该项目的经费部分来自国家过敏和传染性疾病研究所、国家卫生研究所、卫生和人类服务部的联邦基金, CEIRS 合同 P01AI097092-04S1 (P.E.L.)。

本手稿中所做的实验是按照伊坎医学院的《道德和法规规范》进行的。
1. 通过转染或感染来表达流感病毒血凝素
染:
<ol><li>平板人胚胎肾脏 (HEK 293T) 细胞密度为 2 x 104细胞/在白色组织培养中处理96井板, 让它在4个°C 孵化器 (与 37 CO5%) 坐在 2 h。</li>
	<li>染细胞与 100 ng DNA 编码的病毒血凝素和 0.2 ul 的转染试剂每井在 50 ul 的总容量。 注: 质粒和 transfectant 试剂均在1x 最小必需培养基中稀释, 降低血清培养基。</li>
	<li>在37个°C 孵化器中孵化出 5% CO2为18小时的模板后, 删除转染介质。</li>
</ol>2. 感染
<ol><li>板 A549 或 Madin 达犬肾细胞密度为 2 x 104细胞/以及在白色组织培养处理96井板和增长至少 18 h 在37个°C 孵化器与 5% CO2。</li>
	<li>在37个°C 孵化器与 5% CO2中, 在1x 的内存中稀释病毒并感染细胞, 其感染的多样性为3、1或 0.33, 1 h。总容积应等于 100 ul。 注意: 10x 内存被水稀释, 使1x 内存工作库存用于上述病毒稀释. </li>
	<li>感染后, 取出接种, 并在没有胰蛋白酶的情况下加入 100 ul 1x 的记忆。</li>
	<li>在 5% CO2的 37 °C 孵化器中, 将受感染的细胞增长18到24小时。</li>
</ol>3. 通过单克隆抗体或血清评估 FcRg/NFAT 介导的荧光素酶活性的活化
<ol><li>首先, 用25µL 的检测介质 (1x RPMI 1640 补充 4% (卷/卷) 低 IgG 胎牛血清) 取代转染或感染细胞的生长培养基。</li>
	<li>在一个单独的96井板, 执行四倍稀释的抗体的兴趣开始在30微克/毫升使用化验媒介。在 triplicates 中执行稀释, 并在图 1A中演示一个示例板布局。请确保包含一个不包括抗体 (无抗体控制) 和背景控制 (单独检测缓冲区) 的控件。 注: 对于 "no 抗体" 和背景控制, 添加25µL 的检测缓冲器, 而不是抗体。</li>
	<li>将25级稀释抗体从步骤3.2 转移到转染细胞或受感染细胞的相应井上。</li>
	<li>孵育板在 37 °C 孵化器 (与 5% CO2) 为15分钟。</li>
	<li>添加 25 ul 的适当修改效应 Jurkat 细胞表达小鼠 FcgRIV 或人类 FcgRIIIa (7.5万细胞/井)。每个井的总容积应为75µL, 抗体的起始最终浓度为10µg/毫升 (图 1B)。 注意: 对于背景控制, 添加25µL 的检测缓冲区, 而不是效应细胞。为了检查抗体依赖性细胞介导的吞噬功能, 可以使用一种表达人 FcgRIIa 的修饰 Jurkat 细胞。</li>
	<li>孵化板在一个37°c 孵化器与 5% CO2为 6 h。</li>
	<li>解冻和温暖的荧光素酶基板缓冲在 37 °C 水约1小时前使用。 注: 为了使荧光素酶基板缓冲, 重建冻干荧光素酶测定基质在化验缓冲区提供。重构的基板缓冲区可存储在-30 °c 到-10 °c 上, 最多六周。</li>
	<li>添加 75 ul/井的荧光素酶基板缓冲除背景控制以外的所有水井。</li>
	<li>读一紫外线。</li>
	<li>用以下方程式计算褶皱感应: 折叠感应 = (诱发值-背景值)/(无单克隆值-背景值)。</li>
</ol>

