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. Isolation and Differentiation of Bone Marrow-derived Macrophages (BMMs)
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	<li>Day 0:&#160;Sacrifice 4 - 6-week-old female&#160;C57BL/6&#160;or&#160;BALB/c&#160;mouse in a CO2&#160;chamber and confirm death via cervical dislocation. Ensure sterilization of scissors and forceps by k...

assessment of leishmania virulence within cultured macrophages

Source: Sarkar, A. et al., <a href="https://app.jove.com/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of&#160;Leishmania&#160;Parasites.</a>&#160;J. Vis. Exp.&#160;(2018)
This video investigates Leishmania&#39;s virulence by treating macrophages with metacyclic promastigotes, resulting in the formation of intracellular amastigotes. The confirmation of Leishmania&#39;s virulence is achieved through nucleic acid fluorescent dye staining. This insight can contribute to the development of new therapies for leishmaniasis.

<img alt="Figure 1" src="/files/ftp_upload/21792/21792_Figure1.jpg" /> 
Figure 1: A representative illustration of BMM infection with virulent axenic amastigotes (A) and non-virulent log-phase promastigotes (B) of&#160;L. amazonensis.&#160;Immunofluorescence images of macrophages isolated from&#160;BALB/c&#160;mice, 1 h and 48 h following infection. Infected macrophages were processed for immunofluorescence as described in the protocol. PV membranes...

Assessment of Leishmania Virulence within Cultured Macrophages

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 multi-well plate containing bone marrow-derived macrophages on coverslips.
Treat the macrophages with Leishmania promastigotes &#8212; the parasite&#39;s infectious stage.
During incubation, the parasites interact with macrophages, facilitating the assembly of F-actin around the interaction site.
Macrophages engulf the parasites in F-actin-surrounded phagosomes, forming parasitophorous vacuoles or PVs &#8212; a replicative niche.
Within PVs, parasites release survival factors, increasing F-actin accumulation around the vacuoles and inhibiting their fusion with lysosomes.
Additionally, parasites upregulate the production of hepcidin &#8212; a macrophage peptide hormone, inhibiting iron release and elevating intracellular iron levels.
The iron influx into vacuoles promotes promastigote differentiation into pathogenic amastigotes and facilitates their multiplication, enhancing virulence.
Post-incubation, wash to remove free parasites, then, fix and permeabilize the infected macrophages.
Introduce a nucleic acid fluorescent dye to stain the nuclei of infected macrophages and amastigotes.
Under a fluorescence microscope, small nuclei clustered around the macrophage&#39;s large nucleus confirm the presence of intracellular parasites, demonstrating Leishmania&#39;s virulence.