DH82 Canine and RAW264.7 Murine Macrophage Cell Lines Display Distinct Activation Profiles Upon Interaction With Leishmania infantum and Leishmania amazonensis.

Leishmaniasis is an anthropozoonotic disease, and dogs are considered the main urban reservoir of the parasite. Macrophages, the target cells of Leishmania sp., play an important role during infection. Although dogs have a major importance in the epidemiology of the disease, the majority of the current knowledge about Leishmania-macrophage interaction comes from murine experimental models. To assess whether the canine macrophage strain DH82 is an accurate model for the study of Leishmania interaction, we compared its infection by two species of Leishmania (Leishmania infantum and L. amazonensis) with the murine macrophage cell line (RAW264.7). Our results demonstrated that L. amazonensis survival was around 40% at 24 h of infection inside both macrophage cell lines; however, a reduction of 4.3 times in L. amazonensis infection at 48 h post-infection in RAW 264.7 macrophages was observed. The survival index of L. infantum in DH82 canine macrophages was around 3 times higher than that in RAW264.7 murine cells at 24 and 48 h post-infection; however, at 48 h a reduction in both macrophages was observed. Surprisingly at 24 h post-infection, NO and ROS production by DH82 canine cells stimulated with LPS or menadione or during Leishmania infection was minor compared to murine RAW264.7. However, basal arginase activity was higher in DH82 cells when compared to murine RAW264.7 cells. Analysis of the cytokines showed that these macrophages present a different response profile. L. infantum induced IL-12, and L. amazonensis induced IL-10 in both cell lines. However, L. infantum and L. amazonensis also induced TGF-ß in RAW 264.7. CD86 and MHC expression showed that L. amazonensis modulated them in both cell lines. Conversely, the parasite load profile did not show significant difference between both macrophage cell lines after 48 h of infection, which suggests that other mechanisms of Leishmania control could be involved in DH82 cells.

In vitro leishmanicidal activity of monoterpenes present in two species of Protium (Burseraceae) on Leishmania amazonensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis is a neglected disease that affects millions of people around the world. Parasite resistance and the toxicity to the current treatments lead to the search for new effective molecules. Plants are widely used in traditional and indigenous medicine to treat different diseases. The oleoresin of the genus Protium, which is rich in volatile compounds active against different microorganisms, is among these plants. AIM: The aim of this study was to evaluate the leishmanicidal potential of Protium altsonii (PaEO) and P. hebetatum (PhEO) (Burseraceae) oleoresins, as well as of three representative monoterpenes in their constitution: α-pinene, p-cymene and 1,8-cineole. MATERIALS AND METHODS: Protium altsonii (PaEO) and P. hebetatum (PhEO) oleoresins and three of their constituents were tested in vitro on promastigotes and amastigotes-infected macrophages in different concentrations. Their toxicity for macrophages was analyzed by XTT assay and phagocytic ability. It was evaluated the ability of the compounds to induce NO production on treated-macrophages using Griess reaction and the effect of them in lipid profile on treated-parasite through Thin Layer Chromatography. RESULTS: Our data showed that both essential oils have toxic effect on promastigotes and amastigotes of L. amazonensis in vitro in a dose-dependent manner. PaEO IC50 were 14.8 µg/mL and 7.8 µg/mL and PhEO IC50s were 0.46 µg/mL and 30.5 µg/m for promastigotes and amastigotes, respectively. Toxicity to macrophages was not observed at 50 µg/mL with both EOs. The compounds 1,8- cineole, α-pinene, and p-cymene inhibited amastigotes survival in a dose-dependent manner with IC50s of 48.4 µg/mL, 37 µg/mL, 46 µg/mL, respectively. Macrophage viability was around 90% even at 200 µg/mL and the phagocytic capacity was not altered in the treated-macrophages to up 50 µg/mL. The compounds were not able to modulate the nitric oxide production either at rest or LPS-activated macrophages. In addition, treated promastigote revealed an important change in their lipid profile after 48 h at 50 µg/mL in the presence of the compounds. CONCLUSIONS: The results indicate that oleoresins of Protium genus are potent against Leishmania and α-pinene, p-cymene and 1,8-cineole have anti-Leishmania properties that could be explored in synergistic assays in order to develop new drug candidates.

The in vitro antileishmanial activity of essential oil from Aloysia gratissima and guaiol, its major sesquiterpene against Leishmania amazonensis.

