Abstract

GAMBOA MARIN, Yuly Andrea  and  DELGADO MURCIA, Gabriela. Study of Leishmania Viannia infection by means of flow cytometry and Giemsa stain using human and murine macrophage lines (U-937 and J-774). Rev. colomb. cienc. quim. farm. [online]. 2008, vol.37, n.1, pp.84-95. ISSN 0034-7418.

The treatment for Leishmaniasis has presented some difficulties related with adverse effects and resistance. For these reasons it is important to search therapeutic alternatives which must be analyzed using adequately standardized in vitro and in vivo models. With this purpose, we implemented an in vitro model for leishmania infection using U-937 and J-774macrophages, and evaluating promastigote internalization at consecutive time spans (from 2 to 6 hours). The first approximation assayed involves the flow cytometric (CF) analysis for invasion quantification by measuring the fluorescence emitted by parasites previously transfected with green fluorescence protein (GFP). In the alternative strategy, parasitized cells were subjected to Giemsa stain and CF was applied to measure the increase of macrophages cytoplasm density owed to internalized parasites. Giemsa stain also allowed us to estimate the number of parasites within each cell. We report that the presence of 35 parasites per macrophage produces an increase in cytoplasmic density enough to be detected by CF so that infection can be clearly reported. We also found that J-774 macrophages internalize Leishmania promastigotes more efficiently than U-937 cells (P value: 0.0006). Cells of the murine line were infected by a higher number of parasites than the human counterparts used in this study (P value 0.0038). From the resultas, we conclude: (i) the change in macrophage cytoplasm density demonstrated by CF after Giemsa stain are sufficient to estimate the percentage of infection. (ii) Giemsa stain provides a less expensive strategy to evaluate Leishmania infection than the fluorescence based option, although the intra and inter observer variability (semi-quantitative procedure) makes it less precise; (iii) quantitative results obtained by both techniques correlate to each other, suggesting that these two tools can be considered complementary; and (iv) both the percentage of infection and the number of internalized parasites are higher for J-774 than for U-937 macrophages. This probably suggests the presence of more receptor molecules (binding targets) for Leishmania ligands on the murine cell membrane determining a more efficient internalization rate than in human macrophages.

Keywords : In vitro Leishmania infection; flow cytometry (CF); Giemsa stain (CG); macrophages; promastigotes.

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