流感病毒在 LM 实验室的研究部分由纽约流感卓越中心 (NYICE) 提供, 这是 NIAID 流感研究和监测卓越中心 (CEIRS) 的成员。我们感谢温迪. 贝茨对手稿的修正表示支持。

<ol>
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作者没有什么可透露的。

IAV 的小鼠模型广泛应用于体内的 IAV 病机、免疫原性和保护功效的研究。与其他动物模型 (如雪貂或豚鼠) 相比, 小老鼠可以使它们易于操作和储存。此外, 在动物成本、住房和生殖方面的便利使它们能够用于需要大量动物的前体免疫试验。值得注意的是, 由于小鼠已被用于多个研究学科, 一些分子和免疫学鼠试剂可以方便用于 IAV 研究。此外, 最小的寄主变异性和存在的免疫系统, 在进化上类?...

IAVs 被封装在Orthomyxoviridae系列3中的病毒分类。他们包含八单 RNA 分子与阴性极性3。在人类中, IAVs 导致季节性流行病和偶然大流行的重要后果, 当新的病毒在人类人口中引入4。此外, 季节性 IAVs 是高度和迅速传播之间的人产生了高的经济损失世界各地每年2,5。IAV 症状包括咳嗽、鼻塞、发热、不适、头痛、厌食和肌痛, 但病毒也会在免疫缺陷患者中产生更严重的疾病6。事实上, 世界卫生组织 (WHO) 计算, 季节性流感病毒每年造成30万至50万人死亡, 在世界各地1。目前只有两类药物被食品和药物管理局 (FDA) 批准用于人类的流感预防和治疗: 神经氨酸酶 (NA) 抑制剂 (例如, 奥司他韦) 和阻断剂的 M2 离子通道 (例如,金刚烷胺);然而, 耐药性病毒变种的出现日益引起人们的关注。因此, 疫苗接种仍然是保护人类免受 IAVs 感染的最佳医疗选择。迄今为止, 有三种类型的流感疫苗是由 FDA 授权供人使用的: 重组病毒血 (HA) 蛋白疫苗、灭活流感疫苗 (IIV) 和活减流感疫苗 (LAIV)5,7. 三种疫苗的设计目的是诱导对病毒 HA 蛋白的适应性免疫反应, 这是中和抗体对抗 IAVs 的主要目标。
一种验证的小鼠模型, 用于研究 IAV 感染在体内
动物模型被用于研究, 除其他以外, IAV 发病机制8,9,10,11, 导致疾病的病毒因素12和/或病毒传输13 ,14, 并测试新疫苗或抗病毒药物的功效9,10,15。老鼠 (小家鼠) 是最广泛使用的动物模型为 IAV 研究有几个原因: 1) 免疫系统在进化上类似于人类的存在;2) 低成本, 包括动物购买、住房和繁殖;3) 体积小, 易于操作和贮存;4) 最小主机的变异性, 以获得均匀的反应和结果;5) 大鼠生物学知识, 包括基因组序列;6) 许多可用的分子生物学和/或免疫学试剂;7) 可用的敲出 (KO) 小鼠研究某一宿主蛋白对病毒感染的贡献;和, 8) 多小鼠菌株, 可以利用, 以评估遗传基础的感染。
目前有几个老鼠菌株可用于研究 IAV在体内。年龄、免疫状况、性别、遗传背景和小鼠品系以及感染途径、剂量和病毒株均影响小鼠 IAV 感染的结果。IAV 研究中使用的最常见的小鼠菌株是 C57BL/6、balb/c/C 和最近的 DBA. 2 只小鼠, 因为它们比两个前菌株更容易 IAV 疾病16,17,18,19,20. 重要的是, 免疫应答也可以根据鼠标的应变情况而有所不同18,19,20。因此, 恢复所有关于鼠标和 IAV 应变的可用信息是非常重要的, 以选择最佳的实验方法进行。
虽然鼠标是一个良好的动物模型感染的在体内研究与 IAV, 他们有几个限制, 需要考虑的实验设计。例如, 使用小鼠进行体内研究的一个主要限制是, IAVs 不在小鼠之间传播。因而, 为传输研究, 更加被接受的动物模型 (例如, 雪貂或试验品) 使用16,17,21。此外, IAV 在小鼠和人类中的表现也存在着一些差异。与人类不同, 老鼠在 IAV 感染时不会发热;相反, 他们目前与低温16,17。在小鼠中, IAV 的复制集中在下呼吸道 (肺部) 而不是上层气管。因此, IAV 在小鼠中的毒力并不总是与人类所看到的相关联。总之, 由于优势大于有限的劣势, 鼠标代表了第一个动物模型, 用于评估流感病毒的发病机制, 免疫原性和保护功效的疫苗和抗病毒研究。此外, 在 IAV 感染的小动物模型中, 使用大型动物模型进行 IAV 研究, 并没有以前的证据, 在道德上是不能接受的。在这篇手稿中, 我们描述了如何感染小鼠鼻 (.) 与 IAV, 如何监测病毒感染的严重性和进展, 以及如何进行的实验, 以评估体液免疫反应和保护功效。

