We thank Dr. Antonio Virgilio&#160;Failla for providing the FRET acquisition quantification algorithm and Erwin Bohn for providing the pMK-bla and pMK-ova constructs.

1.峰值发射和串扰测定（参见图2） <ol><li>为了确定该发射峰和施主之间的串扰（香豆素衍生物V450）和受体（荧光素）的染料量，执行标记的细胞都在405nm激发个体荧光团的光谱扫描。 </li><li>确定每种染料的将被用于供体和受体通道（这里455-465纳米和525-535纳米）的峰在10纳米的带宽。积归一化强度在波长，并计算在受体通道，反之亦然（图2）的串扰供体染料的平均百分比。 </li></ol> 2.准备Y的菌菌株用于细胞培养的感染<ol><li>使用下面的毒株：WA-314 PMK-OVA（WA-314转化的质粒PMK-BLA 17和PMK-OVA 17，分别）和WA-314ΔYopEPMK-BLA（WA-314ΔYopE用该质粒转化PMK-BLA 17） </li><li>感染连胜Y.前至少两天菌菌株WA-314 PMK-OVA，WA-314 PMK-BLA和WA-314ΔEPMK-BLA从包含所需的抗生素（卡那霉素WA-314 PMK-BLA和WA-314 PMK-OVA LB琼脂平板冷冻库存;卡那霉素和四环素WA-314ΔEPMK-BLA）和成长O / N在27℃。此后保持板长达一个星期4℃。 </li><li>感染前一天接种含有所需抗生素3 ml的LB培养基的与各自的菌株的一个菌落，并培育O / N在27℃在摇床以180rpm。 </li><li>关于感染当天接种2毫升的O / N培养的成40毫升的新鲜LB培养基无抗生素并在摇床孵育90分钟，在37℃下以180rpm以引起III型分泌系统的表达。 </li><li>转移培养物到合适的管中，保持培养物在冰上或在4℃下今后。离心培养物10分钟，在5000×g离心和4℃。 </li><li>重悬细菌沉淀在2-3毫升冰冷的PBS。通过使用分光光度计稀释细菌悬浮液，以8×10 8 CFU / ml的浓度。分别确定相应的光密度。保持这个悬挂在冰上，直到感染的HeLa细胞。 </li></ol> 3.电镀HeLa细胞<ol><li>一天感染种子前1.5×10 4个细胞的HeLa细胞中含有在96孔板的孔中的10％热灭活胎牛血清（FCS），在37℃下培养在5％CO 2培养箱200μl的DMEM中。种子三眼井每一株（三个不同的培养时间）。 </li></ol> 4.感染HeLa细胞与CCF4加载<ol><li>感染HeLa细胞通过加入3.5微升细菌悬浮于培养基中，并轻轻吹打培养基两次混合。让细菌沉积物上到细胞中，以每细胞约100细菌的MOI（感染部分）。对于时间过程开始感染在-90分钟，-60分钟，-30分钟，并在37℃下在5％CO 2培养箱直到0分钟以实现共同的终点。 </li><li>当孵化完成后，小心吸媒体不去除HeLa细胞，并加入50微升PBS含20微克/毫升氯霉素停止效应和2.5mM丙磺舒的表达，减少CCF4出口。 </li><li>在这一点上96孔板转移到显微镜载物台，并定义合适的斑点在每个以后的分析孔。在10分钟内完成这个步骤（对收购事项之详情参见5.1节）。 </li><li>每孔中，加70微升CCF4 / AM加载溶液（0.24微升溶液A，1.2微升B溶液，18.56微升溶液C（溶液A，B和C提供的范围内CCF4 / AM装载套件）和50微升的PBS（含20微克/ ml氯霉素和2.5mM丙磺舒）使用多pipette孵育5分钟，在室温。从现在开始，工作不中断。 </li><li>吸加载溶液，并添加含有20微克/ ml氯霉素和2.5mM丙磺舒的使用多移液管将100μlPBS中。然后迅速在96孔板转移到显微镜载物台，并从加载溶液的吸液在10分钟内开始采集。 </li></ol> 5.激光扫描显微镜和数据分析<ol><li>图像采集<ol><li>设定成像条件的方式以最大化总光子计数和最小化光毒性，漂白和交叉激发。 <ol><li>在现代激光共聚焦显微镜，使用20X高数值孔径浸泡的目的，打开探测器针孔2.5艾里单位（ 即宽光学切片），选择高灵敏度的GaAsP探测器同时与主动供体和受体通道，8位深度，〜750nm的像素大小，3倍帧平均和EXCITe为405nm的二极管激光器的10％。 </li><li>为了确保正确的量化设置两个通道的检测器增益为相同的值，并避免任何饱和的像素。 注意：在此，增益被设定为150％。 </li></ol></li><li>保证连续浸没用1ml注射器扩频浸没介质到孔的底部，例如，避免任何气泡的俘获。 </li><li>激活的透射光，并找到在每个井的视图的代表字段。使用正确校准自动阶段，标志着该软件的位置。 </li><li>去除板染色后（参见4.3节），重新插入板和顶部增加的重量，以避免焦点漂移。回顾与激光扫描方式保存的位置，并适当调整的重点和侧卧位。 </li><li>设置的时间间隔为2分钟，持续时间为2小时，并开始收购。 </li></ol></li><li>图像量化（例子是位平面软件选装定是诸如了Imaris） <ol><li>导入数据集到软件，使像素矩阵的算术运算。通过拖放含有供体和软件图标受体通道的源文件执行此操作。 </li><li>使用相同的偏移减去在这两个信道的背景。要做到这一点，请单击菜单，然后单击图像&gt;处理&gt;基线扣除。选择每个通道和输入基准值。 </li><li>纠正渗出通过供给方信道（​​CH1）到受体通道（CH2）用的预先确定的校正系数f（参见1.1）的，创建一个新的信道： CH2 = CH 2 - 通道1 *˚F 注：渗出通过受体进入施主信道的则没有检测到过度的噪音水平。 <ol><li>点击菜单&gt;图像处理&gt;通道运算在和进入ABS（CH 2 - （CH1 * 0.33））。随后删除频道2通过单击菜单&gt;编辑&gt;删除频道。确保渗出通过修正受体通道保持作为新的渠道2.可选：通过进入菜单&gt;编辑&gt;图像属性更改从灰色渠道颜色与原来的颜色。选择4频道&gt;映射颜色&gt;基础颜色和改变它，例如，绿色环保。 </li></ol></li><li>创建具有以下操作来确定相对FRET系数的新渠道： CH 3 = CH 2&#39;/（CH1 + CH2“） <ol><li>进入菜单&gt;图像处理&gt;通道运算在和输入CH2 ./（CH1 + CH2） </li></ol></li><li>对于一个8位图像的FRET通道进行适当的可视化，创建第四个通道： CH4 = 255 * CH3 <ol><li>进入菜单&gt;图像处理&gt;通道运算在并输入255 * CH 3。通过进入菜单&gt;编辑&gt;图像属性改变通道的颜色。选择4频道&gt;映射颜色&gt;颜色表文件&gt;喷射。 </li></ol></li><li>应用一个3x3的中值滤波通道CH3和CH4塔吨将减少图像中的噪声。要做到这一点，请单击菜单&gt;图像处理&gt;平滑&gt;中值滤波器。选择这两个渠道，应用过滤器大小“3x3x1”。 </li><li>对于蜂窝信号定量，检测的细胞在通道4通过适当地设置偏移和过滤结构由尺寸排除太小的结构。 <ol><li>选择超越查看并点击图标“ 添加新的表面 ”上。限定在表面窗口的检测算法参数。为了更快的处理，激活两个复选框“段利息只有一个区域”和“处理整个图像最后”。通过编辑其尺寸限定感兴趣的区域。 </li><li>选择通道4作为信源通道和阈值&gt;背景减法（局部对比度）设置的阈值，并设置直径10微米。设置最小强度阈手动0和最大阈值的最大的IntensiTY。过滤结构由区，并设置最小值例如，187平方微米。完成表面检测。 </li></ol></li><li>提取的平均值为供体荧光强度（CH1）和相对的FRET系数（CH3）和在蜂窝实体它们的标准偏差。要做到这一点，去表面性能。通过选择面风格/质量&gt;表面改变结构的外观。通过单击过滤器选项卡上选择的所有结构。通过将流程选择&gt;统一统一的检测机构。导出通过点击统计标签表面的统计数据为MS Excel&gt;导出所有统计到文件。 </li><li>随着时间的推移绘制这些值。确定的信道供体荧光增加为每个条件的合理参数来表示易位效应物的量最大斜率的10分钟时间间隔。计算斜率为m =Δ供体荧光强度/Δ时间（分钟）。 </li></ol></l我&gt; </ol>