Leishmaniases is a tropical disease caused by protozoa of the genus Leishmania for which the current treatment is expensive, besides increasing reports of parasite resistance. This study investigated the anti-Leishmania amazonensis activity of the essential oil from Aloysia gratissima (AgEO) and guaiol, the major sesquiterpene constituent in the oil. Our results showed that AgEO killed promastigotes and intracellular amastigotes at an IC50 of 25 and 0·16 µg mL-1, respectively, while guaiol killed amastigotes at an IC50 of 0·01 µg mL-1. Both AgEO and guaiol were safe for macrophages up to 100 µg mL-1, as evaluated by the dehydrogenase activity, membrane integrity and phagocytic capacity. AgEO and guaiol did not induce nitrite oxide (NO) in resting macrophages and inhibited the production of NO in lipopolysaccharide-stimulated macrophages. The ultrastructural analysis suggested that AgEO and guaiol act directly on parasites, affecting promastigotes kinetoplast, mitochondrial matrix and plasma membrane. Together, these results pointed out that AgEO and guaiol could be promising candidates to develop anti-Leishmania drugs.

Antileishmanial Thioureas: Synthesis, Biological Activity and in Silico Evaluations of New Promising Derivatives.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites belonging to the genus Leishmania. Currently, the drugs available for treatment of this disease present high toxicity, along with development of parasite resistance. In order to overcome these problems, efforts have been made to search for new and more effective leishmanicidal drugs. The aim of this study was to synthesize and investigate the leishmanicidal effect of N,N'-disubstituted thioureas against Leishmania amazonensis, with evaluation of their in silico pharmacokinetics and toxicity profiles. Our results showed that different thioureas could be obtained in high to moderate yields using simple reaction conditions. Nine thiourea derivatives (3e, 3i, 3k, 3l, 3p, 3q, 3v, 3x and 3z) were active against parasite promastigotes (IC50 21.48-189.10 µM), with low cytotoxicity on mice peritoneal macrophages (CC50>200 µM), except for thiourea 3e (CC50=49.22 µM). After that, the most promising thioureas (3k, 3l, 3p, 3q and 3v) showed IC50 ranging from 70 to 150 µM against L. amazonensis amastigotes in infected macrophages. Except for thiourea 3p, the leishmanicidal activity of the derivatives were independent of nitric oxide (NO) production. Thioureas 3q and 3v affected promastigotes cell cycle without disturbing the mitochondrial membrane potential. Furthermore, our derivatives showed satisfactory theoretical absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. These data indicate that thiourea derivatives are good candidates as leading compounds for the development of new leishmanicidal drugs.

The Brown Alga Stypopodium zonale (Dictyotaceae): A Potential Source of Anti-Leishmania Drugs.

This study evaluated the anti-Leishmania amazonensis activity of a lipophilic extract from the brown alga Stypopodium zonale and atomaric acid, its major compound. Our initial results revealed high inhibitory activity for intracellular amastigotes in a dose-dependent manner and an IC50 of 0.27 µg/mL. Due to its high anti-Leishmania activity and low toxicity toward host cells, we fractionated the lipophilic extract. A major meroditerpene in this extract, atomaric acid, and its methyl ester derivative, which was obtained by a methylation procedure, were identified by nuclear magnetic resonance (NMR) spectroscopy. Both compounds inhibited intracellular amastigotes, with IC50 values of 20.2 µM (9 µg/mL) and 22.9 µM (10 µg/mL), and selectivity indexes of 8.4 µM and 11.5 µM. The leishmanicidal activity of both meroditerpenes was independent of nitric oxide (NO) production, but the generation of reactive oxygen species (ROS) may be at least partially responsible for the amastigote killing. Our results suggest that the lipophilic extract of S. zonale may represent an important source of compounds for the development of anti-Leishmania drugs.

Oleanolic acid (OA) as an antileishmanial agent: Biological evaluation and in silico mechanistic insights.