<table border="0" cellpadding="0" cellspacing="0" width="656">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Madin-Darby Canine Kidney (MDCK) epithelial cells</td>
			<td style="width:119px;">ATCC</td>
			<td style="width:123px;">CCL-34</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Six- to eight-week-old female C57BL/6 mice</td>
			<td style="width:119px;">National Cancer Institute (NCI)</td>
			<td>01XBE</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Turckey red blod cells</td>
			<td style="width:119px;">Biolink Inc</td>
			<td style="width:123px;"></td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:119px;">Corning Cellgro</td>
			<td style="width:123px;">15-013-CV</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:119px;">Seradigm</td>
			<td style="width:123px;">1500-050</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Penicillin/Streptomycin/L-Glutamine (PSG) 100X</td>
			<td style="width:119px;">Corning</td>
			<td style="width:123px;">30-009-CI</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Penicillin/Streptomycin (PS) 100X</td>
			<td style="width:119px;">Corning</td>
			<td style="width:123px;">30-00-CI</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Bovin Albumin solution (BA)</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:123px;">A7409</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Bovin Serum Albumin (BSA)</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:123px;">A9647</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:123px;">T8802</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Neutral Buffered Formalin 10%</td>
			<td style="width:119px;">EMD</td>
			<td style="width:123px;">65346-85</td>
			<td style="width:121px;">Store at RT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Triton X-100</td>
			<td style="width:119px;">J.T.Baker</td>
			<td style="width:123px;">X198-07</td>
			<td style="width:121px;">Store at RT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Monoclonal Antibody anti-NP Influenza A Virus HB-65</td>
			<td style="width:119px;">ATTC</td>
			<td style="width:123px;">H16-L10-4R5</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Polyclonal rabbit anti-mouse immunoglobulins/FITC</td>
			<td style="width:119px;">Dako</td>
			<td style="width:123px;">F0261</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:295px;">ECL Anti-mouse IgG, Horseradish Peroxidase linked whole antibody</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:123px;">LNA931V/AG</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">TMB substrate set</td>
			<td style="width:119px;">BioLegend</td>
			<td style="width:123px;">421101</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Vmax Kinetic plate reader</td>
			<td style="width:119px;">Molecular Devices</td>
			<td style="width:123px;"></td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Dounce Tissue Grinders</td>
			<td style="width:119px;">Thomas Scientific</td>
			<td style="width:123px;">7722-7</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Receptor destroying enzyme, RDE (II)</td>
			<td style="width:119px;">Denka Seiken Co.</td>
			<td style="width:123px;">370013</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Crystal Violet</td>
			<td style="width:119px;">Fisher Scienctific</td>
			<td style="width:123px;">C581-100</td>
			<td style="width:121px;">Store at RT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">96-well Cell Culture Plate</td>
			<td style="width:119px;">Greiner Bio-one</td>
			<td style="width:123px;">655-180</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Cell Culture dishes 100mm</td>
			<td style="width:119px;">Greiner Bio-one</td>
			<td style="width:123px;">664-160</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Nunc MicroWell 96-Well Microplates</td>
			<td style="width:119px;">Thermo Fisher Scienctific</td>
			<td style="width:123px;">269620</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Nunc 96-Well Polystyrene Conical Bottom MicroWell Plates</td>
			<td style="width:119px;">Thermo Fisher Scienctific</td>
			<td style="width:123px;">249570</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Puralub Vet Ointment</td>
			<td style="width:119px;">Dechra</td>
			<td style="width:123px;">9N-76855</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Fluorescent microscope</td>
			<td style="width:119px;">Olympus</td>
			<td>Olympus IX81</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

