我们感谢亚历杭德罗·布伦来自中钢协，老龄研究所提供的抗裂谷热病毒血清。在我们的实验室工作是由Forschungsförderung宝石的支持。 §2绝对值。 3 KooperationsvertragUniversitätsklinikum吉森UND马尔堡，莱布尼茨研究生院新生病毒性疾病（EIDIS），东风集团Sonderforschungsbereich（SFB）1021和东风集团Schwerpunktprogramm（SPP）1596。

1，播种A549细胞的感染<ol><li>培养的A549细胞的T75烧瓶，在37℃和5％CO 2在细胞培养基（DMEM补充有10％FCS，526.6毫克/升L-谷氨酰胺，50.000 U /升青霉素和50毫克/升的链霉素）。 </li><li>开始收获细胞前，热身细胞培养基，PBS和0.05％胰蛋白酶-EDTA中加热至37℃的水浴</li><li>除去培养基并用10ml PBS洗细胞。再次取出PBS。 </li><li>加3毫升胰蛋白酶-EDTA，并在烧瓶中分发平分。传输烧瓶在培养箱37℃和5％CO 2。 </li><li>当所有的细胞分离，增加7 ml的细胞培养液中，悬浮细胞，并且将细胞悬浮液转移入15ml Falcon管中。 </li><li>离心细胞，在800×g离心5分钟，在室温，除去上清液和重悬沉淀在10毫升新鲜的细胞培养基中。 </li><li>计数细胞计数室。 </li><li>添加2.5×10 6细胞在5毫升细胞培养基中每两个T25烧瓶。孵育16小时，在37℃和5％CO 2。一个烧瓶提供的模拟控制，一个用于氯13的感染。 </li></ol> 2，感染裂谷热病毒克隆13（以Cl 13） <ol><li>注：氯13是在德国可以BSL-2的条件下进行处理的减毒病毒突变体。请参阅相关的国家指导方针。其他典型的干扰素诱导剂是仙台病毒（株Cantell）或新城疫病毒。 </li><li>含5％FCS的预温PBS，无血清培养基和细胞培养液中。 </li><li>制备1.25×10 7 PFU /氯13的无血清培养基毫升感染2.5×10 6个A549细胞用感染复数5（MOI）。准备一个比需要考虑若干（大约10％）更大的量移液器错误。 </li><li>根据1.3所述，用PBS洗涤细胞。 </li><li>加入1 ml的13氯稀释或无血清培养基（未感染的对照，模拟）到细胞中，并孵育1小时，在37℃和5％CO 2。将烧瓶仔细每隔15分钟，以确保氯13稀释和无血清培养基中均匀分布，分别为。 </li><li> 1小时感染后，除去接种物，加入5 ml预热的细胞培养基，用5％FCS，并孵育5小时，在37℃和5％CO 2。 </li></ol> 3，制备细胞裂解液的<ol><li>准备PBS / 0.5％的Triton X-100在4°C不要添加丝氨酸蛋白酶抑制剂。 </li><li>用冷PBS洗涤细胞，并加入10ml新鲜的PBS中。 </li><li>刮去细胞脱落，转移细胞悬液在一个falcon管中，并离心分离，在800×g离心5分钟，在室温。 </li><li>去除上清，重悬细胞沉淀在30微升PBS / 0.5％的Triton X-100。转移溶解物成新的1.5毫升管孵育至少10分钟，在4℃下</li><li>离心裂解物10.000×g下在4℃下10分钟，将澄清的细胞裂解液（上清液）转移到新的管中。 </li><li>通过Bradford法测定蛋白浓度如别处33所述。 </li><li>储存在-20°C或进行胰蛋白酶消化（4.1）或变性PAGE（5.1）。 </li></ol> 4，测定的模式识别受体构象变化的<ol><li>细胞裂解液的TPCK-胰蛋白酶处理<ol><li>稀释L-1-tosylamido -2 - 苯基乙基氯甲基酮处理（TPCK）胰蛋白酶的PBS中至最终工作的2微克/微升的浓度。 </li><li>调整两个新管中的最终蛋白质25微克每蛋白裂解液（模拟或Cl 13）中的9微升用PBS的终体积浓度。因此，它应该是四根管与各25微克的溶胞产物，2次模拟和2倍氯13感染。一个设置为输入控制（未处理）和一组与TPCK-胰蛋白酶处理。 </li><li>加入1μl的PBS（未处理）或1微​​升的2微克/微升TPCK-胰蛋白酶（终浓度为0.2微克/微升）的细胞裂解物，并通过移液混合的反应。不冻结和解冻TPCK-胰蛋白酶等分，因为它会影响消化的效率。 </li><li>孵育的裂解液在37℃下25分钟。停止反应，加入5×变性样品缓冲液（250mM的Tris-盐酸pH为6.8，10％SDS，50％甘油，25％β-巯基乙醇，0.5％溴酚蓝）并通过5分钟，在95℃下沸腾它不延长胰蛋白酶孵育时间是很重要的。在任何情况下，抗蛋白酶片段检测，胰酶消化的时间必须缩短。 </li><li>煮沸后，该样品可以贮存于-20℃。 </li></ol></li><li> SDS-聚丙烯酰胺凝胶电泳（PAGE）和Western印迹<ol><li>加载样品上的十二烷基硫酸钠含有5％堆积在12％分离胶（SDS）聚丙烯酰胺凝胶。分离蛋白质，每凝胶25毫安直到对溴酚蓝用完。 </li><li>激活一聚偏氟乙烯（PVDF）膜，持续30秒，用甲醇，放入转移缓冲液（48毫摩尔Tris，39 mM的甘氨酸，1.3 mM的SDS，20％甲醇）。 </li><li>制备的印迹用半干印迹系统和允许蛋白质在10 V转移1小时。就拿膜出来，用水简单地冲洗，并让它干燥。 </li><li>通过不久转移到甲醇中重新激活该膜。洗5分钟，用TBS。用10％脱脂牛奶的TBS为1小时，在室温或4℃的CO / N方框洗膜3次为每个用TBS 5分钟。 </li><li>制备抗体稀释液的建议，在表1和孵育膜1小时，在室温或4℃的CO / N。 </li><li>洗膜3次为每个用TBS-T 10分钟。加偶联有辣根过氧化物酶在1:20,000稀释在1％脱脂牛奶的TBS适当的二级抗体。孵育45分钟，在RT。 </li><li>洗膜3次为每个用TBS-T 10分钟，和邻NE更多的时间用TBS。 </li><li>用于信号检测使用商业化学发光试剂盒和数字凝胶成像系统。 </li></ol></li><li>考马斯亮蓝G-250染色<ol><li>如在4.2.1中所述进行SDS-PAGE电泳。在SDS聚丙烯酰胺凝胶负载的样品，并在每个凝胶25毫安运行凝胶，直到溴酚蓝用完。 </li><li>在RT下进行考马斯亮蓝G-250染色。做下不断晃动的所有孵化。 </li><li>将凝胶转移到含有40％甲醇和10％乙酸中30分钟固定的解决方案。 </li><li>交换缓冲至脱色液（25％乙醇和8％乙酸）并孵育5分钟。 </li><li>染色的凝胶用0.2％考马斯亮蓝G-250在40％的甲醇和10％乙酸1小时。 </li><li>脱色的凝胶用脱色液和10分钟，30分钟和60分钟后，更换缓冲液。 </li><li>存储该凝胶在25％乙醇，8％醋酸，4％甘油，在4°C。 </l我&gt; <li>根据4.2.8所述进行成像和分析。 </li></ol></li></ol>低聚国家模式识别受体5。分析<ol><li>天然PAGE <ol><li>制备50微克细胞裂解物中的10微升的PBS使终体积，并添加5×原生样品缓冲液（250mM的Tris-盐酸的pH 6.8，1％脱氧胆酸钠，50％甘油，0.5％溴酚蓝）至终浓度1X。 </li><li>装入样品立即在原生聚丙烯酰胺凝胶用5％的堆放和8％的分离胶。任何延误将导致本地络合物34的损失。 </li><li>用50mM的Tris-氢氧化钠pH值为9.0，384 mM的甘氨酸运行的凝胶以每凝胶20毫安在4℃下以作为阳极和50mM的Tris pH值8.3，384 mM的甘氨酸，1％脱氧胆酸钠作为阴极缓冲。后1.5-2小时（溴酚蓝乐队已经离开了凝胶约45分钟更早）电泳结束。 </li></ol></li><li>蛋白质印迹<ol><li>激活polyvinylidene氟乙烯（PVDF）膜，持续30秒，用甲醇，放入Towbin等缓冲液（25mM的Tris，192 mM的甘氨酸，0.1％SDS，20％甲醇）。 </li><li>根据制造商的说明组装的湿印迹腔和填充罐致远缓冲区。 </li><li> 250毫安1.5小时，在4℃下进行杂交</li><li>当杂交完成后请按从4.2.3中所述的。 </li></ol></li></ol>

没有利益冲突的声明。

感病毒的存在和活化的抗病毒I型IFN系统是成功的先天免疫反应22是至关重要的。病毒检测是由病原体识别受体（模式识别受体），如RIG-I和PKR介导的，从而，实现了快速反应和激活抗病毒防御机制。在这里，我们描述了两种方法来直接评估RIG-I和PKR的激活状态。 有限公司蛋白酶消化作为一种工具来监控RIG-I和PKR首次由M.大风小和T.藤田6,17，和JL科尔30，?...