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The authors declare that they have no competing financial interests.

我们在这里成功地应用于基于TEM-1β-内酰胺酶报告基因分析的效应易位中用 Y定量分析菌 。这种敏感，特异的和相对简单的技术的许多不同的变化，在文献中已经描述。在这项研究中的激光扫描显微镜进行最灵敏和最精确的检测荧光信号的进行。专门校正的供体和受体染料和单独调节检测的带宽之间的串扰允许测量更高的精度相比该方法的其它变型。结果清楚地表明，该方法是能够?...

III型分泌系统，它们由不同的属的革兰氏阴性菌，直接递送细菌编码的效应蛋白到真核靶细胞利用专门蛋白出口的机器。而分泌机制本身是高度保守的，专门集效应蛋白进化的不同细菌种类中，以操纵细胞信号途径和促进特定细菌的毒力的策略1。万一耶尔森氏菌，最多七个效应蛋白，所谓Yops（ 耶尔森氏菌外蛋白），被易位在宿主细胞接触并一起行动颠覆免疫细胞反应，如吞噬作用和细胞因子产生， 即，以允许细菌的胞外生存2-4。易位的处理严格控制在不同阶段5。它被确定，T3SS初级活化通过它的接触到目标策触发LL 6。然而，这种引发的精确机制仍有待阐明。在耶尔森所谓微调易位的第二电平由上调或下调细胞的Rho GTP结合蛋白的Rac1或RhoA的活性实现的。 Rac1的激活例如通过侵袭素或细胞毒性坏死因子Y（CNF-Y）导致增加的易位7-9，而GAP（GTP酶活化蛋白质）易位YopE的功能下调Rac1的活性，因此通过一个负反馈型降低易位机制10,11。 有效和精确的方法是前提，以研究如何易位是在小肠结肠炎耶尔森氏宿主细胞相互作用调控。许多不同的系统已被用于这个目的，每个具有特定的优点和缺点。一些方法依赖于裂解感染的细胞，但不细菌由不同的去污剂随后免疫印迹分析。钍这些方法Ë共同的缺点是次要的，但不可避免的细菌裂解潜在污染的细胞裂解液与细菌相关的效应蛋白。然而，提出了治疗细胞用蛋白酶K以降解细胞外的效应蛋白和后续使用毛地黄皂苷为真核细胞的选择性裂解，以尽量减少此问题12。重要的是，这些检测主要依赖于高品质抗效应的抗体，其中大多是尚未投放市场。企图利用效应蛋白翻译融合和荧光蛋白GFP的像并监控易位没有成功可能是由于该荧光蛋白的球状三级结构和分泌装置的无法分泌13之前展开它们。 百日咳毒素然而，有几个不同的记者的标签，如CYA（钙调蛋白依赖的腺苷酸环化酶）域cyclolysin 14或闪存标签被成功用于分析易位。在前者的测定CYA的酶活性被用于扩增胞内融合蛋白的信号，而闪光标签，在很短的四半胱氨酸（4Cys）基序标签，允许与biarsenical染料的Flash标签，而不会干扰分泌的过程中15。 这里所应用的方法是由夏邦杰等人报道首次并基于对细胞内的荧光共振能量转移（FRET）染料CCF4通过易位效应TEM-1β-内酰胺酶融合体16（图1A）的转换。 CCF4 / AM是其中香豆素衍生物（供体）和荧光素部分（受体）由头孢菌素核连接的细胞穿透物化合物。在被动进入真核细胞，所述非荧光酯化CCF4 / AM化合物由蜂窝酯酶处理以带电和荧光CCF4从而被困内的小区。激发香豆素结构部分在405nm处的结果的FRET的荧光素部分，其在530nm发射的绿色荧光信号。头孢菌素核的β-内酰胺酶的FRET的裂解之后被破坏，香豆素结构部分的激励导致的蓝色荧光发射at460纳米。该方法的不同应用在文献中突出了其通用性已被描述。该方法允许易位体外的分析以及在体内，例如，该技术被用来在小鼠感染模型来识别体内 17-19针对易位的白细胞群。信号读出可使用酶标仪，流式细胞仪或荧光显微镜进行。值得注意的是，该方法还提供了感染过程20,21期间监视易位实时由活细胞显微术的可能性。这里激光扫描荧光显微镜涂敷了用于readou荧光信号吨，因为它提供最高的灵敏度和精确度。特别是，能力调整与纳米精度与高敏感检测器组合的发射窗有助于优化荧光检测和最小化串扰。此外，该显微镜的设置可适于易位实时监测和有可能允许对宿主 - 病原体相互作用的在细胞水平上同时分析。 在这项研究中易位Y.率菌野生型菌株和YopE缺失突变体表现出hypertranslocator型10,11示范性地进行了分析。