influenza a virus studies in a mouse model of infection

流感病毒在全球范围内造成超过50万人死亡1 , 并与每年的费用 12-140亿 USD 在美国单独考虑直接医疗和住院费用和工作缺勤的2。动物模型是关键的流感 A 病毒 (IAV) 研究评估病毒发病机制, 宿主-病原体相互作用, 免疫反应, 以及目前和/或新的疫苗方法的功效, 以及抗病毒药物。小鼠是一种有利的小动物模型, 因为它们的免疫系统在进化上与人类相似, 它们可从商业供应商那里获得, 作为基因上相同的学科, 有多种菌株可以利用来评估感染的遗传基础, 它们相对便宜, 易于操作。为了总结 IAV 感染的人通过呼吸道, 小鼠首先被麻醉之前, 鼻腔接种与传染性 IAVs 在适当的生物安全控制下。感染后, IAVs 的发病机制是通过每日监测发病率 (体重损失) 和死亡率 (存活率) 来确定的。此外, 病毒病机还可以评估病毒复制在上 (鼻黏膜) 或较低 (肺) 呼吸道感染小鼠。对 IAV 感染的体液反应可以通过无创性出血和二级抗体检测方法快速评估, 目的是检测总或中和抗体的存在。在这里, 我们描述了常用的方法感染小鼠鼻 (i. n) 与 IAV 和评估发病机制, 体液免疫反应和保护功效。

小鼠病毒病机的特征
IAV 的发病机制与其感染引起的发病率和死亡率有关。这两个参数可以很容易地在小鼠中进行评估: IAV 发病率与受感染小鼠的体重下降有关, 存活率的百分比将表明死亡率 (图 1)。IAV 感染小鼠的体重和生存率通常在感染8、9、<sup class="xr...

Watch this Scientific Journal Video about Influenza A Virus Studies in a Mouse Model of Infection at JoVE.com

小鼠感染模型中的甲型流感病毒研究免疫学和感染|视频

Influenza A Virus Studies in a Mouse Model of Infection

甲型流感病毒 (IAVs) 是人类重要的呼吸道病原体。为了了解 IAVs 的致病性和对新疫苗方法进行临床前测试, 需要模仿人体生理学的动物模型。在这里, 我们描述的技术, 以评估 IAV 病机, 体液反应和疫苗的效果使用小鼠感染模型。

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

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28.0K 次观看.  University of Rochester School of Medicine and Dentistry. 甲型流感病毒 (IAVs) 是人类重要的呼吸道病原体。为了了解 IAVs 的致病性和对新疫苗方法进行临床前测试, 需要模仿人体生理学的动物模型。在这里, 我们描述的技术, 以评估 IAV 病机, 体液反应和疫苗的效果使用小鼠感染模型。

视频: Influenza A Virus Studies in a Mouse Model of Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

通过流式细胞仪测量附件和流感的内在病毒在A549细胞

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...