在抗病毒的宿主防御的一个关键事件是由所谓的模式识别受体（模式识别受体）1,2的快速检测病原体。细胞内检测的RNA病毒感染是依赖于两个的细胞质RNA解旋酶，RIG-I（视黄酸诱导基因I）和MDA5（黑色素瘤分化相关蛋白5）3-5。 RIG-I是由两个N-末端caspase募集结构域（卡），一个中央DECH箱型RNA解旋酶结构域和C-末端结构域（CTD）的4,6。而CTD与解旋酶结构域都需要认识到非自（病毒）的RNA，卡介导的下游信号传导，导致建立抗病毒主机的状态。 如果RIG-I是在无声状态下， 即在没有特定的RNA配体，第二张牌交互与中央解旋酶结构域，并保持RIG-I在自动抑制构象7-11。 RIG-I结合，以短双链（DS）的RNA带有5&#39;-三磷酸（5&#39;PPP），长的dsRNA，和理/ UC-丰富的RNA，经典签名结构，其存在于许多RNA病毒12-16的基因组。 RIG-I激活的两个主要特征是一个开关为闭合构象6,17和均聚的低聚6,18,19。构象开关提高了RNA结合，公开卡下游信号，并重新组成一个活跃的ATP酶活性位点8,9,11,20。低聚RIG-I复合物的形成导致下游信号接头分子的增强招募以形成用于抗病毒的信号转导11的一个平台。该RIG-I调节信号链，最终激活转录因子IRF-3的上调对于一个完整的抗病毒反应21,22干扰素（IFN-alpha/beta）基因和干扰素刺激基因，因此该基因的表达（的ISG）的。一个最佳表征ISG的是RNA活化proteiÑ ​​激酶（PKR）23。 PKR属于真核翻译起始因子2 - α（eIF2α蛋白）激酶家族，它由N-末端的双链RNA结合域和C-末端激酶结构域。激酶结构域构成PKR的激活的二聚化界面的关键，并进行了蛋白质的催化功能。 PKR结合dsRNA病毒导致其构象变化，允许二聚化和自身磷酸化苏氨酸在446等残基中。 PKR介导再eIF2α蛋白磷酸化，从而阻断病毒的mRNA 23-27的翻译。 这两个RIG-I和PKR进行重大的结构重组，形成寡聚复合物，并翻译后磷酸化/去磷酸化和泛素化10,11,19,23,24,26-29修改。为了更好地理解其中的病毒RNA结构被激活RIG-I和PKR（以及在什么阶段病毒拮抗剂可以bë干扰），它精确地确定的激活状态是很重要的。对于这两种的PRR它先前描述的活化导致胰蛋白酶抗性蛋白片段6,17,30和更高阶低聚物6,18,19的出现。然而，由于在抗病毒宿主反应1,2,24这些关键因素的丰富文献，应用直接方法似乎比较罕见的。在刺激更广泛使用的希望，我们提供方便，灵敏协议，以稳健分析RIG-I和PKR的激活状态。干扰素主管人A549细胞感染了RIG-I和PKR，减毒裂谷热病毒突变克隆13（以Cl 13）31,32的既定激活。一个简单的裂解过程后，被感染的细胞的提取物通过有限的胰蛋白酶消化/免疫印迹分析测试，以评估构象转换，并通过蓝色非变性聚丙烯酰胺凝胶电泳（PAGE）/蛋白质印迹analys是测量形成的低聚物。

