这项工作得到了国家自然科学基金（第81973919号）、广东省国家自然科学基金（No.2019A1515011299）和广东省教育厅创新强校项目（No.2016KTSCX015）的支持。

1. 协议注册
<ol><li>注册系统评价。 注:PROSPERO（材料表）由美国国家卫生研究所（National Institute for Health Research）建立，用于系统评价的注册。该系统审查协议已在编号CRD42022301182下注册。</li>
</ol>2. 软件安装
<ol><li>EndNote 软件安装<ol><li>购买EndNote软件，并从EndNote的官方网站（材料表）下载exe文件作为安装程序。</li>
		<li>解压ZIP文件。双击以启动安装程序。</li>
	</ol></li>
	<li>Stata 软件安装<ol><li>从 Stata 官网（材料表）购买 stata 软件。</li>
		<li>双击 Stata 应用程序以启动安装程序。</li>
	</ol></li>
	<li>TSA 软件安装<ol><li>前往哥本哈根审判组网站（材料表）进行注册并下载ZIP文件。</li>
		<li>双击 TSA 可执行 jar 文件进行安装。</li>
	</ol></li>
</ol>3. 数据库检索
<ol><li>在PubMed、Cochrane Library、Embase、中国生物医学文献服务系统（SinoMed）、中国知网（CNKI）、中国科学期刊数据库（VIP）和万方数据库（WildFang）数据库（目录）中构建检索策略。</li>
	<li>检索时间:自数据库启动至2023年7月。以 PubMed 为例:<ol><li>使用 PubMed 高级搜索构建器（补充图 1A）。</li>
		<li>将搜索词组合如下:安公牛黄或安宫丸或牛黄丸）和（病毒性脑炎。</li>
		<li>下载所有检索到的文献: 发送到>引文管理器>创建文件 （补充图1B）。</li>
	</ol></li>
</ol>4. 纳入和排除标准
<ol><li>纳入标准<ol><li>研究设计:纳入随机对照试验（randomized controlled trials， RCTs）。</li>
		<li>干预措施:包括安公牛黄丸 （ANP） 联合常规治疗与单独常规治疗之间的比较。 注意:常规疗法涉及抗生素、抗病毒药物、颅内减压、退热药和维持水电解质平衡等。</li>
		<li>患者:根据诊断标准纳入病毒性脑炎患者。<ol><li>精神状态改变:包括未发现意识改变、嗜睡或持续 ≥24 小时的人格改变的其他原因的患者。</li>
			<li>确保至少存在以下相关表现中的 3 种:（a） 发病前后 72 小时内体温高于 38 °C;（b） 不能完全归因于原有癫痫的全身性或部分性癫痫发作;（c） 神经系统缺陷的新症状;（d） 脑脊液中白细胞计数≥ 5/mm2 ;（e） 神经影像学检查显示脑实质异常;（f） 与脑炎相符的脑电图异常，不能归因于其他原因。</li>
		</ol></li>
		<li>结果:包括总有效率、退热时间、意识恢复时间、头痛消失时间和抽动消失时间等结果。</li>
	</ol></li>
	<li>排除标准<ol><li>完成文献检索并下载全文。阅读全文并排除符合排除标准的研究。记录每个被排除在外的研究的原因。<ol><li>附加药物:排除除 ANP 外，治疗组使用与对照组不同的其他药物的研究。</li>
			<li>数据缺失:排除数据不完整的文献。</li>
		</ol></li>
	</ol></li>
</ol>5. 文献管理与筛选
<ol><li>导入文献并删除重复项<ol><li>打开 Endnote 并通过单击 "我的组">"创建组"来创建一个新组（补充图 2A）。</li>
		<li>通过单击 "文件">"导入>选项">"导入选项">"EndNote 导入">导入 "来导入检索到的文献（补充图2B）。</li>
		<li>单击 "引用">"查找重复项">"保留此记录 "（补充图 2C）来删除重复项。</li>
	</ol></li>
	<li>筛选文献和数据提取<ol><li>创建 Excel 提取电子表格。</li>
		<li>根据纳入和排除标准，由两位作者独立筛选纳入文献并提取纳入文章的数据，具体如下:1）第一作者;2）出版年份;3） VE 患者的基线数据（年龄、性别、ANP 使用、持续时间）;3）结局测量。 注意:记录每个被淘汰研究的原因。</li>
		<li>数据类型:从使用样本量和组成比率的二分类变量（有效率）和使用均值和标准差的连续变量（退热时间、意识恢复时间、头痛消失时间、抽动消失时间）中提取数据。</li>
		<li>创建系统评价和荟萃分析的首选报告项 （PRISMA） 流程图，直观显示每个步骤处理的研究数量以及删除它们的原因。</li>
	</ol></li>
</ol>6. 质量评估
<ol><li>使用Cochrane偏倚风险工具评估该方法。包括偏倚评估的术语:随机序列生成、分配隐藏、受试者和人员的盲法、结果数据不完整的、选择性报告、结果评估的盲法和其他偏倚。 注意:第三方将进行干预，通过讨论解决分歧。</li>
</ol>7. 数据综合与分析
<ol><li>使用STATA软件构建森林图<ol><li>对于连续数据，请按照步骤 7.1.2-7.1.4 操作。</li>
