本研究由重庆市自然科学基金重点项目（CSTC2020jcyj-zdxmX0027）和国家自然科学基金项目（第32270988号）资助。

动物实验按照《实验动物使用和护理指南》进行，并经第三军医大学实验动物福利和伦理委员会批准。
1.纳米乳液（NEs）的制备
<ol><li>对于水相制备，在40°C下搅拌时，将0.15g聚山梨酯80溶解在28.2mL磷酸盐缓冲盐水（PBS）中。</li>
	<li>对于油相制备，使用 表1所示的纳米乳液的油相配方。在40°C下搅拌时，将sornitan trileate 85，DOTAP和RA溶解在角鲨烯中。</li>
	<li>对于初级 O/W 乳液制备，以 1400 rpm 的转速磁性搅拌油相，同时以 1 mL/min 的速率缓慢滴加水性水溶液。使用高剪切乳化剂以 30,000 rpm 的速度预乳化混合物 6 分钟。</li>
	<li>对于高压均质化 （HPH），用高压均质器在 900 bar 下处理上述制备的初级 O/W 乳液 12 个循环，以获得 160-190 nm 范围内的均相纳米乳液。</li>
	<li>在过程结束时，将乳液收集在 50 mL 烧瓶中，并通过 0.2 μm PES 滤膜对乳液进行过滤灭菌。</li>
	<li>通过导管将烧瓶连接到舒伦克管线，旋转三通旋塞阀将系统连接到真空管，然后打开真空泵以在烧瓶中产生真空。然后，旋转三通旋塞阀，将系统连接到氩气管并用氩气填充。重复此步骤 3 次，以确保烧瓶充满氩气。</li>
	<li>更换软木塞并用paraffine薄膜密封，用锡箔包裹烧瓶并将其储存在4°C。</li>
</ol>2. 纳米乳液的表征
<ol><li>粒径和 zeta 电位检测<ol><li>打开仪器并等待 30 分钟，让激光光源稳定下来。</li>
		<li>用超纯水将 NE 稀释 50 倍，并将 1 mL 样品添加到相应的样品池中。</li>
		<li>要测量颗粒大小，请将温度设置为25°C，选择水作为分散介质，并选择一次性塑料盘子作为测量池。加载样品并自动测量。将每个样本的三次重复测量的平均值报告为最终结果。</li>
		<li>要测量 zeta 电位，请将温度设置为 25 °C。 由于选择水相作为分散介质，选择Smoluchowsi模型，折叠毛细管池作为测量池。加载样品并自动测量。将每个样品的三次重复测量的平均值报告为最终结果。</li>
	</ol></li>
	<li>包封率和载药率的测定<ol><li>为了确定 NE 中负载的 RA 量，使用无水乙醇破乳并从 NE 中提取 RA。向 5 μL 样品中加入 995 μL 乙醇，并使用 355 nm 的分光光度计测定溶液的 RA 含量。将吸光度与标准曲线进行比较。使用以下公式： 包封 = 实际 RA 载量/添加药物的重量 载药率=实际RA载量/样品质量</li>
	</ol></li>
</ol>3. 免疫程序和样本采集
<ol><li>免疫程序和分组<ol><li>根据以下实验组，将年龄为6-8周龄，体重约为18-21g，每组5只的雌性Balb / C小鼠分组，在第0天，第14天，第28天进行免疫：（1）PBS组，（2）卵清蛋白（OVA）组，（3）OVA+ NE-RA（OVA/NE-RA）组，（4）OVA + CNE-RA（OVA/CNE-RA）组。</li>
		<li>通过肌肉注射到股四头肌上部用200μL的不同疫苗制剂免疫所有小鼠：100μL乳剂和15μgOVA吸收在100μLPBS中，在给药前混合。注射 100 μL/后腿。对于对照组的小鼠，单独给予PBS。</li>
		<li>安乐死：在完成三次免疫后2周，通过腹膜内注射100mg / kg的1%戊巴比妥钠对所有小鼠实施安乐死。</li>
		<li>样本采集和处理：安乐死后，用剪刀切开小鼠的胸部，将注射器直接插入心脏，吸出约0.5毫升的全血。让血液在室温下静置2小时，然后在4°C下以1800× g 离心5分钟。 收集血清，等分试样并储存在-80°C以进行进一步分析。</li>
		<li>收集全血、脾脏、阴道灌洗液 （VLF）、支气管肺泡灌洗液 （BALF）、鼻灌洗液 （NLF） 和小肠灌洗液 （SILF）。</li>
	</ol></li>
	<li>脾淋巴细胞的分离<ol><li>将小鼠浸泡在75%（v / v）的乙醇中进行短暂灭菌，将小鼠转移到干净的工作台上。用剪刀剪开左腹部的皮肤，露出并切除脾脏。</li>
		<li>将脾脏放入含有 3 mL PBS 的培养皿中，研磨并使用筛子（200 目，70 μm）和研磨棒过滤到 15 mL 离心管中。</li>
		<li>在室温下以650× g 离心细胞5分钟。弃去上清液，用 3 mL 红细胞裂解液悬浮沉淀物，在室温下孵育 5 分钟。</li>
		<li>向试管中加入 10 mL PBS 并摇匀以终止反应，然后在室温下以 650 x g 离心 5 分钟。</li>
		<li>弃去上清液，将细胞重悬于 10 mL PBS 中，将 20 μL 细胞稀释液加入自动细胞计数仪进行细胞计数。</li>
		<li>在室温下以650× g 离心细胞5分钟。弃去上清液，用 1640 完全培养基（1% 青霉素 - 链霉素，10% 胎牛血清）将细胞稀释至 1 x 106 个细胞/mL。</li>
	</ol></li>
	<li>VLF收集：用75μL 1%BSA / PBST冲洗阴道4次，混合灌洗溶液并收集在1.5mL离心管中，在4°C下以5000× g 离心20分钟，并用移液管收集上清液并储存在-80°C。</li>
