Name: immunostimulatory agent evaluation: lymphoid tissue extraction and injection route-dependent dendritic cell activation
Uploaded: 2018-09-16T14:00:00
Description: Watch this Scientific Journal Video about Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation at JoVE.com

This research was supported by Creative Materials Discovery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2017M3D1A1039421) and by Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Republic of Korea and a grant (20150220).

All experimental procedures including animal handling, sacrifice, and organ isolation were performed in strict accordance with the rules of the International Animal Care and Use Committee at Shanghai Public Health Clinical Center and Asan Medical Center. The study protocol was approved by the respective committees on the Ethics of Animal Experiments at Shanghai Public Health Clinical Center (Mouse Protocol Number: SYXK-2010-0098) and Asan Medical Center (2016-02-168).
1. Preparation of Material<...

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Many advances in nanotechnology and immunology have been achieved through therapeutic research of drug delivery and immunostimulation. Careful selection of the injection method is known to be important for immunostimulation, which was the focus of the present study.
Different injection routes were evaluated for a naturally non-toxic and biodegradable DNA-based material, INP (immunostimulatory nanoparticle), to determine which route yielded the best outcome. This approach is also relevant for d...

Advances in immunology and nanomaterials have led to an abundance of potential new therapeutic strategies for applications in biomedicine, including drug delivery and immunostimulation. Optimization of the administration route is a vital aspect affecting the efficacy of immunostimulatory agents. An immunostimulatory nanoparticle (INP) consisting of DNA is a newly developed nano-immune adjuvant self-assembled by microphase separation because of the amphiphilic structure of lipid-DNA1. Therefore, protocols for INP involving administration of the material1&#160;in vivo via different routes, and three procedures for harvesting appropriate tissues such as the inguinal lymph node (iLN), mediastinal LN (mLN), and spleen, are described. Finally, these tissues were analyzed for dendritic cell (DC) activation, the most powerful antigen-presenting cells in the immune system. This protocol can also be applied for evaluating antigens, antibodies, or other immune adjuvants2.
We tested the INP formulation because it is an agent that has shown great promise. INP is a Toll-like receptor 9 (TLR9) adjuvant material that contains nucleic acids, for which assessment of immunostimulation efficacy is required to test different injection methods3. In this context, the stimulation of DCs is a potent endpoint for in vivo evaluation. After antigen or immunostimulatory molecules are phagocytosed by DCs in the peripheral tissues or blood, these cells migrate to lymphoid organs such as the spleen and LNs4,5. Thus, DC activation was analyzed in the spleen, iLN, and mLN of the injected animals. Properly harvesting of these tissues is therefore also crucial for evaluating the immune response to a novel adjuvant or pathogens5. Such tissue harvesting is also important for developing a novel immunological methodology as a cancer therapy. Furthermore, this protocol can be used to verify the efficiency of other drugs, such as anti-human immunodeficiency virus therapeutics6.

<table border="0" cellpadding="0" cellspacing="0" style="width:707px;" width="707">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:325px;">Material</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate buffer saline</td>
			<td rowspan="2" style="width:96px;">Corning</td>
			<td rowspan="2" style="width:119px;">21-040-CVR</td>
			<td rowspan="2">Washing organs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(PBS, pH 7.4)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">isoflurane solution&#160;</td>
			<td style="width:96px;">Aesica Queenborough limited</td>
			<td style="width:119px;">26675-46-7</td>
			<td>Anesthesia process</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tuberculin 1mL syringe -</td>
			<td style="width:96px;">Junglim</td>
			<td style="width:119px;">N/A</td>
			<td>Injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tube&#160;</td>
			<td style="width:96px;">S.P.L</td>
			<td style="width:119px;">50050</td>
			<td>Anesthesia process</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;">1mL Insulin Syringe&#160;</td>
			<td style="width:96px;">(BD Ultra-FineTM&#173;II)_short needle</td>
			<td style="width:119px;">324826</td>
			<td>Intramuscular Injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose</td>
			<td style="width:96px;">Hyclone</td>
			<td style="width:119px;">SH30081.01</td>
			<td>Storing organs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Histopaque</td>
			<td style="width:96px;">&#160;Sigma-Aldrich</td>
			<td style="width:119px;">10771</td>
			<td>FACS analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ethyl alcohol anhydrous 99.5 %&#160;</td>
			<td style="width:96px;">Daejung</td>
			<td style="width:119px;">4022-4110</td>
			<td>Disinfectant</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:325px;">Equipments</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">FineCycler C100 (Thermocycler)</td>
			<td style="width:96px;">Ssufine</td>
			<td style="width:119px;">-</td>
			<td>Anealing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Centrifuge</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS tube&#160;</td>
			<td style="width:96px;">FALCON</td>
			<td style="width:119px;">2052</td>
			<td>FACS analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:325px;">Automated High-performance Flow Cytometer</td>
			<td style="width:96px;">BD (USA), FACSVerse</td>
			<td style="width:119px;">-</td>
			<td>FACS analysis</td>
		</tr>
	</tbody>
</table>

immunostimulatory agent evaluation: lymphoid tissue extraction and injection route-dependent dendritic cell activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

To evaluate appropriate injection routes of INP for lymphatic tissue DC activation, the DC population as lineage was defined as -CD11c+ cells in the spleen, iLN, and mLN and analyzed the expression levels of co-stimulatory molecules. Treatment of INP by subcutaneous (s.c.) and intravenous (i.v.) injection promoted substantial increases in CD40, CD80, and CD86 expression in the spleen and iLN DCs (Figure 2B and 2...