		<li>对于数据输入，请单击 User > Meta-Analysis of Binary and Continuous > Main > Continuous。设置处理组的样本量、均值和标准差为 n1、 mean1和 sd1，对照组的样本量、mean和标准差为 n2、 mean2和 sd2 （补充图3A）。</li>
		<li>效应模型:在报告的试验中应用基于I2统计量的效应模型（固定/随机）。I2 > 50% 表示高度异质性;选择随机模型。单击"用户>二元和连续>连续>随机 I-V 异质性的元分析。如果 I2 ≤ 50%，则使用固定反向方差。</li>
		<li>计算相同单位的加权均数差 （WMD） 的结果;在 Statistics 下，选择 noStandard。对于不同的结果单位，使用标准化平均差 （SMD）;unser Statistics，选择 Cohen （补充图 3B）。</li>
		<li>对于二分类数据，请按照步骤 7.1.6-7.1.7 操作。</li>
		<li>对于数据输入，请使用风险比率 （RR）。点击 用户>二元和连续>主要>数的荟萃分析。设置治疗组的有效数和非有效数为 E1 和 NE1，对照组为 E2 和 NE2 （补充图3C）。</li>
		<li>效应模型:在报告的试验中应用基于I2统计量的效应模型（固定/随机）。I2 > 50% 表示高度异质性;选择随机模型。单击"用户>二元和连续>二元>的元分析"。如果 I2 ≤ 50%，则使用 Fixed 逆方差。 注意:在数据量小且研究较少的情况下，M-H 测试更稳健。但是 M-H 只能处理二进制变量。I-V 既可用于连续变量，也可用于二元变量。</li>
		<li>使用STATA软件进行灵敏度分析<ol><li>如果我在异质性检验中为 2 > 50%，则进行敏感分析以确定异质性的可能原因。单击"用户>元分析">影响分析，基于metan-based（metainf）>连续（补充图3D）。</li>
		</ol></li>
		<li>使用STATA软件检查发表偏倚<ol><li>对于连续数据，选择 Egger 线性回归检验来评估连续变量的发表偏差。使用命令 db metabias。单击 "Main"，选择" _ES _seES"，然后选择" Egger （补充图3E）"。</li>
			<li>对于二元数据，使用 RR 或 OR 作为二元变量 Harbord 加权线性回归检验的效应指标。使用命令 metabias e1 ne1 e2 ne2 或 harbord。</li>
		</ol></li>
		<li>使用STATA软件进行修整和填充程序<ol><li>通过漏斗图可视化，采用修剪和填充程序来检验效应量估计的稳健性，以评估发表偏倚。使用命令 db metatrim。</li>
			<li>单击 "主要 "，然后选择" _es_sees"。然后，单击 "线性>固定>漏斗 "（补充图 3F）。 注意:二进制变量应进行对数变换以生成 metatrim 的新数据集，例如 gen logES = log（_ES）。</li>
		</ol></li>
		<li>试验序贯分析<ol><li>构建新的荟萃分析。单击" 文件">"New Meta-Analysis"。选择 "数据类型"，导入" 名称"、" Group1"和" Group2 "，然后选择" 结果类型 "（补充图4A）。 注:对于二分类数据，负面结果指标，如死亡、癌症发病率;积极的结果指标，如生存率、病毒清除率或戒烟。对于连续变量，负平均效应增加代表负事件（例如，抑郁评分增加），平均效应的正平均增加代表正事件（例如，血小板计数）。</li>
			<li>根据森林图结果设置效应度量和模型。使用 常数 连续校正方法， 值 = 0.5（补充图 4B）。</li>
			<li>点击"试验"，填写"研究"、"年份"、"效果"和"干预组和对照组的总数"。然后，单击"添加试验"（补充图4C）。 注: 补充图 4D 显示了用于区分连续变量和二元变量的数据输入选项卡。</li>
			<li>单击 Alpha-Spending Boundary > Tsa。将 边界类型 设置为 双侧， 类型 <img alt="Equation 11" src="/files/ftp_upload/65969/65969eq11.jpg"> 错误 率 α = 0. 05， 功效 = 0.80。单击"执行计算"，然后单击"图形"以查看图。</li>
			<li>对于二分类结局的meta分析，填写预期事件发生率和干预效果，包括对照组的相对风险降低 （RRR） 和发病率。根据珍贵林地图的总有效率，计算RRR（补充图4E）。</li>
			<li>对于连续数据，单击"元分析"，然后根据森林图的previos结果选择"均值差"和"Randon效应（BT）"。</li>
			<li>点击"试验"，填写干预组和对照组的研究、年份、平均反应、标准差和组大小。然后，单击"添加试用"。</li>
			<li>单击 Tsa。选择 Alpha-Spending 边界。然后，将边界类型设置为双侧，类型 I.错误率α为 0.05，功率为 0.8。在均值差值和方差中选择经验。单击"执行计算"，然后单击"图形"以查看图。</li>
		</ol></li>
	</ol></li>
</ol>