	<li>BALF 和 NLF 集合<ol><li>处死后用75%（v / v）乙醇消毒小鼠。抬起鼠标肚子并伸直脖子。用剪刀在小鼠颈部的皮肤上做一个纵向切口，然后用镊子分离肌肉并充分暴露气管。</li>
		<li>通过一个小的横向开口切开气管，将软管插入肺部，将注射器连接到软管上，将0.5mL PBS注射到肺部并退出，重复冲洗3次，并在4°C下以650× g 离心5分钟，将上清液（BALF）储存在-80°C。</li>
		<li>将软管反转到鼻腔，将注射器连接到软管上并注射 0.5 mL PBS，液体将通过鼻腔流入 1.5 mL 离心管。从离心管中吸出洗涤液并重复冲洗2x，然后在4°C下以650× g 离心5分钟，将上清液（NLF）储存在-80°C。</li>
	</ol></li>
	<li>SILF 系列<ol><li>制备含有0.1 mg/mL胰蛋白酶抑制剂，50 mM / L EDTA-2Na，1 mM / L苯甲基磺酰氟（PMSF）的PBS溶液作为小肠灌洗液。在室温下搅拌溶解并储存在4°C。</li>
		<li>沿肠管切开小肠，浸入 3 mL 小肠灌洗液中。通过在4°C下以15rpm垂直振荡混合30分钟。 在4°C下以1440× g 离心20分钟，并用移液管收集上清液，然后在4°C下以5000× g 离心20分钟。将上清液（SILF）储存在-80°C。</li>
	</ol></li>
</ol>4.肌内给药后OVA特异性抗体反应的评估
<ol><li>通过酶联免疫吸附测定 （ELISA） 测定血清 IgG、IgG1、IgG2a 亚类以及 VLF、BALF、NLF 和 SILF 中特异性 sIgA 的水平。</li>
	<li>对于抗原包被，用包被缓冲液将OVA稀释至5μg/ mL，并在4°C下用每孔100μLova溶液包被96孔ELISA板过夜。</li>
	<li>用 PBST 洗涤 ELISA 板 5 次，并通过在 37 °C 下与 250 μL 1%BSA/PBST 孵育封闭 2 小时。</li>
	<li>用PBST洗涤ELISA板5次，然后加入100μL稀释的样品，如下所述进行测试，并在37°C下孵育1小时。用 0.5% BSA/PBST 稀释血清、VLF、BALF、NLF，用 20% 胎牛血清 （FCS）/PBST 稀释 SILF。<ol><li>首先使用两步稀释程序将血清稀释 1000 倍：将 20 μL 血清稀释至 1 mL，然后将 25 μL 稀释的血清稀释至 500 μL。 使用这种稀释的血清检测 IgG1、IgG2a。</li>
	</ol></li>
	<li>将 200 μL 以 1000x 稀释的血清加入到包被板的第一行中，并将 100 μL 0.5% BSA/PBST 加入到除第一行以外的所有孔中。将 100 μL 从第一排转移到第二排，并用移液器混合 10x。重复此步骤至第 8 行并丢弃 100 μL 液体，已获得不同浓度的血清用于检测 IgG。</li>
	<li>进行 BALF、NLF 和 SILF 的 1：1 稀释以检测 IgA。对 VLF 使用与血清相同的稀释程序。</li>
	<li>用 PBST 洗涤 ELISA 板 5 次。将 100 μL HRP 偶联的山羊抗小鼠 IgG/IgG1/IgG2a/IgA（用 PBST 1：10000 稀释）加入适当的孔中，并在 37 °C 下孵育 40 分钟。</li>
	<li>用 PBST 洗涤 ELISA 板 5 次，加入 100 μL TMB ELISA 底物（高度敏感），并将板转移到避光的地方 5 分钟。立即加入 100 μL 450 nm 终止溶液用于 TMB 底物以停止反应。</li>
	<li>测量每个孔在450nm处的吸光度（OD），并通过取空白组小鼠血清值的2.1倍作为阳性反应值来计算抗体滴度。</li>
</ol>5. ELISpot 检测
<ol><li>按照 ELISpot Plus 的指南，使用试剂盒中的小鼠 IFN-γ （ALP） 进行检测。</li>
	<li>从密封包装中取出板，用无菌 PBS（200 μL/孔）洗涤 4 次。用 1640 完全培养基（200 μL/孔）调节板，并在室温下孵育 30 分钟。</li>
	<li>取出培养基，将步骤3.2.2中制备的调节浓度的淋巴细胞悬浮液（100μL/孔）加入板中，然后加入100μL刺激物，即含有20μg/ mL OVA257~264或OVA323~339或1640完全培养基的1640完全培养基。将板置于37°C，5%CO2 的加湿培养箱中48小时。用铝箔包裹板，以避免蒸发。</li>
	<li>弃去细胞悬浮液并用 PBS（200 μL/孔）洗涤板 5 次。在含有0.5%胎牛血清（PBS-0.5%FCS）的PBS中将检测抗体（R4-6A2-生物素）稀释至1μg/ mL。加入 100 μL/孔，在室温下孵育 2 小时。</li>
	<li>按照步骤 5.2 清洗板。在PBS-0.5%FCS中稀释链霉亲和素-ALP（1：1000），加入100μL/孔，在室温下孵育1小时。</li>
	<li>按照步骤 5.2 清洗板。通过 0.45 μm 过滤器过滤即用型底物溶液 （BCIP/NBT-plus），并加入 100 μL/孔。发展直到出现明显的斑点。</li>
	<li>在自来水中大量清洗以停止发色，取下柔软的塑料，让板子晾干。</li>
	<li>在 ELISpot 读数仪中检查和计数斑点。</li>
</ol>6. 统计分析
<ol><li>采用单因素方差分析法分析4组数据间的差异，然后进行Tukey多重比较后测。将所有结果表示为均值 ± SD.，小于 0.05 的 P 值被认为显著。</li>
</ol>