Although a worldwide health problem, leishmaniasis is considered a highly neglected disease, lacking efficient and low toxic treatment. The efforts for new drug development are based on alternatives such as new uses for well-known drugs, in silico and synthetic studies and naturally derived compounds. Oleanolic acid (OA) is a pentacyclic triterpenoid widely distributed throughout the Plantae kingdom that displays several pharmacological activities. OA showed potent leishmancidal effects in different Leishmania species, both against promastigotes (IC(50 L. braziliensis) 30.47 ± 6.35 µM; IC(50 L. amazonensis) 40.46 ± 14.21 µM; IC(50 L. infantum) 65.93 ± 15.12 µM) and amastigotes (IC(50 L. braziliensis) 68.75 ± 16.55 µM; IC(50 L. amazonensis) 38.45 ± 12.05 µM; IC(50 L. infantum) 64.08 ± 23.52 µM), with low cytotoxicity against mouse peritoneal macrophages (CC(50) 235.80 ± 36.95 µM). Moreover, in silico studies performed to evaluate OA molecular properties and to elucidate the possible mechanism of action over the Leishmania enzyme sterol 14α-demethylase (CYP51) suggested that OA interacts efficiently with CYP51 and could inhibit the ergosterol synthesis pathway. Collectively, these data indicate that OA is a good candidate as leading compound for the development of a new leishmaniasis treatment.

Leishmanicidal Effect of Synthetic trans-Resveratrol Analogs.

BACKGROUND: Stilbene-based compounds show antitumoral, antioxidant, antihistaminic, anti-inflammatory and antimicrobial activities. Here, we evaluated the effect of the trans-resveratrol analogs, pterostilbene, piceatannol, polydatin and oxyresveratrol, against Leishmania amazonensis. METHODOLOGY/PRINCIPAL FINDINGS: Our results demonstrated a low murine macrophage cytotoxicity of all four analogs. Moreover, pterostilbene, piceatannol, polydatin and oxyresveratrol showed an anti-L. amazonensis activity with IC50 values of 18 µM, 65 µM, 95 µM and 65 µM for promastigotes, respectively. For intracellular amastigotes, the IC50 values of the analogs were 33.2 µM, 45 µM, 29 µM and 30.5 µM, respectively. Among the analogs assayed only piceatannol altered the cell cycle of the parasite, increasing 5-fold the cells in the Sub-G0 phase and decreasing 1.7-fold the cells in the G0-G1 phase. Piceatannol also changed the parasite mitochondrial membrane potential (ΔΨm) and increased the number of annexin-V positive promastigotes, which suggests incidental death. CONCLUSION/SIGNIFICANCE: Among the analogs tested, piceatannol, which is a metabolite of resveratrol, was the more promising candidate for future studies regarding treatment of leishmaniasis.

IL-27 enhances Leishmania amazonensis infection via ds-RNA dependent kinase (PKR) and IL-10 signaling.

The protozoan parasite Leishmania infects and replicates in macrophages, causing a spectrum of diseases in the human host, varying from cutaneous to visceral clinical forms. It is known that cytokines modulate the immunological response against Leishmania and are relevant for infection resolution. Here, we report that Interleukin (IL)-27 increases Leishmania amazonensis replication in human and murine macrophages and that the blockage of the IL-10 receptor on the surface of infected cells abolished the IL-27-mediated enhancement of Leishmania growth. IL-27 induced the activation/phosphorylation of protein kinase R (PKR) in macrophages, and PKR blockage or PKR gene deletion abrogated the enhancement of the parasite growth driven by IL-27, as well as the L. amazonensis-induced macrophage production of IL-27. We also observed that L. amazonensis-induced expression of IL-27 depends on type I interferon signaling and the engagement of Toll-like receptor 2. Treatment of Leishmania-infected mice with IL-27 increased lesion size and parasite loads in the footpad and lymph nodes of infected animals, indicating that this cytokine exerts a local and a systemic effect on parasite growth and propagation. In conclusion, we show that IL-27 is a L. amazonensis-enhancing factor and that the PKR/IFN1 axis and IL-10 are critical mediators of this IL-27 induced effect.

Resveratrol is active against Leishmania amazonensis: in vitro effect of its association with Amphotericin B.

Resveratrol is a polyphenol found in black grapes and red wine and has many biological activities. In this study, we evaluated the effect of resveratrol alone and in association with amphotericin B (AMB) against Leishmania amazonensis. Our results demonstrate that resveratrol possesses both antipromastigote and antiamastigote effects, with 50% inhibitory concentrations (IC50s) of 27 and 42 µM, respectively. The association of resveratrol with AMB showed synergy for L. amazonensis amastigotes, as demonstrated by the mean sums of fractional inhibitory index concentration (mean ΣFIC) of 0.483, although for promastigotes, this association was indifferent. Treatment with resveratrol increased the percentage of promastigotes in the sub-G0/G1 phase of the cell cycle, reduced the mitochondrial potential, and showed an elevated choline peak and CH2-to-CH3 ratio in the nuclear magnetic resonance (NMR) spectroscopy analysis; all these features indicate parasite death. Resveratrol also decreased the activity of the enzyme arginase in uninfected and infected macrophages with and without stimulation with interleukin-4 (IL-4), also implicating arginase inhibition in parasite death. The anti-Leishmania effect of resveratrol and its potential synergistic association with AMB indicate that these compounds should be subjected to further studies of drug association therapy in vivo.