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作者声明没有利益冲突。

此处描述的方法允许用户测量 HA 特定单克隆抗体与小鼠 FcγRIV 的接触能力。靶抗原、流感病毒血凝素, 在病毒感染或质粒 DNA 转染后在细胞表面表达。这项试验还可以与其他 FcγRs (人类或小鼠) 结合使用不同的表面表达的病毒蛋白。此外, 我们使用血清样本来评估抗体的能力, 在多克隆的情况下, 以诱发 Fc 效应函数 (数据未显示), 这可能提供更多的相关信息, 当研究感染诱发的适应性免疫反应或接种....

季节性流感疫苗提供的免疫传统上是通过血凝抑制 (HI) 检测方法1来评估的, 它测量了针对血凝素受体结合部位的抗体的存在。这些高活性抗体可以赋予杀菌免疫, 但在其保护范围内一般是狭窄的, 只对少数几株流感病毒提供免疫力。广泛反应性单克隆抗体的分离和鉴定血凝素 (HA) 表明, 普遍流感疫苗的发展范围内可达2, 3, 4,5,6,7,8. 普遍流感疫苗的主要目标之一是对流感病毒的保守区域(9、10、11、12 )产生强烈的抗体应答。,13,14,15,16. 有广泛的反应性抗体识别这些保守的表位, 由中和和非中和抗体17,18, 被证明需要 Fc FcγR 的相互作用, 以优化保护在体内并突出显示 Fc 介导的免疫对流感病毒的整体免疫应答的贡献19,20。
保护的相关性对于评估感染的免疫力至关重要。这些指标允许科学家和临床医生评估疫苗的功效、数据的重要性和治疗过程。预防流感病毒感染的唯一确定的相关性是血凝抑制试验。1:40 的滴度与患病风险的50% 降低有关 (121 ), 并反映了抗体的存在, 通过靶向位于 HA 球状头的受体结合点来抑制凝集。然而, 也应该指出, T 细胞反应可能是更好的相关性预防流感病毒感染的老年人。有趣的是, 最近的一份报告表明, 神经氨酸酶抑制活性可能是更好的预测流感免疫的22。幸运的是, 典型的体外检测方法, 如中和或神经氨酸酶抑制试验可以测量 HA 茎或神经氨酸酶特异抗体。然而, 大多数这些常规的体外化验只考虑抗体的抗原结合区的功能, 而不测量 Fc 区域的作用。此外, 未检测到通过 Fc 受体参与在体内中保护的非中和抗体的贡献17,18。为了测量诱发 Fc 介导免疫的抗体所提供的保护, 如抗体依赖性细胞介导的毒性 (ADCC), 需要一个健壮的体外检测。
下面描述的方法评估小鼠单克隆抗体的能力, 通过使用基因修饰的 Jurkat 细胞系, 表达激活鼠型 1 FcR, FcγRIV, 诱导 Fc 介导的功能。抗体参与 FcγR transduces 细胞内信号, 触发核因子的活化 T 细胞介导的荧光素酶活性。该方法优于传统技术, 需要分离和培养原效应细胞, 使用流式细胞仪检测 Fc 介导的效应函数的激活19,23,24 ,25,26。首先, 这里描述的协议可以很容易地被改编成包含不同病毒的目标, FcγRs 和不同物种 (人和小鼠) 的抗体。其次, 使用修饰的 Jurkat 细胞系表达 FcγR 与荧光素酶报告基因在活化 T 细胞 (NFAT) 核因子下, 允许一个大的格式分析, 可以很容易地用一个平板阅读器测量发光。在这里, 传统的激活的主要效应细胞被取代诱导 NFAT-荧光素酶的抗体接触 FcγR 表面上的 Jurkat 细胞。这96井板格式化验允许测量多达四个不同的样品在 triplicates 与七稀释-许多样品可能是繁琐的使用传统技术。另外还有一点需要考虑, 这可能会影响实验设计和数据的解释。病毒靶必须在细胞表面表达, 以便抗体识别。为了减轻这种情况, 目标抗原 (例如内部病毒蛋白) 可以直接涂在盘子的表面上。然而, 这还没有经过严格的测试。此外, 修改后的 Jurkat 细胞系只表达一种激活 FcγR 的类型, 不表达任何抑制 FcγRs, 而主细胞系表达所有受体在生理环境中。
我们以前用这种方法来证明多克隆语境中的表位特异性在调节 Fc 介导的效应函数方面起着关键作用, 而抗体依赖性细胞介导反应的最佳活化需要两点联系27,28。在这里, 我们描述一个方法, 评估的能力, 茎特定单克隆抗体参与和激活小鼠激活 FcγRIV。尽管有异常29、30, 但大量广泛的反应性抗体瞄准了血凝素的抗原保守的茎区, 从而在普遍流感的发展中起着重要作用。疫苗.