病毒性脑炎治疗的系统评价和荟萃分析

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<li>Yu, P. L., Yan, S. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+observation+of+adjuvant+therapy+with+Angong+Niuhuang+pill+in+the+treatment+of+viral+encephalitis%5BTitle%5D)+AND+%22Hubei+Zhongyi+zazhi%22%5BJournal%5D)">Clinical observation of adjuvant therapy with Angong Niuhuang pill in the treatment of viral encephalitis.</a> Hubei Zhongyi zazhi. 28 (6), 33(2006).</li>
<li>Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+observation+of+adjuvant+therapy+with+Angong+Niuhuang+Pill+in+the+treatment+of+42+children+with+severe+viral+encephalitis%5BTitle%5D)+AND+%22Zhongguo+Zhongxiyi+jiehe+jijiu+zazhi%22%5BJournal%5D)">Clinical observation of adjuvant therapy with Angong Niuhuang Pill in the treatment of 42 children with severe viral encephalitis.</a> Zhongguo Zhongxiyi jiehe jijiu zazhi. (11), 43(1997).</li>
<li>Cao, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+adjunctive+effect+of+Xing+Nao+Jing+Injection+for+viral+encephalitis%3A+A+systematic+review+and+meta-analysis+of+randomized+controlled+trials%5BTitle%5D)+AND+%22Medicine+%28Baltimore%29%22%5BJournal%5D)">Evaluation of the adjunctive effect of Xing Nao Jing Injection for viral encephalitis: A systematic review and meta-analysis of randomized controlled trials.</a> Medicine (Baltimore). 98, 15181(2019).</li>
<li>Song, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Downregulation+of+cellular+c-Jun+N-terminal+protein+kinase+and+NF-%CE%BAB+activation+by+berberine+may+result+in+inhibition+of+herpes+simplex+virus+replication%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Downregulation of cellular c-Jun N-terminal protein kinase and NF-κB activation by berberine may result in inhibition of herpes simplex virus replication.</a> Antimicrob Agents Chemother. 58, 5068-5078 (2014).</li>
<li>Qin, S., Huang, X., Qu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Baicalin+induces+a+potent+innate+immune+response+to+inhibit+respiratory+syncytial+virus+replication+via+regulating+viral+non-structural+1+and+matrix+RNA%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Baicalin induces a potent innate immune response to inhibit respiratory syncytial virus replication via regulating viral non-structural 1 and matrix RNA.</a> Front Immunol. 13, 907047(2022).</li>
<li>Li, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Borneol-modified+Schisandrin+B+Micelles+cross+the+blood-brain+barrier+to+treat+Alzheimer%27s+disease+in+aged+mice%5BTitle%5D)+AND+%22ACS+Chem+Neurosci%22%5BJournal%5D)">Borneol-modified Schisandrin B Micelles cross the blood-brain barrier to treat Alzheimer's disease in aged mice.</a> ACS Chem Neurosci. , (2024).</li>
<li>Chang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Borneol+is+neuroprotective+against+permanent+cerebral+ischemia+in+rats+by+suppressing+production+of+proinflammatory+cytokines%5BTitle%5D)+AND+%22J+Biomed+Res%22%5BJournal%5D)">Borneol is neuroprotective against permanent cerebral ischemia in rats by suppressing production of proinflammatory cytokines.</a> J Biomed Res. 31 (4), 306-314 (2017).</li>
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这项荟萃分析提供的证据表明，辅助 ANP 可将 VE 患者的总有效率提高 17%。ANP辅助治疗可显著缩短阳性病理体征和症状的消失时间。从ANP中提取的Xingnaojing（XNJ）注射液广泛用于与脑病相关的意识异常。最近的一项荟萃分析验证了 XNJ 治疗 VE 的辅助作用，有助于增加治愈病例数并缩短症状缓解时间25.XNJ 的组件与 ANP 并不完全相同。根据中华人民共和国药典（2020年版）），ANP由牛黄...

