This work was supported by the National Natural Science Foundation of China (No. 81973919), the National Natural Science Foundation of Guangdong Province (No. 2019A1515011299), and the Innovation and Strong School project of the Department of Education of Guangdong Province (No. 2016KTSCX015).

1.&#160;Protocol registration
<ol>
	<li>Register the systemic review. 
	NOTE: PROSPERO (Table of Materials) was built by National Institute for Health Research for the registration of systematic review. This systemic review protocol has been registered under the number CRD42022301182.</li>
</ol>
2. Software installation
<ol>
	<li>EndNote software installation
	<ol>
		<li>Purchase EndNote software and download the exe&#160;file as an installation program&#160;from EndNote&#39;s official website (Table of Materials).</li>
		<li>Decompress the ZIP file. Double-click to start the installer.</li>
	</ol>
	</li>
	<li>Stata software installation
	<ol>
		<li>Purchase stata software from the Stata official website (Table of Materials).</li>
		<li>Double-click the Stata application program to start the installer.</li>
	</ol>
	</li>
	<li>TSA software installation
	<ol>
		<li>Go to the website of The Copenhagen Trial Unit (Table of Materials) for registration and downloading a ZIP file.</li>
		<li>Double-click the TSA executable jar file for installation.</li>
	</ol>
	</li>
</ol>
3. Database retrieval
<ol>
	<li>Construct retrieval strategy in the following databases: PubMed, Cochrane Library, Embase, Chinese Biomedical Literature Service System (SinoMed), China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Database (VIP), and WanFang database (Table of Materials).</li>
	<li>Retrieval time: From the inception of the database to July 2023. Take PubMed as an example:
	<ol>
		<li>Use PubMed advanced search builder (Supplementary Figure 1A).</li>
		<li>Combine the search terms as follows: Angong Niuhuang OR Angong pill OR Bezoar pill) AND (Viral encephalitis.</li>
		<li>Download all the retrieved literature: Send To &#62; Citation Manager &#62; Create File (Supplementary Figure 1B).</li>
	</ol>
	</li>
</ol>
4. Inclusion and exclusion criteria
<ol>
	<li>Inclusion criteria
	<ol>
		<li>Study design: Include randomized controlled trials (RCTs).</li>
		<li>Interventions: Include the comparison between Angong Niuhuang Pill (ANP) plus conventional therapy versus conventional therapy alone. 
		NOTE: Conventional therapies involved antibiotics, antiviral drugs, intracranial decompression, antipyretics, and maintaining water-electrolyte balance, etc.</li>
		<li>Patients: Include patients with viral encephalitis based on the diagnostic criteria.
		<ol>
			<li>Altered mental status: Include patients with no other cause found for altered consciousness, drowsiness, or personality changes lasting &#8805;24 h.</li>
			<li>Ensure at least 3 of the following associated manifestations are present: (a) Body temperature higher than 38 &#176;C within 72 h before or after onset; (b) Generalized or partial seizures that cannot be entirely attributed to pre-existing epilepsy; (c) New symptoms of neurological defects; (d) White blood cell count &#8805; 5/mm2 in cerebrospinal fluid; (e) Neuroimaging showed abnormal brain parenchyma; (f) EEG abnormalities consistent with encephalitis that cannot be attributed to other causes.</li>
		</ol>
		</li>
		<li>Outcomes: Include outcomes such as total effective rate, time of defervescence, time of consciousness recovery, time of headache disappearance, and time of tic disappearance.</li>
	</ol>
	</li>
	<li>Exclusion criteria
	<ol>
		<li>Complete the literature retrieval and download the full texts. Read the full texts and exclude studies that meet the exclusion criteria. Record the reasons for each excluded study.
		<ol>
			<li>Additional drug: Exclude studies in which, besides ANP, the treatment group uses additional drugs different from the control group.</li>
			<li>Data missing: Exclude literature with incomplete data.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Literature management and screening
<ol>
	<li>Importing literature and removing the duplicates
	<ol>
		<li>Open Endnote and create a new group by clicking My Group &#62; Create Group&#160;(Supplementary Figure 2A).</li>
		<li>Import the retrieved literature by clicking on File &#62; Import &#62; Options &#62; Import Options &#62; EndNote Import &#62; Import (Supplementary Figure 2B).</li>
		<li>Remove the duplicates by clicking Reference &#62; Find Duplicates &#62; Keep this Record (Supplementary Figure 2C).</li>
	</ol>
	</li>
	<li>Screening literature and data extraction
	<ol>
		<li>Create an Excel extraction spreadsheet.</li>
		<li>According to the inclusion and exclusion criteria, have two authors screen the included literature and extract data of included articles independently as follows: 1) first author; 2) publication year; 3) baseline data of a patient with VE (age, gender, ANP usage, duration); 3) outcome measures. 
		NOTE: Record the reasons for each eliminated study.</li>
		<li>Data types: Extract data from dichotomous variables (effective rate), which used sample size and composition ratio, and continuous variables (time of defervescence, time of consciousness recovery, time of headache disappearance, time of tic disappearance), which used mean and standard deviation.</li>
		<li>Create a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart, visualizing the number of studies processed at each step and the reasons for removing them.</li>
	</ol>
	</li>
</ol>
6. Quality assessment
<ol>
	<li>Use the Cochrane risk of bias tool to evaluate the methodology. Include the terms of bias assessment: random sequence generation, allocation concealment, blinding of participants and personnel, incomplete outcome data, selective reporting, blinding of outcome assessment, and other biases. 