<table border="0" cellpadding="0" cellspacing="0" width="921">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">A549 cells&#160;</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-185</td>
			<td style="width:353px;">Adenocarcinomic human alveolar basal epithelial cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HEK 293T cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CRL-3216</td>
			<td>Human embryonic kidney cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lipofectamine 2000</td>
			<td style="width:141px;">Life Technologies&#160;</td>
			<td style="width:184px;">11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Opti-MEM Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">51985-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White tissue culture treated 96-well plate</td>
			<td>Corning, Inc.&#160;</td>
			<td>3917</td>
			<td>Assay plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">10X MEM</td>
			<td style="width:141px;">Gibco</td>
			<td style="width:184px;">11430-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing murine FcgRIV</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">M1201</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7010</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G9901</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luminometer</td>
			<td>BioTek</td>
			<td>Synergy H1 Multi-Mode reader</td>
			<td>Luminescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bio-Glo Luciferase Assay System</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7940</td>
			<td>Contains luciferase assay buffer and luciferase assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Madin Darby canine kidney cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-34</td>
			<td>Canine kidney cells</td>
		</tr>
	</tbody>
</table>

a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies

抗体通过其抗原结合域和 Fc 区域, 在耦合病毒病原体的先天和适应性免疫应答中起着至关重要的作用。在这里, 我们描述如何测量 Fc 效应函数的活化作用的单克隆抗体靶向流感病毒血凝素与使用基因工程的 Jurkat 细胞系表达活化型 1 Fc FcγR. 使用此方法,免疫球蛋白所赋予的特定 Fc-FcγR 相互作用的贡献可以通过体外测定来确定。

我们证明, 一个茎特定的单克隆, 6F12, 但不是头部特异的单克隆, PY102, 能够激活主要的自然杀伤细胞植入 CD107a 和干扰素γ19。为了模拟茎特异性抗体激活原发性 NK 细胞的能力, 我们表明, 一个茎特异性的单克隆抗体, 6F12, 可以有力地激活修饰的 Jurkat 细胞, 表达小鼠 FcγRIV 14 倍。另一方面, 头部特定的单克隆抗体, PY102 和对照 IgG 不 (图 2)28。</p...

Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

我们描述了一种方法来衡量 Fc 介导的效应函数的活化作用的抗体, 针对流感病毒血凝素。这种检测也可以适应评估单克隆抗体或多克隆血清靶向其他病毒表面糖蛋白, 以诱导 Fc 介导的免疫能力。

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

a-method-to-assess-fc-mediated-effector-functions-induced-influenza

Research

JoVE Journal

Immunology and Infection

7.6K 次观看.  Icahn School of Medicine at Mount Sinai. 我们描述了一种方法来衡量 Fc 介导的效应函数的活化作用的抗体, 针对流感病毒血凝素。这种检测也可以适应评估单克隆抗体或多克隆血清靶向其他病毒表面糖蛋白, 以诱导 Fc 介导的免疫能力。

视频: A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

科研

免疫与感染

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

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

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

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

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

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

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

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

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 ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...