脑炎被定义为脑实质炎症，死亡率高，以神经系统病理体征、发热、精神状态异常以及脑电图 （EEG） 和神经影像学改变为特征 1,2。脑炎的发病率为每 100,000 人 7 至 15 例 3,4,5。大约一半的脑炎病例的病因尚不清楚，病毒性脑炎 （VE） 约占诊断病例的 20%-50% 5,6。常见病毒包括人肠道病毒、人疱疹病毒、单纯疱疹病毒、人免疫缺陷病毒和巨细胞病毒7型。VE患者需要密切监测和支持措施，以维持生命体征和缓解脑部炎症8.阿昔洛韦是所有 VE9 病例的经验性治疗。使用糖皮质激素、免疫球蛋白和干扰素α的证据不一致8。安公牛黄丸（ANP）是著名的开孔药之一，广泛用于重大疾病引起的精神和意识异常。中国医学教育协会推荐ANP为A级推荐强度和II级证据质量（来自非随机研究或随机对照试验的证据）10。
系统评价 （SR） 是对符合纳入标准的所有临床研究的全面收集和严格评估。Meta分析是SR中一种定量分析的方法。 与基本meta分析方法不同，采用修剪和填充程序来检验效应量估计对发表偏倚评估的稳健性11。少数研究被删除，直到新的效应量使情节对称。根据插补的镜像研究，将其他研究重新添加到图中。如果修整和填充程序前后重新计算的合并估计值（PE）的差值小于0.05，则发表偏倚的效应量估计值是稳健的。使用TSA 0.9.5.10软件降低了假阳性结果的风险，并计算了所需信息量（RIS），这有利于使我们的结果更加稳健。鉴于没有对ANP治疗VE的症状、体征、生化指标和不良反应改善的数据进行系统评价，因此我们对现有的随机对照试验进行了这项meta分析。

<table><tbody><tr><td>PROSPERO</td><td>National Institute for Health and Care Research</td><td>https://www.crd.york.ac.uk/PROSPERO/</td><td></td></tr><tr><td>STATA</td><td>&amp;nbsp;StataCorp</td><td>https://www.stata.com/</td><td></td></tr><tr><td>TSA</td><td>Copenhagen Trial Unit, Centre for Clinical Intervention Research</td><td>https://ctu.dk/tsa/downloads/</td><td></td></tr><tr><td>PubMed</td><td>National Library of Medicine</td><td>https://pubmed.ncbi.nlm.nih.gov/</td><td></td></tr><tr><td>Cochrane Library</td><td>Cochrane Library</td><td>https://www.cochranelibrary.com/</td><td></td></tr><tr><td>Embase</td><td>Elsevier</td><td>https://www.embase.com/search/quick</td><td></td></tr><tr><td>SinoMed</td><td>SinoMed</td><td>http://www.sinomed.ac.cn/index.jsp</td><td></td></tr><tr><td>CNKI</td><td>China National Knowledge Infrastructure</td><td>https://www.cnki.net/</td><td></td></tr><tr><td>VIP</td><td>Chongqing VIP Information Co., Ltd.</td><td>http://www.cqvip.com/</td><td></td></tr><tr><td>WanFang database</td><td>Wanfang Data Knowledge Service Platform</td><td>https://www.wanfangdata.com.cn/index.html</td><td></td></tr></tbody></table>

the adjuvant efficacy of angong niuhuang pill in the treatment of viral encephalitis: a meta-analysis of randomized controlled trials