评估纳米乳化包埋的维甲酸在小鼠中的免疫原性

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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+coating+approach+to+overcome+mucosal+entrapment+of+DNA+nanoparticles+for+oral+gene+delivery+of+glucagon-like+peptide+1.">Surface coating approach to overcome mucosal entrapment of DNA nanoparticles for oral gene delivery of glucagon-like peptide 1.</a> Acs Appl Mater Inter. 11 (33), 29593-29603 (2019).</li><li>Lou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+self-amplifying+mRNA+vaccines+by+cationic+lipid+nanoparticles:+The+impact+of+cationic+lipid+selection.">Delivery of self-amplifying mRNA vaccines by cationic lipid nanoparticles: The impact of cationic lipid selection.</a> J Cont Release. 325, 370-379 (2020).</li></ol>

作者声明他们在这项工作中没有利益冲突。

在该协议中，我们开发了一种阳离子纳米乳液包封的视黄酸，用作佐剂以促进抗原特异性全身和粘膜反应。与传统的NE佐剂相比，它具有以下两个优点。首先，一般来说，O/W NEs的表面具有高负电荷，这使得直接负载抗原变得困难。阳离子NEs能有效吸附肽或蛋白抗原，增强特异性免疫原性。其次，传统疫苗研究的经验表明，皮下注射或肌肉注射很难刺激粘膜反应5。通过将 FDA 批准?...