	NOTE: The third party would intervene to resolve discrepancies by discussions.</li>
</ol>
7. Data synthesis and analysis
<ol>
	<li>Constructing Forest plots using STATA software
	<ol>
		<li>For continuous data, follow steps 7.1.2-7.1.4.</li>
		<li>For data entry, click User &#62; Meta-Analysis of Binary and Continuous &#62; Main &#62; Continuous. Set the sample size, mean, and standard deviation of the treatment group as n1, mean1, and sd1, and set the sample size, mean, and standard deviation of the control group as n2, mean2, and sd2 (Supplementary Figure 3A).</li>
		<li>Effect model: Appy the effect model (fixed/random) based on I2 statistics among the reported trials. I2&#160;&#62; 50% indicates a high heterogeneity; choose the Random model. Click User &#62; Meta-Analysis of Binary and Continuous &#62; Continuous &#62; Random I-V Heterogeneity. Use Fixed Inverse Variance if I2&#160;&#8804; 50%.</li>
		<li>Calculate the outcomes of the same unit as weighted mean difference (WMD); under Statistics, choose noStandard. For different units of outcomes, use the standardized mean difference (SMD); unser Statistics, choose Cohen (Supplementary Figure 3B).</li>
		<li>For dichotomous data, follow steps steps 7.1.6-7.1.7.</li>
		<li>For data entry, use Risk ratios (RR). Click on User &#62; Meta-Analysis of Binary and Continuous &#62; Main &#62; Count. Set the effective number and non-effective number of the treatment group as E1 and NE1, and for the control group as E2 and NE2 (Supplementary Figure 3C).</li>
		<li>Effect model: Apply the effect model (fixed/random) based on I2 statistic among the reported trials. I2&#160;&#62; 50% indicates a high heterogeneity; choose the Random model. Click User &#62; Meta-Analysis of Binary and Continuous &#62; Binary &#62; Fixed. Use Fixed Inverse Variance if I2&#160;&#8804; 50%. 
		NOTE: With small data and few studies, the M-H test is more robust. But M-H can only process binary variables. I-V can be adopted for both continuous and binary variables.</li>
		<li>Performing sensitivity analysis using STATA software
		<ol>
			<li>If I2&#160;&#62; 50% in heterogeneity test, perform sensitive analysis to identify the possible cause of heterogeneity. Click on User &#62; Meta-Analysis &#62; influence analysis, metan-based (metainf) &#62; Continuous (Supplementary Figure 3D).</li>
		</ol>
		</li>
		<li>Checking publication bias using STATA software
		<ol>
			<li>For continuous data, select Egger&#39;s linear regression test to assess the publication bias of continuous variables. Use the command db metabias. Click Main, choose _ES _seES, and select Egger (Supplementary Figure 3E).</li>
			<li>For binary data, use RR or OR as the effect indicators of binary variable Harbord&#39;s weighted linear regression test. Use the command metabias e1 ne1 e2 ne2, or harbord.</li>
		</ol>
		</li>
		<li>Trimming and filling procedure using STATA software
		<ol>
			<li>Visualized by a funnel plot, adopt the trim and fill procedure to test the robustness of the effect size estimate to assess publication bias. Use the command db metatrim.</li>
			<li>Click Main and choose _es_sees. Then, click Linear &#62; Fixed &#62; Funnel (Supplementary Figure 3F). 
			NOTE: Binary variables should make a logarithmic transformation to generate a new dataset for metatrim, such as gen logES = log(_ES).</li>
		</ol>
		</li>
		<li>Trial sequential analysis
		<ol>
			<li>Build a new meta-analysis. Click on File &#62; New Meta-Analysis. Choose Data Type, import Name, Group1, and Group2 and choose the Outcome Type (Supplementary Figure 4A). 
			NOTE: For dichotomous data, negative outcome measures such as death, cancer incidence; positive outcome measures such as survival, viral clearance, or smoking cessation. For continuous variables, a negative mean effect increase represents a negative event (e. g., increase in depression score), and a positive mean increase in mean effect is a positive event (e.g., platelet count).</li>
			<li>Set effect measure and model according to Forest plot results. Use the Constant continuous correction method and Value = 0.5 (Supplementary Figure 4B).</li>
			<li>Click on Trial, fill in the Study, Year, and Effect and Total number of Intervention and Control groups. Then, click Add Trial (Supplementary Figure 4C). 
			NOTE: Supplementary Figure 4D shows the data entry tab that is used to distinguish between continuous and binary variables.</li>
			<li>Click Tsa &#62; Alpha-Spending Boundary. Set Boundary Type as Two-Sided, the Type <img alt="Equation 11" src="/files/ftp_upload/65969/65969eq11.jpg" /> Error rate &#945; = 0. 05 and Power = 0.80.&#160;Click Perform Calculation, then Graph to see the figure.</li>
			<li>For the meta-analysis of dichotomous outcome, fill the expected event rate and intervention effect, including Relative Risk Reduction (RRR) and Incidence in the control group. According to precious forest plot of total effective rate, calculate RRR (Supplementary Figure 4E).</li>
			<li>For continuous data, click Meta-analysis and choose Mean difference and Randon-effect(BT) according the previos results of forest plot.</li>
			<li>Click on Trial, fill in the Study, Year, Mean response, Standard Deviation and Group size of Intervention and Control groups. Then, click Add Trial.</li>
			<li>Click on Tsa. Select Alpha-Spending Boundary. Then, setting the Boundary Type as Two-Sided, the Type &#8544;Error rate &#945; to 0.05 and Power to 0.8. Choose Empirical in mean difference and variance.Click Perform Calculation, then click Graph to see the figure.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Systematic Review and Meta-Analysis on Viral Encephalitis Treatments

