Adenosine reverses metacyclogenesis <i>in vitro</i> and reduces <i>in vivo</i> development of metacyclic promastigotes.

<p>(A) Metacyclic promastigotes from CGS-treated cultures, obtained by Ficoll gradient centrifugation, were cultivated in “spent” medium (late stationary phase medium from control cultures) or in “spent” medium plus adenosine (Ado). Parasite growth was evaluated by hemocytometer counting. (B) Metacyclic promastigotes from CGS-treated cultures, obtained by Ficoll gradient centrifugation followed by complement mediated lysis (as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001833#pntd-0001833-g002" target="_blank">Figure 2D</a>), were cultured in Grace's insect medium without supplements or in this medium plus adenosine. Parasite growth was evaluated by hemocytometer counting. (C) General aspect of metacyclic promastigotes after double purification (Ficoll + complement) (upper left). Cells after 72 hr of incubation in Grace's insect medium without supplements (upper right), or in the presence of 100 µM (lower left) or 500 µM of adenosine (lower right). Pictures taken with an Axio Cam MR3 attached to a Carl-Zeiss Axio Imager M2 microscope. Adult insects of <i>Lutzomyia longipalpis</i>, majority of females, were allowed to feed in a naturally <i>Leishmania infantum chagasi</i>-infected dog for 30 min. Insects were fed for 8 to10 days with 30% sucrose solution with or without adenosine (5 mM). Each midgut was evaluated by light microscopy for the total amount of parasites (D) and for the percentage of metacyclic promastigotes (E). (F) Image of a metacyclic (upper panel) and non-metacyclic promastigote (lower panel) of <i>L. infantum chagasi</i> from sand fly midgut. Pictures taken with a DFC300FX camera attached to a Leica DM5000B microscope. Means and standard deviations (or medians in graph E) from at least two independent experiments are plotted; *p<0.05, ***p<0.0001 determined by two-tailed Student's t-test indicate significant difference from control group.</p

Presence of CGS 15943 in <i>Leishmania amazonensis</i> culture halts parasite growth and enhances metacyclogenesis.

<p>(A) Evaluation of <i>L. amazonensis</i> growth in Grace's insect medium containing different concentrations of CGS as determined by hemocytometer counting. (B) Viability of parasites was assessed by flow cytometry using propidium iodide incorporation after 72 hr of culture. Phase contrast image of DMSO-treated (C) and CGS-treated (D) cultures of <i>L. amazonensis</i> promastigotes (picture taken with an Axio Cam MR3 attached to a Carl-Zeiss Axio Imager M2 microscope). Percentage of metacyclic promastigotes in control (Medium; DMSO) and CGS-treated cultures was evaluated by differential counting in optical microscopy (E) and Ficoll density gradient purification (F). Means and standard deviations from at least three independent experiments are plotted; *p<0.05, **p<0.01 determined by two-tailed Student's t-test indicate significant difference from control group.</p

Control of metacyclogenesis <i>in vitro</i>.

<p>(A) Uptake of [³H]adenosine by mid-log phase promastigotes of <i>L. amazonensis</i> in presence of CGS or dipyridamole (DIP). Experiments were performed at pH 7.4. # indicates p<0.01 when we compared DIP with CGS groups using the two-tailed Student's t-test. (B) <i>L. amazonensis g</i>rowth curve and metacyclogenesis (C) in the presence of DIP (please see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001833#pntd-0001833-g001" target="_blank">Figure 1</a> for details). (D) Growth evaluation of <i>L. amazonensis</i> in presence of N⁶methyladenine (N⁶-MA) followed by metacyclogenesis assessment (E). (F) Assessment of metacyclogenesis in cultures stimulated with CGS and CGS plus adenosine (Ado), inosine (Ino), adenine (Ade) or hypoxanthine (Hyp). CGS was always used at concentration of 50 µM and for nucleosides/nucleobases, the concentration is indicated in parentheses (µM). Experiments were performed in Grace's culture medium supplemented with fetal bovine serum, glutamine and penicillin, pH 6.5. (G) Compilation of results from experiments evaluating growth curve and metacyclogenesis in different conditions. In all experiments metacyclogenesis was evaluated by Ficoll density gradient. Means and standard deviations from at least three independent experiments are plotted; *p<0.05, **p<0.01 determined by two-tailed Student's t-test indicate significant difference from control group.</p