Watch this Scientific Journal Video about Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation at JoVE.com

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

The overall goal of this procedure is to familiarize the unique technique required in isolating mediastinal lymph node from mice. The operation was conducted after injecting immunostimulant material on six different routes in mice. In this study, we used a new class of material forming nano-sized structures.Surfactants consist of hydrophilic head group and hydrophobic tail. Those form spherical micelle or other secondary structure due to microphage separation. We utilized this property to a bio macromolecule that is DNA.DNA amphiphile, shown here, as regular nucleotides such as A, C, G, T, has hydrophilic blocks while lipid modified neuro-cell nucleobases act as the hydrophobic units. The modified oligo was synthesized by DNA synthesizer in large scale octograms. In equi-media the amphiphilic lipid DNAs spontaneously form nano-sized spherical micelles.When micelle is formed, the DNA remains single-stranded as an empty carrier with about 10 nanometer in diameter. To equip the carrier with immune-stimulant, it is simple matter of extending the DNA sequence with toll-like receptor nine adjuvant namely eCpG. Lipid DNA and eCpG base here therefore the outer surface of micelle is degraded with CpG sequence.We then name this Immune-Stimulating Nano particle as the INP. To determine the proper way of INPs for immune response we injected INPs to mice in 64 injection routes and consequently a proper lymph node tissue were analyzed. For more detailed injection instruction please find a JOB article published by Charles River in 2012.The main advantage of this video is visually showing how to obtain mediastinal lymph node. The video explicitly shows the location of mediastinal lymph node which a lot of people find a difficulty in isolating the organs. All experiments were carried out under the guidelines of the Institutional Animal Care and Use Committee at the Shanghai Public Health Clinical Center, or University of Ulsan College of Medicine.This part will show the specific location of organs or tissues, which are crucial for immunological analysis. Spleen and two lymph nodes will be isolated. First of all, mice were sacrificed by the carbon dioxide inhalation euthanasia and all efforts were made to minimize suffering as following the guidance of Institutional Animal Care and Use Committee.From now on let's find the organs and isolate them. First, properly fix the mouse on the operating desk with four pins on the palm of the mouse. Sterilize the dissection area with alcohol.Dissect the outer cover the bottom of the mouse abdomen with a big-sized scissor. Use forceps to open the both sides of attachment and fix it with pins on the operating table. The location of inguinal lymph node is exposed by finding the conjunction of vessel going down toward the left leg.The shape of the vessel looks like tilted alphabet letter Y.Uncover the layer with the forceps and pull it from the skin. Cut the peritoneum with smaller scissor to expose internal organs. Move intestine to the right by using forceps then wrap in shaped spleen, which show on the left, upper left side of the abdomen.Additional attention is required during the procedure since spleen is easy to disrupt. Finally the key point of this video:harvesting mediastinal lymph node. It is located underneath the upper side of the lung, which is hard to see due to its small size and somewhat hidden location.Carefully expose the area using small scissors and forceps. Cut both sides of the ribs and the diaphragm as well. Clip the cut off ribs above the chest.During the procedure avoid damaging heart and blood vessels since it might blind the site, finding the translucent mediastinal lymph node. If by accident the blood vessel burst, gently wipe out the blood with gauze. Grab the left side of the lung and softly turn it over to the other side to see underneath the lung.Pull up the mediastinal lymph node using forceps. After harvesting the mediastinal lymph node, make sure to perfectly remove all the fat and blood surrounding the lymph node since undesired tissue may influence the analysis. Finally, all the isolated organs on a petri dish filled with media.Total 0.1 times 10 to the sixth cells were analyzed on this flow cytometry. Based on force getter and size getter leukocyte population was gated and that cells were excluded by dark staining. 24 hours after INP injection by 64 injection routes, spleen, inguinal lymph node, and mediastinal lymph node were harvested and analyzed DC activation by flow cytometry.Based on the strategy of flow cytometry DC population was defined as CD11C positive and lineage negative cells in the live leukocyte. Notably, intranasal injection of INP promoted significant increases in the CD40, 80, 86 expression in the mediastinal lymph node compared to PBS for the control. Therefore, this data suggested that intranasal injection of INP can be used as a mucosal adjuvant for enhancement of immunity in the lung.

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