Dolabelladienetriol, a compound from Dictyota pfaffii algae, inhibits the infection by Leishmania amazonensis.

BACKGROUND: Chemotherapy for leishmaniasis, a disease caused by Leishmania parasites, is expensive and causes side effects. Furthermore, parasite resistance constitutes an increasing problem, and new drugs against this disease are needed. In this study, we examine the effect of the compound 8,10,18-trihydroxy-2,6-dolabelladiene (Dolabelladienetriol), on Leishmania growth in macrophages. The ability of this compound to modulate macrophage function is also described. METHODOLOGY/PRINCIPAL FINDINGS: Leishmania-infected macrophages were treated with Dolabelladienetriol, and parasite growth was measured using an infectivity index. Nitric oxide (NO), TNF-α and TGF-ß production were assayed in macrophages using specific assays. NF-kB nuclear translocation was analyzed by western blot. Dolabelladienetriol inhibited Leishmania in a dose-dependent manner; the IC(50) was 44 µM. Dolabelladienetriol diminished NO, TNF-α and TGF-ß production in uninfected and Leishmania-infected macrophages and reduced NF-kB nuclear translocation. Dolabelladienetriol inhibited Leishmania infection even when the parasite growth was exacerbated by either IL-10 or TGF-ß. In addition, Dolabelladienetriol inhibited Leishmania growth in HIV-1-co-infected human macrophages. CONCLUSION: Our results indicate that Dolabelladienetriol significantly inhibits Leishmania in macrophages even in the presence of factors that exacerbate parasite growth, such as IL-10, TGF-ß and HIV-1 co-infection. Our results suggest that Dolabelladienetriol is a promising candidate for future studies regarding treatment of leishmaniasis, associated or not with HIV-1 infection.

Leishmanicidal activity of a supercritical fluid fraction obtained from Tabernaemontana catharinensis.

The branches and leaves of Tabernaemontana catharinensis were extracted with supercritical fluid using a mixture of CO(2) plus ethanol (SFE), and the indole alkaloid enriched fraction (AF3) was selected for anti-Leishmania activity studies. We found that AF3 exhibits a potent effect against intracellular amastigotes of Leishmania amazonensis, a causative agent of New World cutaneous leishmaniasis. AF3 inhibits Leishmania survival in a dose-dependent manner, and reached 88% inhibition of amastigote growth at 100 microg/mL. The anti-parasite effect was independent of nitric oxide (NO), since AF3 was able to inhibit NO production induced by IFN-gamma plus LPS. In addition, AF3 inhibited TGF-beta production, which could have facilitated AF3-mediated parasite killing. The AF3 fraction obtained from SFE was nontoxic for host macrophages, as assessed by plasma membrane integrity and mitochondrial activity. We conclude that SFE is an efficient method for obtaining bioactive indole alkaloids from plant extracts. Importantly, this method preserved the alkaloid properties associated with inhibition of Leishmania growth in macrophages without toxicity to host cells.

The 3A1-La monoclonal antibody reveals key features of Leishmania (L) amazonensis metacyclic promastigotes and inhibits procyclics attachment to the sand fly midgut.

In this work, we characterise metacyclic promastigotes of Leishmania amazonensis, the causative agent of cutaneous and diffuse cutaneous leishmaniasis in the New World. To purify metacyclics from stationary culture by negative selection, we used the monoclonal antibody 3A1-La produced against procyclic promastigotes. The purified forms named 3A1-La(-) promastigotes, present key metacyclic characteristics: slender cell body and long flagella, ultrastructural features, resistance to complement lysis, high infectivity for macrophages and mice and reduced capacity for binding to the sand fly midgut. Moreover, the epitope recognised by 3A1-La is important for the promastigote attachment to the insect vector midgut epithelium. These results further characterise 3A1-La(-) promastigotes as metacyclic forms of L. amazonensis.