离心15分钟，用0.​​22μm的膜过滤器过滤。以提高慢病毒滴度，我们离心含病毒介质在110000×g离心2.5小时，在4℃下小心重悬病毒颗粒的体积的兴趣介质的小。 </li><li>种子小牛- / - MEF细胞或其它基因敲除的MEF细胞1天befo再感染6孔板在〜30-40％汇合。 </li><li>感染MEF细胞用1ml的慢病毒和2ml新鲜完全DMEM，补充用聚凝胺至10微克/毫升的最终浓度。 </li><li>离心板在500×g离心，在30℃30分钟，并在板传送到组织培养箱中。 </li><li>在6小时后感染更换培养基用新鲜完全DMEM。 </li><li>分裂MEF细胞在24小时后的感染，并加入嘌呤霉素的1微克/毫升按48小时后的感染的终浓度来选择稳定表达细胞MAVS。 </li><li>保持MEF细胞中含有嘌呤霉素的培养基，并通过与相应的抗体免疫印迹验证蛋白的表达。 </li><li>细胞可用于病毒感染，细胞因子基因表达和分泌实验，在方案2中描述。 </li></ol>

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	<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124, 783-801 (2006).</li><li>Kato, H., Takahasi, K., Fujita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIG-I-like+receptors:+cytoplasmic+sensors+for+non-self+RNA.">RIG-I-like receptors: cytoplasmic sensors for non-self RNA.</a> Immunol. Rev. 243, 91-98 (2011).</li><li>Kawai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPS-1,+an+adaptor+triggering+RIG-I-+and+Mda5-mediated+type+I+interferon+induction.">IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.</a> Nat. Immunol. 6, 981-988 (2005).</li><li>Seth, R. B., Sun, L., Ea, C. K., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+MAVS,+a+mitochondrial+antiviral+signaling+protein+that+activates+NF-kappaB+and+IRF+3.">Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.</a> Cell. 122, 669-682 (2005).</li><li>Xu, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VISA+is+an+adapter+protein+required+for+virus-triggered+IFN-beta+signaling.">VISA is an adapter protein required for virus-triggered IFN-beta signaling.</a> Mol. Cell. 19, 727-740 (2005).</li><li>Meylan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardif+is+an+adaptor+protein+in+the+RIG-I+antiviral+pathway+and+is+targeted+by+hepatitis+C+virus.">Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.</a> Nature. 437, 1167-1172 (2005).</li><li>Speck, S. H., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+viral+genetics+of+chronic+infection:+a+mouse+model+of+gamma-herpesvirus+pathogenesis.">Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.</a> Curr. Opin. Microbiol. 2, 403-409 (1999).</li><li>Dong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma-herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+initiate+lytic+replication.">Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.</a> PLoS Pathog. , (2010).</li><li>Dong, X., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma+herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+abrogate+NFkappaB+activation+and+antiviral+cytokine+production.">Murine gamma herpesvirus 68 hijacks MAVS and IKKbeta to abrogate NFkappaB activation and antiviral cytokine production.</a> PLoS Pathog. 7, (2011).</li><li>Dong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gammaherpesvirus+68+evades+host+cytokine+production+via+replication+transactivator-induced+RelA+degradation.">Murine gammaherpesvirus 68 evades host cytokine production via replication transactivator-induced RelA degradation.</a> J. Virol. 86, 1930-1941 (2012).</li><li>Dong, X., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Host-virus+Interaction+in+Lytic+Replication+of+a+Model+Herpesvirus.">Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus.</a> J. Vis. Exp. , (2011).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137, 1100-1111 (2009).</li><li>Sun, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specific+and+essential+role+of+MAVS+in+antiviral+innate+immune+responses.">The specific and essential role of MAVS in antiviral innate immune responses.</a> Immunity. 24, 633-642 (2006).</li></ol>

作者宣称没有利益冲突。

病毒免疫逃避是最有活力和迷人的交互接口病毒进攻和宿主防御9之一。宿主先天免疫成分的结构，使得信号转导被有效地启动和忠实传送。划定信号级联的层次结构和调控是先天免疫的一个主要话题。在这里，我们介绍一个协议，以确定一种天生的免疫成分，MAVS，在细胞因子产生的病毒逃避的调控作用。该协议包括一种方法确定细胞因子产生的病毒感染的小鼠和评估的MEF细胞因子基因表...

最近的研究已经安装在宿主先天免疫反应中概述的整体信号级联。不同的隔间内居住，模式识别受体（的PRRs）检测病原体相关分子模式不同来源的（的PAMP）来触发先天免疫信号1。视黄酸诱导基因I（RIG-I）和黑色素瘤分化抗原5（MDA5）蛋白是细胞内的传感器能够识别RNA分子具有特定的结构特征2。在激活时，RIG-I与其下游MAVS（也称为IPS-1，VISA和CARDIF）适配器，反过来，激活IKK（IKKαβγ）和IKK-相关激酶（TBK1和IKKε，也称为一骑交互）配合物3-6。激活先天免疫激酶磷酸化的基因表达的关键调节剂，包括转录因子和其抑制剂，以及使宿主的抗病毒基因（ 如 IL6，TNF＆＃转录激活945，CCL5和IFNβ）。这些信号级联构成了建立抗微生物状态，在感染的早期阶段，以限制病原体传播烈性固有先天免疫反应。 鼠γHV68是密切相关的人类致癌KSHV和EB病毒。因此，γHV68感染的小鼠提供了一种易于处理的小动物模型，研究对γ疱疹病毒感染的体内7的宿主免疫应答。使用γHV68，我们的实验室已经发现了一个复杂的策略，即γHV68劫持宿主先天免疫信号，使病毒感染。一方面，γHV68激活MAVS-IKKβ途径通过指导激活IKKβ磷酸化的复制式激活（RTA），该病毒的转录因子密钥γHV68复制8，以促进病毒的转录激活。在另一方面，本IKKβ介导的磷酸化素数的RelA降解和术语inatesNFκB激活9。因此，γHV68感染有效地避免了抗病毒细胞因子的产生。有趣的是，利用γHV68的表达文库筛选鉴定RTA作为一个E3连接酶诱导的RelA退化和废除NFκB激活10。这些发现揭示了复杂的免疫逃避策略，即上游免疫信号事件是由γHV68利用，以否定的终极抗病毒细胞因子的产生。 在这里，我们描述了一个协议来衡量抗病毒细胞因子的产生在体内和前VIVØ感染γHV68小鼠。在协议中，我们通过慢病毒转导，从而精确定位特定的固有免疫分子在调节抗病毒细胞因子产生的作用进一步探讨在淘汰赛胚胎成纤维细胞的天然免疫成分的“复原”的表情。这个协议可以很容易地适应其它维鲁SES和信号通路。