<table border="0" cellpadding="0" cellspacing="0" width="320">
	<tbody>
		<tr height="43" style="height:32.25pt">
			<td class="xl66" height="43" style="height:32.25pt;width:60pt" width="80">Name</td>
			<td class="xl67" style="width:60pt" width="80">Company</td>
			<td class="xl67" style="width:60pt" width="80">Catalog Number</td>
			<td class="xl67" style="width:60pt" width="80">Comments</td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl68" height="22" style="height:16.5pt;width:60pt" width="80">cell culture</td>
			<td class="xl76" colspan="3" style="border-right:1.0pt solid black;
 border-left:none;width:180pt" width="240"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl69" height="106" style="height:79.5pt;width:60pt" width="80">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td class="xl70" style="width:60pt" width="80">Gibco</td>
			<td class="xl65" style="width:60pt" width="80">21969-035</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl72" height="22" style="height:16.5pt;width:60pt" width="80">OptiMEM</td>
			<td class="xl70" style="width:60pt" width="80">Gibco</td>
			<td class="xl65" style="width:60pt" width="80">31985-47</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl72" height="22" style="height:16.5pt;width:60pt" width="80">L-glutamine</td>
			<td class="xl70" style="width:60pt" width="80">PAA</td>
			<td class="xl65" style="width:60pt" width="80">25030-024</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl72" height="61" style="height:45.75pt;width:60pt" width="80">penicillin-streptomycin</td>
			<td class="xl70" style="width:60pt" width="80">PAA</td>
			<td class="xl65" style="width:60pt" width="80">15070-063</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl73" height="64" style="height:48.0pt;width:60pt" width="80">fetal calf serum (FCS)</td>
			<td class="xl70" style="width:60pt" width="80">PAA</td>
			<td class="xl65" style="width:60pt" width="80">10270</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl69" height="64" style="height:48.0pt;width:60pt" width="80">0.05% trypsin-EDTA</td>
			<td class="xl70" style="width:60pt" width="80">Gibco</td>
			<td class="xl65" style="width:60pt" width="80">25300-054</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl68" height="22" style="height:16.5pt;width:60pt" width="80">chemicals</td>
			<td class="xl76" colspan="3" style="border-right:1.0pt solid black;
 border-left:none;width:180pt" width="240"></td>
		</tr>
		<tr height="190" style="height:142.5pt">
			<td class="xl73" height="190" style="height:142.5pt;width:60pt" width="80">L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated (TPCK) trypsin</td>
			<td class="xl65" style="width:60pt" width="80">Sigma Aldrich</td>
			<td class="xl65" style="width:60pt" width="80">T1426</td>
			<td class="xl65" style="width:60pt" width="80"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl68" height="22" style="height:16.5pt;width:60pt" width="80">antibodies</td>
			<td class="xl78" colspan="3" style="border-right:1.0pt solid black;
 border-left:none;width:180pt" width="240"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl69" height="106" style="height:79.5pt;width:60pt" width="80">mouse monoclonal anti-RIG-I antibody (ALME-1)</td>
			<td class="xl74" style="width:60pt" width="80">Enzo Life Sciences</td>
			<td class="xl65" style="width:60pt" width="80">ALX-804-849-C100</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:500 in 1% skim milk in TBS</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl69" height="85" style="height:63.75pt;width:60pt" width="80">mouse monoclonal anti-PKR (B10)</td>
			<td class="xl65" style="width:60pt" width="80">Santa Cruz</td>
			<td class="xl65" style="width:60pt" width="80">sc-6282</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:500 in 1% skim milk in TBS</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl69" height="85" style="height:63.75pt;width:60pt" width="80">rabbit monoclonal anti-P-PKR (Thr446)</td>
			<td class="xl65" style="width:60pt" width="80">Epitomics</td>
			<td class="xl65" style="width:60pt" width="80">1120-1</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:1.000 in 5% BSA in TBS</td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl69" height="64" style="height:48.0pt;width:60pt" width="80">rabbit polyclonal anti-IRF-3</td>
			<td class="xl65" style="width:60pt" width="80">Santa Cruz</td>
			<td class="xl65" style="width:60pt" width="80">sc-9082</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:500 in 1% skim milk in TBS</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl69" height="85" style="height:63.75pt;width:60pt" width="80">rabbit monoclonal anti-P-IRF-3 (Ser386)</td>
			<td class="xl65" style="width:60pt" width="80">IBL</td>
			<td class="xl65" style="width:60pt" width="80">og-413</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:100 in 1% skim milk in TBS</td>
		</tr>
		<tr height="126" style="height:94.5pt">
			<td class="xl81" height="211" rowspan="2" style="border-bottom:1.0pt solid black;
 height:158.25pt;border-top:none;width:60pt" width="80">rabbit anti-RVFV hyperimmune serum &#34;C2&#34; (MP-12)</td>
			<td class="xl82" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:60pt" width="80"></td>
			<td class="xl82" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:60pt" width="80"></td>
			<td class="xl75" style="width:60pt" width="80">Kindly provided by Alejandro Brun (CISA-INIA)</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl65" height="85" style="height:63.75pt;width:60pt" width="80">WB: 1:2.000 in 1% skim milk in TBS</td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl69" height="106" style="height:79.5pt;width:60pt" width="80">mouse monoclonal anti-beta-actin (8H10D10)</td>
			<td class="xl65" style="width:60pt" width="80">Cell Signalling</td>
			<td class="xl65" style="width:60pt" width="80">3700</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:1.000 in 1% skim milk in TBS</td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl69" height="106" style="height:79.5pt;width:60pt" width="80">polyclonal peroxidase-conjugated goat anti-rabbit</td>
			<td class="xl65" style="width:60pt" width="80">Thermo Fisher</td>
			<td class="xl65" style="width:60pt" width="80">0031460 1892914</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:20.000 in 1% skim milk in TBS</td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl64" height="106" style="height:79.5pt;width:60pt" width="80">polyclonal peroxidase-conjugated goat anti-mouse</td>
			<td class="xl63" style="width:60pt" width="80">Thermo Fisher</td>
			<td class="xl63" style="width:60pt" width="80">0031430 1892913</td>
			<td class="xl63" style="width:60pt" width="80">WB: 1:20.000 in 1% skim milk in TBS</td>
		</tr>
	</tbody>
</table>