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The authors declare that there are no conflicts of interest.

This meta-analysis provides evidence that the adjunctive ANP can improve the total effective rate by 17% in patients with VE. Adjuvant therapy with ANP can significantly shorten the disappearance time of positive pathological signs and symptoms. Xingnaojing (XNJ) injection extracted from ANP is widely used for abnormal consciousness associated with encephalopathy. A recent meta-analysis verified the adjunctive effect of XNJ for treating VE, contributing to an increase in the number of cure cases and shortening the time o...

Encephalitis is defined as the inflammation of the brain parenchyma with high mortality, characterized by neurological pathological signs, fever, abnormal mental status, and altered electroencephalographic (EEG) and neuroimaging1,2. The incidence of encephalitis ranges between 7 and 15 cases per 100,000 people3,4,5. The cause of approximately half of encephalitis cases is unknown, and viral encephalitis (VE) accounts for about 20% to 50% of diagnosed cases5,6. Common viruses include human enterovirus, human herpesvirus, herpes simplex virus, human immunodeficiency virus, and cytomegalovirus7. Patients with VE require intensive monitoring and supportive measures to maintain vital signs and alleviate cerebral inflammation8. Acyclovir is an empiric treatment for all cases of VE9. Evidence for the use of glucocorticoids, immunoglobulins, and interferon alfa are inconsistent8. Angong Niuhuang Pill (ANP), one of the well-known orifice-opening medicines, is widely used in mental and conscious abnormalities due to critical illness. China Medical Education Association recommended ANP as an A-level strength of recommendation and II-level quality of evidence (evidence from non-randomized studies or randomized controlled trials)10.
A systematic review (SR) is a comprehensive collection and rigorous evaluation of all clinical studies that meet the inclusion criteria. Meta-analysis is a methodology for quantitative analysis in SR. Distinguishing from the methods of basic meta-analysis, a trim and fill procedure was adopted to test the robustness of the effect size estimate to the assessment of publication bias11. A small number of studies were removed until a new effect size made the plot symmetrical. Additional studies were added back to the plot based on imputed mirror-image studies. If the difference of the recalculated pooled estimate (PE) before and after the trim and fill procedure was less than 0.05, the effect size estimate of publication bias was robust. TSA 0.9.5.10 software was used to reduce the risk of false-positive results and calculate the required information size (RIS), which was beneficial to make our findings more robust. Given that no systematic review of the data on the improvement of symptoms, signs, biochemical indicators, and adverse reactions of ANP in the treatment of VE, we therefore performed this meta-analysis of the available randomized controlled trials.

