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Detection of Leishmania Parasites via
Flow Cytometry
Analiese Wenger
What are Leishmania?♦ Single-cell parasite♦ Infect humans and other mammalian
hosts
Life Cycle
Leishmaniasis♦ Manifestations:• Cutaneous
• Mucosal
• Visceral
♦ 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
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
Scientific Question
Can macrophages infected with Leishmania be detected using a flow
cytometer?
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
Flow Cytometry
♦ Counts individual cells using laser technology• Forward-scattered light (FSC)• Side-scattered light (SSC)
♦ Fluorescence dyes (fluorochromes) for sensing target proteins
♦ Counts individual cells using laser technology• Forward-scattered light (FSC)• Side-scattered light (SSC)
♦ Fluorescence dyes (fluorochromes) for sensing target proteins
Flow Cytometry Data
Experimental Design
♦ Macrophage Cells• Mouse cell line
J774A.1
♦ Leishmania species• L. major - cutaneous• L. infantum - visceral
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
Techniques♦ Preliminary experimentation• FSC & SSC of macrophages
♦ Photomicroscopy♦ Intracellular staining• Modified protocol
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
Techniques♦ Preliminary experimentation• FSC & SSC of macrophages
♦ Photomicroscopy♦ Intracellular staining• Modified protocol
Photomicroscopy Results
L. major
L. infantum
Techniques♦ Preliminary experimentation• FSC & SSC of macrophages
♦ Photomicroscopy♦ Intracellular staining• Modified protocol
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
Intracellular Staining Results
Figure 6 L. major infected macrophages
Figure 7 L. infantum infected macrophagesFigure 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 macrophagesFigure 8 L. infantum infected macrophages mixed with control
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
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
ReferencesColomer - 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).
Questions?