Leishmanicidal effect of 1,3,4-thiadiazolium mesoionic salts on Leishmania amazonensis in vitro.

Leishmaniasis is a neglected broad clinical spectrum disease caused by protozoa of the genus Leishmania, which affect millions of people annually in the world and the treatment has severe side effects and resistant strains have been reported. Mesoionic salts are a subclass of the betaine group with extensive biological activity such as microbicide and anti-inflammatory In this work, we analyze the cytotoxic effects of mesoionic salts, 4-phenyl-5-(X-phenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (X = 4 Cl; 3,4 diCl and 3,4 diF), on Leishmania amazonensis in vitro. Initially, Mesoionic salts toxicity were evaluated by XTT assay on L. amazonensis promastigotes. Our results show that the mesoionic salts MI-3,4 diCl, MI-4 Cl and MI-3,4 diF were toxic to the promastigote parasite with IC50 values of 14.3, 40.1 and 61.8 µM, respectively. The amastigote survival was evaluated in treated infected-macrophages, and the results demonstrate that MI-4 Cl (IC50 = 33 µM) and MI-3,4 diCl (IC50 = 43 µM) have a toxic effect against these forms. None of the mesoionic compounds tested present host cell toxicity up to the tested concentration of 100 µM. The selectivity index for MI-3,4 diCl and MI-4 Cl were 3.94 and 6.97, respectively. Nitric oxide (NO) production assayed by Griess reagent, in LPS-activated macrophages or not, in the presence of the salts showed that only the MI-3,4 diCl compound reduced NO levels. Lipid profile analysis of treated-promastigotes showed no alteration of neutral lipids. Evaluation of mitochondrial membrane potential (∆Ψm) showed that the MI-4Cl compound was able to reduce (∆Ψm) by 50%. Therefore, our results suggest that the chlorinated compounds are promising biomolecules, which cause inhibition of L.amazonensis promastigotes, amastigotes, leading to mitochondrial damage.

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.

ETosis: A Microbicidal Mechanism beyond Cell Death.

Netosis is a recently described type of neutrophil death occurring with the release to the extracellular milieu of a lattice composed of DNA associated with histones and granular and cytoplasmic proteins. These webs, initially named neutrophil extracellular traps (NETs), ensnare and kill microorganisms. Similarly, other cell types, such as eosinophils, mast cells, and macrophages, can also dye by this mechanism; thus, it was renamed as ETosis, meaning death with release of extracellular traps (ETs). Here, we review the mechanism of NETosis/etosis, emphasizing its role in diseases caused by protozoan parasites, fungi, and viruses.

Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus.

Feline leukemia virus (FeLV), a common, naturally occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the haematopoietic system, immunodeficiency and neoplasia. FeLV infection causes an important suppression of neutrophil function, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils, as well as to evaluate neutrophil function in FeLV naturally infected symptomatic and asymptomatic cats through the phagocytosis process, release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) activity. The results showed that feline neutrophils stimulated with protozoa parasites released structures comprising DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophil stimulation showed a significant increase in NET release by neutrophils from FeLV(-) and FeLV(+) asymptomatic cats compared with FeLV(+) symptomatic cats. Moreover, the number of released NETs and MPO activity in unstimulated neutrophils of FeLV(+) symptomatic cats were higher than those in unstimulated neutrophils from FeLV(-) and FeLV(+) asymptomatic cats. This study reports, for the first time, NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. The results indicate that the NET mechanism appears to be overactivated in FeLV(+) cats and that this feature could be considered a marker of disease progression in FeLV infection.

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.

Flow cytometric assessment of Leishmania spp metacyclic differentiation: validation by morphological features and specific markers.

Characterization of infective metacyclic promastigotes of Leishmania spp can be an essential step in several experimental protocols. Metacyclic forms of all Leishmania species display a typical morphology with short, narrow cell body, and an elongated flagellum. This feature suggests that metacyclics can be distinguished from procyclic forms by non-fluorimetric flow cytometric parameters thus enabling the follow-up of their appearance and acquisition of specific properties, during metacyclogenesis in in vitro cultures. Here we describe the flow cytometric parameters of stage-specific promastigotes of Leishmania major, Leishmania donovani, Leishmania amazonensis, and Leishmania braziliensis. Our findings were validated by optical microscopy morphology and specific procyclic labeling with FITC-peanut agglutinin. Furthermore, we show that parasite's distribution in the plot during differentiation in culture is not species specific and that the parasites displaying low forward-angle light scatter (FSC(low)) are three times more infective than the FSC(high) ones. The method here described can be applied to the identification of metacyclics of different Leishmania spp within the whole stationary population.

Leishmania (Viannia) braziliensis metacyclic promastigotes purified using Bauhinia purpurea lectin are complement resistant and highly infective for macrophages in vitro and hamsters in vivo.

In this study we characterised metacyclogenesis in axenic culture of Leishmania (Viannia) braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World. Metacyclogenesis of other species of Leishmania has been shown by morphological changes as well as molecular modifications in the lipophosphoglycan, the major cell surface glycoconjugate of the promastigotes. In order to obtain metacyclic forms of L. braziliensis we tested a panel of different lectins. Our results showed that Bauhinia purpurea lectin facilitated the purification of metacyclic promastigotes from stationary-phase culture by negative selection. The B. purpurea non-agglutinated promastigotes had a slender short cell body and long flagella, typical of metacyclic morphology. The ultrastructural analysis showed that B. purpurea non-agglutinated promastigotes have a dense and thicker glycocalyx. They are resistant to complement lysis, and highly infective for macrophage in vitro and hamsters in vivo. Contrary to procyclic promastigotes, B. purpurea non-agglutinated forms were poorly recognised by sand fly gut epithelial cells. These results suggest that the B. purpurea non-agglutinated promastigotes are the metacyclic forms of L. braziliensis.