Changes in the ΔΨ_m of macrophage.

<p>Peritoneal hamster macrophages (2×10⁶ cells/mL) were cultured in the presence of LQB-118 for 48 h at 37°C/5%CO₂. The cells were incubated for 10 min with JC-1 and analysed fluorometrically. Results are presented as means ± standard error; <i>n</i> = 3. *<i>P</i><0.05; **<i>P</i><0.01.</p

Activity of LQB-118 on golden hamsters infected with <i>L. braziliensis</i>.

<p>Golden hamsters (5/group) infected with <i>L. braziliensis</i> (10⁷) were treated on the seventh day of infection with LQB-118 intralesional (26 µg/kg/day) three times/week or orally (4,3 mg/kg/day) five times/week during eight weeks. Controls were untreated, treated with intralesional DMSO three times/week or Glucantime five times/week by intraperitoneal route. <b>A</b>) Lesion thickness was measured for nine weeks. The arrow indicates the start of treatment. Mean ± SD, # P<0.002 (in relation to untreated group). <b>*</b> p<0.001 (in relation to intralesional DMSO group); <b>B</b>) Intradermal reaction at <i>L. braziliensis</i> antigen was evaluated on the contralateral foot pad on eight week of infection. The swelling was measured 48 h later in the antigen-injected footpads. * p<0,04; ** p<0,01. Each point represents one animal and the horizontal bar indicates the mean. <b>C</b>) Parasite burden was assessment by limiting dilution at the end of treatment. *** p<0,001. il, intralesional/subcutaneous; ip, intraperitoneal.</p

Evaluation of LQB118 inducing apoptosis on <i>L. braziliensis</i> – A) and B) Phosphatidylserine exposure on promastigotes.

<p>Promastigotes were incubated with 3,5 or 20 µM LQB118 to 24 or 48 h/28°C and then stained with Annexin V-FITC+propidium iodide and analyzed by flow cytometer. Controls were promastigotes incubated with 60 µM Miltefosine or the culture medium supplemented with 20% fetal bovine serum. In <b>A</b> only treatment at 48 h and in <b>B</b> quantitative evaluation of cells stained with annexin V-FITC at 24 and 48 h. (mean ± SD, n = 3). * P<0.05 ** P<0.01. <b>C) ROS generation and D) impairment of ATP production in LQB-118-treated promastigotes</b>. Promastigotes of <i>L. braziliensis</i> were incubated for 48 h in the presence of LQB-118 in Schneider's insect medium plus 10% HIFCS. <b>A</b>) ROS generation was quantified using H₂DCFDA, <b>B</b>) Cellular ATP concentration was measured by bioluminescence assay. Results are presented as means ± standard error; <i>n</i> = 3. *<i>P</i><0.05; **<i>P</i><0.01. <b>E) </b><b><i>In situ</i></b><b> DNA fragmentation of intracellular amastigote</b>. Infected monolayers of hamsters peritoneal macrophages were treated with the indicated concentrations of LQB118 for 48 h. Monolayers were labeled using TUNEL and observated using fluorescence microscopy. Highlight the cells in 400× magnification. Legend Arrows indicate intracellular amastigotes, N – Macrophage nucleus.</p

In vitro Activity of LQB-118 on <i>L. braziliensis</i>-infected macrophages.

<p>Monolayer of hamster peritoneal macrophage were infected with <i>L. braziliensis</i> (ratio of 5 parasites/macrophages) and subjected to treatment with the indicated concentrations of LQB-118 for 48 hours at 37°C/5% CO2. <b>A</b>) Macrophage monolayer was stained and the infection index was determined by counting at least 100 macrophage. Nitric oxide production in the supernatants was measured by assaying for nitrite using Griess method (inset) or <b>B</b>) After 48 h of the treatment the monolayers of infected macrophages were washed twice and incubated with Schneider's medium and 20% fetal bovine serum at 28°C for more 48 h and promastigotes were counted (mean ± SD, n = 3). * p<0, 01, ** p<0, 001(LQB-118 treatment in relation to control).</p