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><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&amp;amp;L (HRP)</td><td>Abcam</td><td>Ab205720-500ug</td><td></td></tr><tr><td>Goat&amp;nbsp;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&amp;ndash;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

536 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 ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

癌症研究

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

医学

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (&#916;F) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

颗粒稳定乳液的细胞亲和力促进抗原内化

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug ...

生物工程

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

鼻自组装纳米乳肿瘤疫苗体外和体内的制备、特性、毒性及疗效评价

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

评估纳米乳剂佐剂疫苗对耐甲氧西林 金黄色葡萄球菌 （MRSA）感染的免疫反应

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

Preparation of a Nanoemulsion Adjuvant Vaccine

Source: Zhang, Z. et al., <a href="https://app.jove.com/v/64299">Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates the production of nanoemulsion tumor vaccines. The stable oil reservoir depots carrying synthetic peptides and adjuvants are produced by low-energy nanoemulsion, offering significant promise for tumor immunotherapy.

纳米乳剂佐剂疫苗的制备

1. Preparation of the I-OVA NE

	Admix 1 mg of monophosphoryl lipid A (MPLA) with 100 &#181;L of dimethyl sulfoxide (DMSO), vortex for 5 min, and allow it ...

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Functionalized Nanoparticle Targeting of Ovarian Cancer: A Technique to Facilitate Folic Acid-conjugated Nanoparticle Contrast Agent Uptake by Ovarian Cancer Cells

First, mix the folic acid-capped copper sulfide nanoparticles at the proper concentration in fresh RPMI media. Add this nanoparticle solution to each well of the prepared 24-well plate that contains SKOV-3 cells at a concentration of 400 micrograms per milliliter. Incubate at 37 degrees Celsius with 5% carbon dioxide for two hours.
After this, trypsinize the cells with 0.5 milliliters of 0.25% trypsin and EDTA. To neutralize the trypsin, add at least 1 milliliter of folic acid-free RPMI 1640 complete growth media and centrifuge the cells at 123 x g for six minutes. To wash the cells, remove the supernatant.
Resuspend the cells in 2 milliliters of PBS and centrifuge at 123 x g for six minutes. Repeat this wash step twice to remove any unbound nanoparticles.
Then, resuspend the cells with 1 to 2 milliliters of a 2% Tween solution in PBS. Count the cells using a hemocytometer and Trypan Blue. Dilute the cells in a solution of 2% Tween and PBS to the chosen concentration for detection.

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

副本

摘要

探索更多视频

此视频中的章节

在细菌粘附的研究剪应力

体外共生分析评估病原体诱导中性粒细胞跨上皮迁移

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

颗粒稳定乳液的细胞亲和力促进抗原内化

鼻自组装纳米乳肿瘤疫苗体外和体内的制备、特性、毒性及疗效评价

体外纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

评估纳米乳剂佐剂疫苗对耐甲氧西林金黄色葡萄球菌（MRSA）感染的免疫反应

纳米乳剂佐剂疫苗的制备

卵巢癌的功能化纳米颗粒靶向:一种促进卵巢癌细胞摄取叶酸偶联纳米颗粒造影剂的技术

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

副本

摘要

探索更多视频

此视频中的章节

在细菌粘附的研究剪应力

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

颗粒稳定乳液的细胞亲和力促进抗原内化

鼻自组装纳米乳肿瘤疫苗体外和体内的制备、特性、毒性及疗效评价

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

评估纳米乳剂佐剂疫苗对耐甲氧西林 金黄色葡萄球菌 （MRSA）感染的免疫反应

纳米乳剂佐剂疫苗的制备

卵巢癌的功能化纳米颗粒靶向:一种促进卵巢癌细胞摄取叶酸偶联纳米颗粒造影剂的技术

体外共生分析评估病原体诱导中性粒细胞跨上皮迁移

体外纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

评估纳米乳剂佐剂疫苗对耐甲氧西林金黄色葡萄球菌（MRSA）感染的免疫反应