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. Infection
<ol>
	<li>Infect Anopheles stephensi mosquitoes with Plasmodium berghei ANKA by feeding them for 15 min on a gametocytemic mouse. Keep the infected mosquitoes at 80% humidity and 21 &#176;C.</li>
	<li>Collect female mosquitoes from their cage 17 to 22 days after the blood meal. Place them on ice to anesthetize them.</li>
	<li>Using forceps, place three to four mosquitoes on a glass slide covered with a drop of cold RPMI medium. Place the slide under a microscope.</li>
	<li>Using forceps, carefully stretch the mosquito between the head and body. Isolate the salivary gland using a syringe and needle. Repeat this procedure with the remaining mosquitoes.</li>
	<li>Collect the salivary glands from the glass slide by sucking them up with a glass pipette and collecting them in a 1.5 mL centrifuge tube. 
	NOTE: Depending on the infection rates, usually 8,000 to 15,000 infectious sporozoites can be obtained per salivary gland.</li>
	<li>For approximately 3 min, smash the isolated salivary glands within the centrifuge tube with a small, plastic stick to isolate the sporozoites from the salivary gland tissue.</li>
	<li>Centrifuge for 3 min at 1,000 x g and 4 &#176;C to purify the sporozoites from the remaining tissue.</li>
	<li>Pipette the supernatant, which contains the sporozoites (SPZ), to a new centrifuge tube and count the purified sporozoites in a Neubauer hemocytometer.</li>
	<li>Adjust the concentration of purified sporozoites to 10,000/mL by adding phosphate-buffered saline.</li>
	<li>Inject a total of 1,000 sporozoites (0.1 mL) into the tail veins of inbred C57BL/6 mice to initiate infection. To facilitate the injections, place the C57BL/6 mice in a restrainer and put the tails into warm (approximately 37 &#176;C) water to assist with the visualization of the tail veins. 
	NOTE: The injection itself is a short procedure that can be performed within a few seconds.</li>
	<li>Once daily, check blood-stage parasitemia on blood smears from day 3 onwards after the SPZ infection.</li>
	<li>Assess the mice once daily with the Rapid Murine Coma and Behavior Scale (RMCBS) score, starting from day 5 after the sporozoite injection.</li>
	<li>Assess the mice with magnetic resonance imaging (MRI) imaging according to the Rapid Murine Coma and Behavior Scale (RMCBS) score and the research question to be addressed.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>1001747</td>
			<td>for anesthesia</td>
		</tr>
		<tr>
			<td>Dotarem</td>
			<td>Guebert</td>
			<td>1086923</td>
			<td>Gd-DTPA contrast agent; 0.5mmol/ml</td>
		</tr>
		<tr>
			<td>Amira (Image Processing Program)</td>
			<td>FEI Group</td>
			<td></td>
			<td>Version Amira 5.3.2</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The MathWorks, Inc.,</td>
			<td></td>
			<td>Release 2012b</td>
		</tr>
		<tr>
			<td>FDT toolbox</td>
			<td>FMRIB&#39;s Software Library</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a mouse model of experimental cerebral malaria using host mosquito-derived sporozoites

Source: Hoffmann, A. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55334">In Vivo&#160;Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging.</a>&#160;J. Vis. Exp.&#160; (2017)
This video demonstrates the generation of an experimental mouse model of cerebral malaria. Sporozoites isolated from Plasmodium berghei-infected female mosquitoes are injected into a restrained mouse&#39;s tail veins, leading to blood-brain barrier disruption and brain inflammation, causing cerebral malaria. This infected mouse model offers a valuable tool to study in vivo microvascular pathology associated with cerebral malaria.

Generation of a Mouse Model of Experimental Cerebral Malaria Using Host Mosquito-Derived Sporozoites

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

Begin with a tube containing salivary glands from female mosquitoes infected with Plasmodium berghei &#160;&#8212; a malaria-causing parasite.
Mechanically disrupt the glands, releasing the sporozoites&#160; &#8212; an infective form of the parasite into the medium.
Pellet the gland fractions and debris. Collect the sporozoite-containing supernatant and inject them into the tail vein of a restrained mouse.
The injected sporozoites travel through the bloodstream and reach the liver, where they infect hepatocytes and multiply, forming merozoites&#160; &#8212; an invasive form of the parasite.&#160;
Over time, hepatocytes release merozoites into the bloodstream, invading the red blood cells, or RBCs.
Inside the RBCs, the merozoites replicate asexually, developing into mature form and expressing parasite-derived antigens on the RBCs&#39; surface.
These antigens bind to endothelial receptors, including those in brain blood vessels, leading to the sequestration of infected and non-infected RBCs.
This abnormal RBC accumulation disrupts the blood-brain barrier, causing fluid accumulation in the brain&#39;s parenchyma and developing cerebral malaria in the mouse.

generation-mouse-model-experimental-cerebral-malaria-using-host

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

97 vistas. Fuente: Hoffmann, A. et al., Seguimiento in vivo del desarrollo de edema y patología microvascular en un modelo de malaria cerebral experimental mediante imágenes de resonancia magnética.J. Vis. Exp.(2017) Este video muestra la generación de un modelo experimental de ratón de malaria cerebral. Los esporozoítos aislados de mosquitos hembra infectados con Plasmodium berghei se inyectan en las venas de la cola de un ratón restringido, lo que provoca la ruptura de la barrera hematoencefálic...

Video: Generación de un modelo murino de malaria cerebral experimental utilizando esporozoítos derivados de mosquitos huéspedes - Concepto

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Análisis fenotípico de las etapas de la sangre asexual y sexual del parásito de la malaria de los roedores y las etapas de los mosquitos

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Investigación

JoVE Journal

Inmunología e Infección

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Evaluación de la función de la vesícula extracelular durante la infección de la Malaria

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

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).

Medición de anticuerpos que inducen la fagocitosis adquirida naturalmente a los parásitos plasmodium falciparum mediante un ensayo basado en citometría de flujo

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

Generation of a Mouse Model of Experimental Cerebral Malaria Using Host Mosquito-Derived Sporozoites

Transcripción

Generation of a Mouse Model of Experimental Cerebral Malaria Using Host Mosquito-Derived Sporozoites