<table border="0" cellpadding="0" cellspacing="0" width="1068">
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		<tr height="21">
			<td height="21" style="height:21px;width:373px;">LB-Agar</td>
			<td style="width:209px;">Roth</td>
			<td>X969.2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Medium</td>
			<td>Roth</td>
			<td>X968.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8662-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate (black, clear bottom)</td>
			<td>Greiner Bio One</td>
			<td>655087</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco/Life Technologies</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>Sigma-Aldrich</td>
			<td>P8761-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>LiveBLAzer FRET-B/G Loading Kit with CCF4-AM</td>
			<td>Life Technologies</td>
			<td>K1095</td>
			<td></td>
		</tr>
		<tr>
			<td>Lectin from Triticum vulgaris (wheat) FITC conjugate</td>
			<td>Sigma-Aldrich</td>
			<td>L4895</td>
			<td>Used for peak emission and cross-talk determination</td>
		</tr>
		<tr>
			<td>V450 Rat anti-Mouse CD8a</td>
			<td>BD Bioscience</td>
			<td>560469</td>
			<td>Used for peak emission and cross-talk determination</td>
		</tr>
		<tr>
			<td>Immersol W immersion oil&#160;</td>
			<td>Zeiss</td>
			<td>444969-0000-000</td>
			<td>Refractive index = 1.3339 @ 23 &#176;C</td>
		</tr>
		<tr>
			<td>TCS SP5 II confocal laser scanning microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>2x GaAsP-Hybrid detectors, 4 channel spectrometer, acusto optical beam spliter, motorized XY stage, adjustable pinhole, objective 20x HC PL APO CS IMM/CORR, 405nm diode laser 50mW</td>
		</tr>
		<tr>
			<td>Imaris 7.6 software</td>
			<td>Bitplane</td>
			<td></td>
			<td>Plugins included ImarisXT and MeasurementPro</td>
		</tr>
		<tr>
			<td>MatLab compiler runtime</td>
			<td>MathWorks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 5</td>
			<td>GraphPad software</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of yersinia enterocolitica effector translocation into host cells using beta-lactamase effector fusions

许多革兰氏阴性细菌包括致病耶尔森菌属使用III型分泌系统，转运的效应蛋白到真核靶细胞。在宿主细胞内的效应蛋白操纵细胞功能的细菌的益处。为了更好地理解III型分泌宿主细胞相互作用，灵敏，准确的检测过程中控制测量易位是必需的。我们在这里描述了基于一小肠结肠炎耶尔森效应蛋白片段的融合的测定法（耶尔森外蛋白; YopE）的应用程序。用TEM-1β-内酰胺酶的易位的定量分析的测定法，依赖于细胞裂解渗透剂FRET染料（CCF4 / AM）通过易位β-内酰胺酶的融合。 CCF4的由β-内酰胺酶的头孢菌素核的裂解之后，从香豆素FRET荧光素被破坏，香豆素结构部分的激励导致的蓝色荧光发射。该方法的不同应用在文献中突出了其通用性已被描述。该方法允许易位体外的分析以及在体内，例如，在小鼠模型。可以用酶标仪，流式细胞仪或荧光显微镜进行检测荧光信号。在由不同耶尔森突变这里描述的，在效应子融合的体外易位至HeLa细胞中设置由激光扫描显微镜监测。通过实时β-内酰胺酶的效应融合录制FRET记者细胞内转化提供了可靠的定量结果。我们在这里展示示范性数据，由Y.展示增加易位菌 YopE突变体比野生型菌株。

为了证明所描述的方法的能力进行定量分析的效应易位至靶细胞，二耶尔森株不同易位动力学进行了研究：Y。菌野生型菌株WA-314（血清型O8，窝藏的毒力质粒pYVO8 22）及其衍生物WA-314ΔYopE（WA-314窝藏pYVO8ΔYopE23）。此前的研究显示，Y.菌突变体缺乏功能YopE表现出显著高于烨转率10。两个菌株用为YopE和TEM-1β-内酰胺酶（PMK-BLA）17的 n-末端分泌信号的...

Watch this Scientific Journal Video about Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions at JoVE.com

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

analysis-yersinia-enterocolitica-effector-translocation-into-host

Research

JoVE Journal

Immunology and Infection

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

8.5K 次观看.  University Medical Center Hamburg-Eppendorf. Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

视频: Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

表面等离子体共振检测到宿主细胞质的毒素易位

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

慢性自身免疫性干眼鼠模型增加效应记忆T细胞在眼球组织

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

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

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

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 ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.