eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Materials and Reagents
<ol>
	<li>Use female Anopheles stephensi mosquitoes (Sda500 strain) that feed on infected mice 3-5 days after emergence and rear as described previously.</li>
	<li>Use parasites Plasmodium berghei ANKA clone expressing the Green Fluorescent Protein (GFP) gene under the control of the constitutive Heat Shock Protein 70 (HSP70) promoter. This yields bright fluorescence throughout the parasite life cycle.</li>
	<li>Use female C57BL/6JRj mice (7 week-old). 
	NOTE: All the reagents used in the described protocol are listed in the Table of Materials/Equipment.</li>
</ol>
2. Radiation-attenuated Sporozoite Isolation from Mosquito Salivary Glands
<ol>
	<li>Between 18-25 days after the infectious blood meal, collect and cold-anesthetize mosquitoes into a 15 ml tube on ice as described previously. Carefully transfer them onto a Petri dish by inverting the tube. Maintain the insects on ice and expose the mosquitoes to a 12 krad dose of &#947;- or x-irradiation.</li>
	<li>Isolate attenuated sporozoites from irradiated mosquito salivary glands as previously described. Collect infected salivary glands in a small volume (around 15 &#956;l) of 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) on ice and gently crush them to release sporozoites. 
	NOTE: The injection of a highly concentrated parasite suspension in a small volume being critical, it is therefore essential to harvest a sufficient number of salivary glands from a high number of preselected well-infected mosquitoes, as evidenced by their robust expression of green-fluorescence.</li>
	<li>Filter the sporozoite suspension through a 35 &#181;m cell strainer cap adapted to a low adhesion 1.5 ml microcentrifuge tube in order to eliminate clumps and mosquito debris.</li>
	<li>Count the total number of isolated sporozoites as previously described and adjust the concentration of parasite suspension with cold 1x DPBS to obtain 83,000 sporozoites/&#956;l. Store the parasites on ice while continuing the next steps.</li>
	<li>Collect the equivalent number of salivary glands from uninfected mosquitoes that will be used as negative control and process the sample as described in steps 2.2 and 2.3. Dilute the sample as indicated above to obtain similar concentration of salivary gland extracts in the suspension. 
	NOTE: Sporozoites can be stored on ice for a maximum of 2 hr. However, ideally they are injected within an hour after crushing the salivary glands.</li>
</ol>
3. Injection of Sporozoites into the Dermal Layer of the Ear
<ol>
	<li>Anesthetize animals by intraperitoneal injection of ketamine (50 mg/kg)/xylazine (5 mg/kg) mixture. Wait 5-8 min for the mouse to be in a state of unconsciousness and apply ophthalmic ointment to the eyes to prevent corneal drying. Evaluate the depth of anaesthesia by performing a gentle toe pinch on both rear feet. 
	NOTE: The dose of anaesthesia lasts for less than 20 min, so the next steps must be done quickly.</li>
	<li>Stabilize the ear pinna of the mouse by fixing the ventral side with clear tape previously placed under a stereomicroscope (Figure 1A). Gently press the ear to avoid damage of the vasculature.</li>
	<li>Load a 10 &#956;l syringe/35 gauge bevelled needle with 0.6 &#181;l of resuspended parasites.</li>
	<li>Deliver the sporozoite suspension in 4 injection sites (0.15 &#181;l per site) by carefully inserting the needle on the dorsal side of the ear (Figure 1A), beneath the epidermis, with the bevel up, taking care to avoid any blood vessels. Allow the needle to remain in the dermis for few sec to prevent backflow of the injectant before removing it. Observe a characteristic papule at each injection site at the end of the procedure (Figure 1B and 1C).</li>
	<li>After injection, carefully remove the ear from the tape.</li>
	<li>Inject the contralateral ear with the same dose of parasites by following the steps 3.2-3.4.</li>
	<li>Inject 2 additional mice with either 0.6 &#181;l of 1x DPBS or 0.6 &#181;l of 1x DPBS + salivary gland extract from uninfected mosquitoes in order to evaluate the inflammatory response mediated by the injection alone and the deposition of mosquito material in the dermis.</li>
	<li>&#160;Monitor mouse recovery from anaesthesia before placing it back into the cage. 
	NOTE: The proper sporozoite deposition in the skin can be monitored by fluorescence microscopy (Figure 2A and 2B), the mouse being prepared as described previously.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ketamine: Imalgene&#174; 1000</td>
			<td>Merial</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylazine: Rompun&#174; 2%</td>
			<td>Bayer</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NanoFil syringe + 35 gauge needle</td>
			<td>World Precision Instruments</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Omnican&#174; 50 Insulin syringe 0,5 ml/50 I.U.</td>
			<td>B. Braun Medical</td>
			<td>9151125</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MultiwellTM 6 well tissue culture plate &#8211; Flat Bottom</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td>-</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2 ml syringe</td>
			<td>Terumo</td>
			<td>SS-02S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 15ml Polypropylene conical tube</td>
			<td>BD Falcon</td>
			<td>352097</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 50ml Polypropylene conical tube</td>
			<td>BD Falcon</td>
			<td>352098</td>
			<td>-</td>
		</tr>
		<tr>
			<td>5ml Polystyrene Round-Bottom Tube with 35&#181;m Cell-Strainer Cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DPBS 1X Cacl2- and MgCl2-free</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DMEM 1X + GlutaMAXTM</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas, type IV</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td>50 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Female C57BL/6JRj mice (7-week-old)</td>
			<td>Janvier Laboratories</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