monitoring activation of the antiviral pattern recognition receptors rig-i and pkr by limited protease digestion and native page

宿主防御病毒感染依赖于快速检测由先天免疫系统的模式识别受体（的PRR）。在细胞质中，PRRS的RIG-I和PKR绑定到特定的病毒RNA配体。该第一介导的构象转换和低聚，然后使活化的抗病毒干扰素应答。而方法来测量抗病毒宿主的基因表达被确立，方法直接监测RIG-I和PKR的激活状态是仅部分和少确立。 在这里，我们描述了两种方法来监测RIG-I和PKR感染刺激后，有一个既定的干扰素诱生剂，裂谷热病毒突变克隆13（以Cl 13）。有限的胰蛋白酶消化使分析改建蛋白酶的敏感性，说明猪蓝耳病的构象变化。裂解液从模拟感染的细胞产生的RIG-I和PKR，wherea的快速降解胰酶消化s氯13感染导致的蛋白酶抗性RIG-I片段的出现。也PKR显示了胰蛋白酶消化，这与其标志的磷酸化苏氨酸在446一致的病毒引起的局部阻力。RIG-I和PKR低聚物是由非变性聚丙烯酰胺凝胶电泳（PAGE）验证的形成。一旦感染，有RIG-I和PKR寡聚物的深厚积累，而这些蛋白仍然是模拟感染样本中单体。 有限蛋白酶消化和天然PAGE，既耦合到免疫印迹分析，允许RIG-I和PKR激活两个不同步骤的敏感和直接测量。这些技术是相对容易和快速执行，并且不需要昂贵的设备。

病毒受体激动剂通过RIG-I或PKR的识别触发构象开关6,17,30和齐聚6,18,27。 ，我们检测这两种活化标志物由有限蛋白酶消化和非变性聚丙烯酰胺凝胶电泳（PAGE）分别。 人类A549细胞被感染裂谷热病毒克隆13（以Cl 13），其特征在于，干扰素拮抗剂奈米35,36的突变。由于缺乏功能性奈米球，克隆13强诱导RIG-I和PKR，导致细胞12,31,32,37建立一个强大的抗病?...

Watch this Scientific Journal Video about Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE at JoVE.com

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

先天防御病毒感染是由模式识别受体（模式识别受体）触发。这两个细胞质模式识别受体RIG-I和PKR结合到病毒签名的RNA，构象变化，低聚，并激活抗病毒信号。方法被描述，其允许方便地监控构象交换和这些细胞质的PRR的低聚。

在抗病毒模式识别受体RIG-I和PKR通过有限蛋白酶消化和Native PAGE监测激活

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

monitoring-activation-antiviral-pattern-recognition-receptors-rig-i

Research

JoVE Journal

Immunology and Infection

12.0K 次观看.  Philipps-University Marburg. 先天防御病毒感染是由模式识别受体（模式识别受体）触发。这两个细胞质模式识别受体RIG-I和PKR结合到病毒签名的RNA，构象变化，低聚，并激活抗病毒信号。方法被描述，其允许方便地监控构象交换和这些细胞质的PRR的低聚。 

视频: Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

在极少数情况下，表皮转谷氨酰胺酶IgA和组织转谷氨酰胺酶2抗体的识别恒河猴皮炎

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

科研

免疫与感染

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

早期的病毒进入测定的鉴定和抗病毒化合物的评价

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

免疫沉淀法研究受体与酶的功能相互作用

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...