Alterations suggestive of autophagy in intracellular parasites treated with 17-AAG.

<p>Transmission electron microscopy was used to investigate ultrastructural morphological alterations in intracellular parasites inside 17-AAG-treated macrophages. (<b>A</b>) Control infected macrophages. After 12 h of treatment, several morphological alterations were seen in intracellular parasites, including: (<b>B</b>) numerous small vesicles some containing cytoplasmic material inside <b>(black arrow)</b>, (<b>C</b>) vacuoles larger in size <b>(black arrow-head)</b>, (<b>D</b>) with intravacuolar materials degraded <b>(white arrow)</b>. After 24 h of treatment, the intracellular parasites presented a large number of vesicles occupying most of the cytoplasm containing well-preserved nuclei, mitochondria, and subpellicular microtubules (<b>E–F</b>). After 48 h of treatment, no preserved parasites inside cells were observed, yet empty vesicles, and membrane-bounded structures with an electron-density similar to parasite cytosol in parasitophorous vacuoles were seen (<b>G</b>). At 12 and 24 h after treatment, several alterations were also visible in parasite cytoplasm, including myelin figures <b>(*)</b> (<b>H–I</b>), vesicles with double-layered membranes <b>(white arrow-head)</b> (<b>J–M</b>)<b>,</b> and portions of mitochondria inside membrane-bounded structures (<b>M</b>). The nuclei (<b>N</b>) and mitochondria (<b>M</b>) and kinetoplast (<b>K</b>) remained intact in all groups.</p

Modulation of mediator production by treatment with 17-AAG.

<p>Mediators released by 17-AAG-treated cells were measured in cell supernatants using an inflammatory CBAKit, as described in Materials and Methods: (<b>A</b>) IL-6; (<b>B</b>) IL-10; (<b>C</b>) IL-12; (<b>D</b>) TNF-α; (<b>E</b>) MCP-1. Bars represent means ± SD of a single experiment performed in sextuplicate (Student’s <i>t</i>-test and Mann-Whitney, **<i>p</i><0.001, ***<i>p</i><0.0001).</p

Irreversibility of treatment with 17-AAG on intracellular <i>Leishmania</i>.

<p>To assessment the reversibility of parasite growth inhibition by treatment with 17-AAG parasite load was determined by quantifying the percentage of infected macrophages (<b>A</b>) and the number of parasites per macrophage (<b>B</b>) as described in Materials and Methods. Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, ***<i>p</i><0.0001, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001).</p

Reduction of parasite intracellular viability by 17-AAG.

<p>Treatment’s effect on parasite viability was assessed after 24 h (<b>A</b>) and 48 h (<b>B</b>) of infection, as described in Materials and Methods. Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, ***<i>p</i><0.0001, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001; Mann Whitney test, **<i>p</i><0.001).</p

Inhibition of axenic growth of <i>Leishmania</i> and reduction in parasite load by 17-AAG.

<p>(<b>A</b>) Axenic promastigotes were exposed to several concentrations of 17-AAG (25, 125, 500 nM) for 48 h and the number of viable parasites was assessed as described in Materials and Methods. Data are presented as the percentage inhibition of parasite growth related to untreated controls (4,754×10⁷). Bars represent means ± SD of one representative out of two experiments performed in sextuplicate (one-way ANOVA, Dunnett’s Multiple Comparison Test, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001). (<b>B, C</b>) Drug effects at early times after infection. Following 6 h of incubation with parasites, macrophage cultures were treated for 6, 24 and 48 h with specific concentrations of 17-AAG (25, 125, 500 nM); (<b>D, E</b>) Drug effects at later stages after infection. Following 6 h of incubation with parasites, macrophage cultures were reincubated for additional 48 h then submitted to treatment with specific concentrations of 17-AAG (25, 125, 500 nM). Bars represent means ± SD of one representative experiment out of two performed in sextuplicate (one-way ANOVA, Dunnett’s Multiple Comparison Test, *<i>p</i><0.05, **<i>p</i><0.001, ***<i>p</i><0.0001, post-test for linear trend, <i>p</i><0.0001).</p

Reduced O₂

<p>⁻<b>and NO production in macrophage cultures treated with 17-AAG.</b> (<b>A</b>) O₂⁻ production was measured at early stages of infection using a lucigenin-derived CL method. Points on the graph represent photon emissions per second by macrophage cultures 2 min prior to the addition of <i>L. amazonensis</i> promastigotes, as well as throughout the incubation period of 20 min. Data are derived from one representative experiment out of four performed in uniplicate (Mann Whitney test, p = 0.028); (<b>B</b>) Intracellular O₂⁻ production was assessed by determining cell fluorescence in the presence of hydroethidine (5 µM) and expressed as MFI using a flow cytometer. The histogram overlay depicts the MFI of hydroethidine-labeled cells (solid lines) in comparison to unlabeled control cells (shaded areas). Data are derived from one representative experiment out of three performed in uniplicate (Mann Whitney test, <i>p</i> = 1). (<b>C</b>) NO production was measured by detecting nitrite in the supernatants of 17-AAG-treated cells, as described in Materials and Methods. Bars represent NO production measurement expressed as means ± SD of one representative experiment out of four performed in triplicate or more (Student’s <i>t</i> test, ***<i>p</i><0.0001).</p

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

<div><p> BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.</p></div

Evaluating the Accuracy of Molecular Diagnostic Testing for Canine Visceral Leishmaniasis Using Latent Class Analysis

<div><p>Host tissues affected by <i>Leishmania infantum</i> have differing degrees of parasitism. Previously, the use of different biological tissues to detect <i>L. infantum</i> DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4–100) in splenic aspirate; 79.2% (CI 68–90.3) in lymph nodes; 77.3% (CI 64.5–90.1) in skin; 75% (CI 63.1–86.9) in blood; 50% (CI 30–70) in bone marrow; 37.5% (CI 24.2–50.8) in left-eye; and 29.2% (CI 16.7–41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7–79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5–96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting <i>L. infantum</i> infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing.</p></div

Prevalence of latent classes and conditional probabilities to the LCA model for <i>L. infantum</i> infection detection in dogs.

<p>Prevalence of latent classes and conditional probabilities to the LCA model for <i>L. infantum</i> infection detection in dogs.</p

Response patterns^a of Camaçari dogs for LCA model with 2 latent classes for diagnosis of CVL.

a<p>Response patterns of all samples tested using the three techniques.</p><p>*Estimation based on only one animal sample presenting this pattern.</p><p>N: Negative; P: Positive.</p