<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

This meta-analysis aims to evaluate the efficacy of Angong Niuhuang Pill (ANP) as an adjuvant therapy in the treatment of viral encephalitis. Seven databases (PubMed, Cochrane Library, Embase, SinoMed, CNKI, VIP and WanFang) were included for literature retrieval from inception to July 2023. Randomized controlled trials comparing ANP plus conventional therapy with conventional therapy alone were eligible. Pooled effect sizes and 95% confidence intervals (CIs) were calculated for evaluating efficacy and safety. Sensitivity analysis and publication bias assessments were performed for analyzing the inconclusiveness of findings. 13 studies involving 1045 cases were included for meta-analysis. Adjuvant treatment with ANP increased the probability of the total effective rate by 17% compared with conventional treatment (Risk ratios (RR) = 1.17, 95%CI [1.08, 1.27]). The disappearance time of clinical syndromes and signs was significantly decreased after adjuvant treatment with ANP, including the time of defervescence (weighted mean difference (WMD) = -1.59, 95%CI [-2.09, -1.09]), the time of consciousness recovery (WMD = -1.79, 95%CI [-2.06, -1.51]), the time of headache disappearance (WMD = -1.51, 95%CI [-1.93, -1.08]), the time of tic disappearance (WMD = -1.88, 95%CI [-2.39, -1.36]). The adjuvant efficacy of ANP for treating viral encephalitis (VE) appears to improve the total effective rate and shorten the disappearance time of clinical syndromes. More high-quality randomized controlled trials (RCTs) are needed to support our findings.

Search results and study characteristics 
Systematic retrieval yielded 122&#160;studies and manual searching added 9 relevant literatures. After removing 49&#160;duplicates, 82&#160;studies remained to be screened by abstract and full-text. Ultimately, 13 studies12,13,14,15,16,17,<sup class="x...

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

Author Spotlight: Evaluating the Adjuvant Efficacy and Safety of Angong Niuhuang Pill in Viral Encephalitis Treatment

Here, we present a protocol to analyze the effectiveness and safety of Angong Niuhuang Pill in the treatment of viral encephalitis.

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

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

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JoVE Journal

Medicine

430 Views.  Guangzhou University of Chinese Medicine. We richten ons op de behandeling van virale encefalitis en presenteren een protocol om de effectiviteit en veiligheid van de Angong Niuhuang-pil bij de behandeling van virale encefalitis te analyseren. De adjuvante werkzaamheid van de Angong Niuhuang-pil bij de behandeling van virale encefalitis omvat het verbeteren van de totale effectieve percentages en het verkorten van de verdwijningstijd van het klinische syndroom. Vanwege de suboptimale kwaliteit van het bewijs zijn onze bevindingen ech...

Video: Auteur in de schijnwerpers: evaluatie van de adjuvante werkzaamheid en veiligheid van de Angong Niuhuang-pil bij de behandeling van virale encefalitis

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.

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

Neuroscience

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.

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

Immunology and Infection

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.

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.

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.

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.

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

Immunogenicity Enhancement by Mycobacterium paratuberculosis in Autoimmune Encephalomyelitis

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.

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

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.

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

Evaluating Immunogenicity of Nanoemulsion&#45;Encapsulated Retinoic Acid in Mice

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.

Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid System for Mucosal Vaccination

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.

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

Meta-Analysis of the Effectiveness and Safety of Shugan Jieyu Capsules for the Treatment of Insomnia

A systematic review and meta-analysis were conducted to evaluate the clinical effectiveness and safety of Shugan Jieyu capsules for treating insomnia by searching seven databases up to February 21, 2022. The study was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The quality of the studies was assessed using the risk of bias assessment tool. This article describes in detail how to retrieve and screen the literature. The detailed steps for conducting the meta-analysis are also included in the protocol. Fourteen studies were found to be eligible, including 1,283 insomnia patients (644 with and 639 without Shugan Jieyu capsules at baseline). The meta-analysis showed a better total clinical effectiveness (odds ratio [OR]: 5.71, 95% confidence interval [CI]: 3.56 to 9.15) and a lower Pittsburgh Sleep Quality Index (PSQI) score (mean difference [MD]: &#8722;2.95, 95% CI: &#8722;4.97 to &#8722;0.93) with combined Shugan Jieyu capsules and Western medicine compared to Western medicine alone. The secondary outcomes showed that the Shugan Jieyu capsule group had significantly reduced adverse reactions and improvements in sleep duration, night awakening, nightmares with excessive dreaming, daytime sleepiness, and low energy. Further multicenter randomized trials must be encouraged to provide more concrete evidence on whether Shugan Jieyu capsules are beneficial in routine clinical practice.

The presented protocol describes an investigation strategy and systematic review to evaluate the clinical effectiveness and safety of Shugan Jieyu capsules for treating insomnia.

A systematic review and meta-analysis were conducted to evaluate the clinical effectiveness and safety of Shugan Jieyu capsules for treating insomnia by ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

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

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