这项荟萃分析旨在评估安公牛黄丸 （ANP） 作为辅助疗法治疗病毒性脑炎的疗效。纳入7个数据库（PubMed、Cochrane Library、Embase、SinoMed、CNKI、VIP和WanFang）进行文献检索，检索时间截至2023年7月。比较ANP联合常规治疗与单独常规治疗的随机对照试验是合格的。计算合并效应量和 95% 置信区间 （CI） 以评估疗效和安全性。进行了敏感性分析和发表偏倚评估，以分析研究结果的不确定性。纳入13项研究，涉及1045例病例进行meta分析。与常规治疗相比，ANP辅助治疗使总有效率的概率增加了17%（RR=1.17,95%CI [1.08,1.27]）。ANP辅助治疗后临床综合征和体征消失时间显著减少，包括退热时间（加权均数差（WMD）=-1.59， 95%CI [-2.09， -1.09]）、意识恢复时间（WMD = -1.79， 95%CI [-2.06， -1.51]）、头痛消失时间（WMD = -1.51， 95%CI [-1.93， -1.08]）、抽动消失时间（WMD = -1.88， 95%CI [-2.39， -1.36]）。ANP辅助治疗病毒性脑炎（VE）的疗效似乎提高了总有效率，缩短了临床综合征的消失时间。需要更多高质量的随机对照试验（RCTs）来支持我们的研究结果。

搜索结果和研究特征 系统检索产生了122项研究，手动检索增加了9篇相关文献。在删除了49篇重复文献后，仍有82项研究有待通过摘要和全文进行筛选。最终，13项研究（12,13,14,15,16,17,18,19,20,21,22,23,24）被纳入meta分析，涉及1045例（图1）。<s...

Read this Scientific Article Protocol about Author Spotlight: Evaluating the Adjuvant Efficacy and Safety of Angong Niuhuang Pill in Viral Encephalitis Treatment at JoVE.com

作者聚焦:评价安宫牛黄丸治疗病毒性脑炎的辅助疗效和安全性

在这里，我们提出了一个方案来分析安宫牛黄丸治疗病毒性脑炎的有效性和安全性。

安公牛黄丸治疗病毒性脑炎的辅助疗效:随机对照试验的Meta分析

This meta-analysis aims to evaluate the efficacy of Angong Niuhuang Pill (ANP) as an adjuvant therapy in the treatment of viral encephalitis. Seven ...

我们专注于病毒性脑炎的治疗，并提出了一个方案来分析安宫牛黄丸治疗病毒性脑炎的有效性和安全性。安公牛黄丸治疗病毒性脑炎的辅助疗效包括提高总有效率、缩短临床综合征消失时间等。然而，由于证据质量欠佳，我们的研究结果不是确定的，需要更多高质量的随机对照试验。我们的实验室将重点研究安宫牛黄丸治疗病毒性脑炎的药理机制。

the-adjuvant-efficacy-angong-niuhuang-pill-treatment-viral

Research

JoVE Journal

Medicine

The Adjuvant Efficacy of Angong Niuhuang Pill in the Treatment of Viral Encephalitis: A Meta-Analysis of Randomized Controlled Trials

481 次观看.  Guangzhou University of Chinese Medicine. 我们专注于病毒性脑炎的治疗，并提出了一个方案来分析安宫牛黄丸治疗病毒性脑炎的有效性和安全性。安公牛黄丸治疗病毒性脑炎的辅助疗效包括提高总有效率、缩短临床综合征消失时间等。然而，由于证据质量欠佳，我们的研究结果不是确定的，需要更多高质量的随机对照试验。我们的实验室将重点研究安宫牛黄丸治疗病毒性脑炎的药理机制。

视频: 作者聚焦:评价安宫牛黄丸治疗病毒性脑炎的辅助疗效和安全性

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

科研

神经科学

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

免疫与感染

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

重组腺相关病毒在成年小鼠中的直接注射

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

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 ...

副结核分枝杆菌在自身免疫性脑脊髓炎中的免疫原性增强

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

作者聚焦: 副结核分枝杆菌在增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原性中的辅助活性  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...

评估纳米乳化包埋的维甲酸在小鼠中的免疫原性

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

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

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

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

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 ...

安公牛黄丸治疗病毒性脑炎的辅助疗效:随机对照试验的Meta分析

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颅内注射腺相关病毒载体

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

重组腺相关病毒在成年小鼠中的直接注射

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副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

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

安公牛黄丸治疗病毒性脑炎的辅助疗效:随机对照试验的Meta分析

副本

摘要

探索更多视频

颅内注射腺相关病毒载体

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

重组腺相关病毒在成年小鼠中的直接注射

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

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

体外纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价