佐剂通常用于通过刺激免疫系统对疫苗做出更强烈的反应来增强疫苗的功效，从而提高对特定病原体的免疫力1.纳米乳液（NE）助剂是指通过乳化一定比例的油相和水相，产生油包水（W/O）或水包油（O/W）2形式的乳液，具有热力学稳定性的胶体分散体系。O/W纳米乳液佐剂可在注射部位产生细胞因子和趋化因子，诱导单核细胞、中性粒细胞、嗜酸性粒细胞等重要免疫细胞的快速聚集和增殖，增强免疫应答，提高抗原的免疫原性3。目前，三种纳米乳剂佐剂（MF59、AS03和AF03）已获准用于疫苗，并显示出良好的安全性和有效性4。
然而，目前在常规胃肠外疫苗接种中许可的佐剂制剂对粘膜免疫问题处理得很差5.据报道，维甲酸 （RA） 可诱导免疫细胞肠道归巢，但其极性低、在水中的溶解度差以及光稳定性和热稳定性差限制了其用于稳健的肠道疫苗接种。纳米乳液为提高高亲脂性药物的生物利用度提供了机会，而 O/W 乳液佐剂的油芯适用于溶解 RA6 等非极性物质。因此，纳米乳剂可以作为RA的载体，以达到全身免疫和黏膜免疫的双重反应效果。
与中性或阴离子递送系统相比，阳离子递送系统具有相对高效的抗原包封和递送能力，可以增强抗原的免疫原性7,8,9。各种佐剂体系的阳离子表面电荷对其佐剂效果很重要 10,11,12。阳离子电荷是延长疫苗在注射部位的保留时间、增加抗原呈递和延长体内细胞免疫刺激的重要因素12.
基于上述考虑，我们开发了一种阳离子纳米乳液，可有效共递送RA和抗原。利用动态光散射（DLS）法测定纳米乳液的粒径和zeta电位，并通过肌内注射13评价纳米乳液联合OVA的全身和粘膜免疫反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1640 medium</td>
			<td>GIBCO, USA</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>450 nm Stop Solution for TMB Substrate</td>
			<td>Abcam</td>
			<td>ab171529-1000 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Countstar, China</td>
			<td>IC1000</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich, USA</td>
			<td>B2064-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf, Germany</td>
			<td>5811000398</td>
			<td></td>
		</tr>
		<tr>
			<td>Danamic Light Scattering</td>
			<td>Malvern</td>
			<td>Zetasizer Nano S90</td>
			<td></td>
		</tr>
		<tr>
			<td>DOTAP</td>
			<td>CordenPharma, Switzerland</td>
			<td>O02002</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISpot Plus: Mouse IFN-gamma (ALP)</td>
			<td>mabtech</td>
			<td>ab205719</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO, USA</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Full-function Microplate Reader</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>VL0000D2</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG1(HRP)</td>
			<td>Abcam</td>
			<td>ab97240-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgA alpha chain (HRP)</td>
			<td>Abcam</td>
			<td>ab97235-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG H&#38;L (HRP)</td>
			<td>Abcam</td>
			<td>Ab205720-500ug</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat&#160;Anti-Mouse IgG2a heavy chain (HRP)</td>
			<td>Abcam</td>
			<td>ab97245-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure homogenizer</td>
			<td>ATS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANE 85 PPI</td>
			<td>SEPPIC, France</td>
			<td>L12910</td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANOX 80 PPI</td>
			<td>SEPPIC, France</td>
			<td>36372K</td>
			<td></td>
		</tr>
		<tr>
			<td>OVA257&#8211;264</td>
			<td>Shanghai Botai Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>OVA323-339</td>
			<td>Shanghai Botai Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>ZSGB-bio</td>
			<td>ZLI-9061</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Solarbio, China</td>
			<td>R1010</td>
			<td></td>
		</tr>
		<tr>
			<td>retinoic acid</td>
			<td>TCI, Japan</td>
			<td>TCI-R0064-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Squalene</td>
			<td>Sigma, USA</td>
			<td>S3626</td>
			<td></td>
		</tr>
		<tr>
			<td>T10 basic Ultra-Turrax</td>
			<td>IKA, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMB ELISA Substrate</td>
			<td>Abcam</td>
			<td>ab171523-1000ml</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>Diamond</td>
			<td>A003570-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Macklin, China</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet spectrophotometer</td>
			<td>Hitachi</td>
			<td>U-3900</td>
			<td></td>
		</tr>
	</tbody>
</table>

cationic nanoemulsion-encapsulated retinoic acid as an adjuvant to promote ova&#45;specific systemic and mucosal responses