<table border="0" cellpadding="0" cellspacing="0" width="684">
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			<td height="20" style="height:20px;width:251px;">Mouse CCL5 ELISA kit</td>
			<td style="width:211px;">R&#38;D systems</td>
			<td style="width:223px;"><a href="http://www.rndsystems.com/Products/DY478">DY478</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">1.0 mm Zirconia/Silica beads</td>
			<td style="width:211px;">BioSpec Products</td>
			<td style="width:223px;">11079110z</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">TRIzol</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:223px;"><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=15596018">15596-018</a></td>
		</tr>
		<tr height="79">
			<td height="98" rowspan="2" style="height:98px;width:251px;">SuperScript II Reverse Transcriptase</td>
			<td rowspan="2" style="width:211px;">Invitrogen</td>
			<td rowspan="2" style="width:223px;">18064-014</td>
		</tr>
		<tr height="19">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"> </td>
			<td></td>
			<td></td>
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			<td colspan="2" height="21" style="height:21px;">CCL5 quantitative real-time PCR primers</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCTGCTGCTTTGCCTACCTCTC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ACACACTTGGCGGTTCCTTCGA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissecting innate immune signaling in viral evasion of cytokine production

响应于病毒感染，宿主先天免疫反应被激活，上调抗病毒细胞因子的基因表达和生产。相反，病毒已经进化复杂的策略，以规避和利用宿主的免疫信号传导的生存和繁殖。病毒免疫逃避，将会导致宿主防御和病毒逃避，提供了最迷人的和动态的接口之一辨别宿主 - 病毒相互作用。这些研究推进我们理解先天免疫调节和我们铺平道路，以开发新的抗病毒治疗。 鼠γHV68鼠是啮齿动物的天然病原体。 γHV68感染小鼠提供了一个听话的小动物模型，研究抗病毒对人类社会的KSHV和EB病毒的体内病毒-宿主相互作用的扰动并不适用。在这里，我们描述了一个协议来确定抗病毒细胞因子的产生。这个协议可以适于其它vir区使用和信号通路。 最近，我们已发现，γHV68劫持MAVS与IKKβ，胞质RIG-I和MDA5的下游键先天免疫信号分量，废除NFκB活化和抗病毒细胞因子的产生。具体来说，γHV68感染激活IKKβ和激活IKKβ磷酸化的RelA加速的RelA退化。因此，γHV68高效解耦从上游激活IKKβNFκB活化，否定了抗病毒细胞因子基因表达。这项研究阐明了一个复杂的策略，即上游先天免疫激活被病毒病原体截获请求撤销的直接下游转录激活和逃避抗病毒细胞因子的产生。

三个有代表性的人物都在这里显示，包括细胞因子产生的γHV68感染小牛肺+ / +和小牛 -/ - / - - γHV68感染的小牛 + / +和小牛的鼠标，细胞因子的分泌和基因表达水平的MEF和γHV68感染小牛的细胞因子mRNA水平- / - MEFs细胞“再造”与MAVS。这些代表性实验利用基因敲除小鼠的研究抗病毒药细胞因子的产生在体内和探索基因敲除...

Watch this Scientific Journal Video about Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production at JoVE.com

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

我们描述了一种协议来测量抗病毒细胞因子的产生在感染了模型疱疹病毒，鼠γ-疱疹病毒68（γHV68）小鼠是密切相关的人类卡波西肉瘤相关疱疹病毒（KSHV）和Epstein-Barr病毒（EBV）。利用转基因小鼠品系和小鼠胚胎成纤维细胞（MEFs细胞）中，我们评估了抗病毒细胞因子的产生在体内和离体 。 “重构”的淘汰赛中胚胎成纤维细胞通过慢病毒转导的天然免疫成分的表达，我们进一步确定具体的固有免疫分子和剖析关键的信号事件差异调节抗病毒细胞因子的产生。

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. ...

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Immunology and Infection

10.4K 次观看.  Keck School of Medicine, University of Southern California. 我们描述了一种协议来测量抗病毒细胞因子的产生在感染了模型疱疹病毒，鼠γ-疱疹病毒68（γHV68）小鼠是密切相关的人类卡波西肉瘤相关疱疹病毒（KSHV）和Epstein-Barr病毒（EBV）。利用转基因小鼠品系和小鼠胚胎成纤维细胞（MEFs细胞）中，我们评估了抗病毒细胞因子的产生在体内和离...

视频: Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

科研

免疫与感染

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

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 ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...