intradermal immunization with live attenuated sporozoites in a mouse model

Source: Mac-Daniel, L., et al. <a href="https://app.jove.com/v/53796">Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites.</a> J. Vis. Exp. (2016).
This video demonstrates an assay for immunizing a mouse model with live attenuated sporozoites. The radiation-attenuated Plasmodium sporozoites are injected into the dorsal side of the mouse&#39;s ear. After injections at multiple locations on both ears, the mouse is allowed to recover.

<img alt="Figure 1" src="/files/ftp_upload/22279/22279_Figure1.jpg" /> 
Figure 1: Intradermal Injection of Sporozoites into the Ear of Mouse. (A) Image showing intradermal injection of sporozoites in the ear pinna of an anesthetized mouse. The ventral side of the ear is stabilized with clear tape previously placed under a dissecting microscope. The needle is carefully inserted on the dorsal side of the ear, beneath the epidermis, wit...

Intradermal Immunization with Live Attenuated Sporozoites in a Mouse Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with an anesthetized mouse under a stereomicroscope, with the underside of one of the mouse&#39;s ears fixed on the stage via adhesive tape.
The eyes are coated with an ointment to prevent them from drying.
Bring a syringe loaded with the sporozoite stage of Plasmodium, a malarial parasite. The live sporozoites are radiation-attenuated to render them non-infectious.
Using the stereomicroscope, inject into the top side of the ear, penetrating the outermost epidermis layer and releasing the sporozoites into the dermis.
Keep the needle in place briefly to prevent backflow of the injected suspension before removing it.
A visible swelling at the injection site, termed a papule, confirms a successful injection.
Perform multiple injections into the same ear.
Release the injected ear and repeat the injections into the other ear.
Post-injection, allow the mouse to recover.
The injected sporozoites trigger an immune response, priming the immune system against the parasite.

intradermal-immunization-with-live-attenuated-sporozoites-mouse

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Immunogenics and Vaccine Development

160 조회수. 출처: Mac-Daniel, L., et al. 생쥐 피부에서 골수성 세포 분리 및 생약독 Plasmodium Sporozoites를 사용한 피내 면역 후 림프절 배출. J. Vis. 특급 (2016). 이 비디오는 생쥐 모델에 살아있는 약독화 포자형성물을 면역시키기 위한 분석을 보여줍니다. 방사선 감쇠된 Plasmodium sporozoites는 쥐 귀의 등쪽에 주입됩니다. 양쪽 귀의 여러 위치에 주사를 맞은 후 마우스가 회복할 수 있습니다. 

비디오: 마우스 모델에서 약독화 포자조산을 이용한 피내 면역 - 개념

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

유동 세포측정 기반 분석체에 의한 플라스모듐 팔시파룸 기생충에 자연적으로 획득된 식세포증-유도 항체 측정

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

연구

JoVE Journal

면역학 및 감염병학

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Anopheles 모기 벡터에서 플라스모듐 팔시파룸 감염 검출을 위한 표준 막 공급 분석

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

체외 세포독성 림프구에 의한 변형체 감염 적혈구 사멸 분석

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

Intradermal Immunization with Live Attenuated Sporozoites in a Mouse Model

내레이션 대본

Intradermal Immunization with Live Attenuated Sporozoites in a Mouse Model