阳离子纳米结构已成为一种佐剂和抗原递送系统，可增强树突状细胞成熟、ROS 生成和抗原摄取，然后促进抗原特异性免疫反应。近年来，视黄酸（RA）因其激活粘膜免疫反应的作用而受到越来越多的关注;然而，为了将RA用作粘膜佐剂，有必要解决其溶解、加载和输送的问题。在这里，我们描述了一种阳离子纳米乳包封的视黄酸（CNE-RA）输送系统，由阳离子脂质1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOTAP）、视黄酸、角鲨烯作为油相、聚山梨醇酯80作为表面活性剂和脱水山梨糖醇三油酸酯85作为辅助表面活性剂组成。利用动态光散射和分光光度计对其物理和化学性质进行了表征。与单独使用OVA相比，用抗原（卵清蛋白，OVA）和CNE-RA的混合物免疫小鼠显着升高了小鼠阴道灌洗液和小肠灌洗液中抗OVA分泌免疫球蛋白A（sIgA）的水平。该协议描述了用于制备，表征和评估CNE-RA佐剂效果的详细方法。

总共制备了四种纳米乳液制剂，其粒径（图1）、zeta电位和包封效率均进行了表征，如 表2所示。粒径集中在160-190nm附近，DOTAP的加入逆转了纳米乳液的Zeta电位。第三次免疫后 2 周检测 OVA 特异性血清 IgG 及其血清亚组抗体水平。纳米乳佐剂疫苗显着增加了血清中OVA特异性IgG，IgG1和IgG 2a抗体滴度（图2）。更重要的是，当OVA与CNE-RA佐剂时，阴?...

Watch this Scientific Journal Video about Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion-Encapsulated Retinoic Acid System for Mucosal Vaccination at JoVE.com

作者聚焦：用于粘膜疫苗接种的阳离子纳米乳封装视黄酸系统的开发和评估

在该方案中，我们开发了一种阳离子纳米乳液包封的视黄酸 （RA），用作佐剂以促进抗原特异性全身和粘膜反应。通过将 FDA 批准的 RA 添加到纳米乳液中，肌肉注射纳米乳剂后，抗原特异性 sIgA 在阴道和小肠中得到促进。

阳离子纳米乳液包封的视黄酸作为佐剂，促进 OVA 特异性全身和粘膜反应

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

我们正在研究一种粘膜疫苗，该疫苗可产生免疫反应作为感染组，具有预防感染和阻断传播的潜力。我们开发了一种紧张性纳米乳剂包封的视黄酸递送系统，通过肌内注射卵清蛋白进行免疫，可触发全身和粘膜免疫反应。在未来的研究中，肠道相关疾病的动物模型可用于进一步评估阳离子纳米乳包封的维甲酸在增强疫苗保护方面的效果和机制。

cationic-nanoemulsion-encapsulated-retinoic-acid-as-an-adjuvant-to

Research

JoVE Journal

Immunology and Infection

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

517 次观看.  Third Military Medical University. 我们正在研究一种粘膜疫苗，该疫苗可产生免疫反应作为感染组，具有预防感染和阻断传播的潜力。我们开发了一种紧张性纳米乳剂包封的视黄酸递送系统，通过肌内注射卵清蛋白进行免疫，可触发全身和粘膜免疫反应。在未来的研究中，肠道相关疾病的动物模型可用于进一步评估阳离子纳米乳包封的维甲酸在增强疫苗保护方面的效果和机制。

视频: 作者聚焦：用于粘膜疫苗接种的阳离子纳米乳封装视黄酸系统的开发和评估

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

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 ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

小印度蒙古狼的异苯抗酸模拟分析(赫皮斯特奥罗普塔图斯)塞拉,用作口服狂犬病疫苗接种生物标记

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

评估对流感嗜血杆菌的呼吸道免疫应答

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

评估假型病毒对 SARS-CoV-2 的血清中和

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

作者聚焦：研究宿主-病毒与假型病毒的相互作用 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

假型病毒作为通过中和测定监测针对 SARS-CoV-2 的体液免疫反应的分子工具

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...