4. ♦ Recognition of lipophosphoglycan (LPG)
♦ Promastigote engulfment
♦ Leishmania live in vesicles
protected from:
• Antibodies
• Killer T cells
♦ Inhibition of phagosome-endosome fusion
♦ Recognition of lipophosphoglycan (LPG)
♦ Promastigote engulfment
♦ Leishmania live in vesicles
protected from:
• Antibodies
• Killer T cells
♦ Inhibition of phagosome-endosome fusion
Immunological Subversion
5. Distribution
♦ 98 endemic countries
• 0.2 to 0.4 million new VL cases; 0.7 to 1.2 million new CL
cases each year worldwide
♦ Restricted to tropical and temperate regions
7. Predictions and Hypotheses
♦ Predictions – The intracellular staining will
enable detection of Leishmania species inside
macrophages
♦ Alternative hypothesis – flow cytometry will
detect Leishmania
♦ Null hypothesis – flow cytometry will not detect
Leishmania
10. Experimental Design
♦ Macrophage Cells
• Mouse cell line J774A.1
♦ Leishmania species
• L. major - cutaneous
• L. infantum - visceral
11. Experimental Design
♦ L. major and L. infantum treatments:
• Control without fluorescein isothiocyanate
(FITC)
• Experimental group with FITC
♦ Anti-Leishmania (GP-63) Monoclonal
Antibody
♦ Visualized parasites following
flow acquisition
13. FSC & SSC Results
Figure 1 Control J774 macrophages Figure 2 Macrophages infected with
L. major
Figure 3 Macrophages infected with
L. infantum
Figure 4 Macrophages infected with L.
infantum mixed with control
Figure 1 Control J774 macrophages Figure 2 Macrophages infected with
L. major
Figure 3 Macrophages infected with
L. infantum
Figure 4 Macrophages infected with L.
infantum mixed with control
17. Parasite Flow Results
Figure 1 L. major: (left) without FITC and
(right) with FITC
Figure 2 L. infantum: (left) without FITC and
(right) with FITC
Figure 3 Histogram plot of L. major: (left)
without FITC and (right) with FITC
Figure 4 Histogram plot of L. infantum: (left)
without FITC and (right) with FITC
FL1 FL1
FL3
FL3
FL1 FL1 FL1 FL1
FL3
FL3
FL1FL1
18. Intracellular Staining Results
Figure 6 L. major infected macrophages
Figure 7 L. infantum infected macrophages Figure 8 L. infantum infected
macrophages mixed with control
Figure 5 Control macrophagesFigure 5 Control macrophages Figure 6 L. major infected macrophages
Figure 7 L. infantum infected macrophages Figure 8 L. infantum infected
macrophages mixed with control
19. Conclusions
♦ Anti-leishmania antibody does attach to the
major surface protease of L. infantum and L.
major
♦ L. infantum binds with higher specificity to
antibody than L. major
♦ Incomplete separation of infected versus
uninfected macrophages
20. Acknowledgements
• Dr. Gabrielle Stryker –
Mentor
• Dr. Blaise Dondji –
Leishmania expert & donor
• W. M. Keck Foundation and
Murdock Fund
• Mark Young – FlowJo
Wizard
• Eric Foss –
Photomicroscopy
Technician
• Heidi Anderson
• Mercedes Cheslock
21. References
Colomer - Gould, V., L. G. Quintao, J. Keithly, N. Nogueira. (1985). A
common surface antigen on amastigoes and promastigotes of
Leishmania species. J. Exp. Med., 162(902).
Desjardins, M., & Descoteaux, A. (1997). Inhibition of phagolysosomal
biogenesis by the Leishmania lipophosphoglycan. The Journal of
Experimental Medicine, 185(12), 2061–2068.
Russel, D.G., and H. Wilhelm. (1986). The involvement of the major
surface glycoprotein (gp63) of Leishmania in attachment to
macrophages. J. Immunol., 136(2616).
Kram, D., Thale, C., Kolodziej, H., and Kiderlen, A. (2008).
Intracellular parasite kill: Flow cytometry and NO detection for rapid
discrimination between anti-leishmanial activity and macrophage
activation. Journal of Immunological Methods, 333(20).
Leishmaniasis is transmitted by the bite of infected female phlebotomine sandflies. The sandflies inject the infective stage (i.e., promastigotes) from their proboscis during blood meals . Promastigotes that reach the puncture wound are phagocytized by macrophages and other types of mononuclear phagocytic cells. Progmastigotes transform in these cells into the tissue stage of the parasite (i.e., amastigotes) , which multiply by simple division and proceed to infect other mononuclear phagocytic cells . Parasite, host, and other factors affect whether the infection becomes symptomatic and whether cutaneous or visceral leishmaniasis results. Sandflies become infected by ingesting infected cells during blood meals (, ). In sandflies, amastigotes transform into promastigotes, develop in the gut (in the hindgut for leishmanial organisms in the Viannia subgenus; in the midgut for organisms in the Leishmania subgenus), and migrate to the proboscis .
The parasites cause a complex of diseases called leishmaniasis
We show that LPG repeating units enable Leishmania to inhibit the phagosome-endosome fusion process efficiently, thereby suggesting a survival strategy during their differentiation into amastigotes.
L inhabit mo intracellularly which protects from other immune respones.
