Complement receptor 3 mediates ruffle-like, actin-rich aggregates during phagocytosis of Leishmania infantum metacyclics.

The parasitic protozoan Leishmania infantum resides primarily in macrophages throughout mammalian infection. Infection is initiated by deposition of the metacyclic promastigote into the dermis of a mammalian host by the sand fly vector. Promastigotes enter macrophages by ligating surface receptors such as complement receptor 3 (CR3), inducing phagocytosis of the parasite. At the binding site of metacyclic promastigotes, we observed large asymmetrical aggregates of macrophage membrane with underlying actin, resembling membrane ruffles. Actin accumulation was observed at the point of initial contact, before phagosome formation and accumulation of peri-phagosomal actin. Ruffle-like structures did not form during phagocytosis of attenuated promastigotes or during phagocytosis of the intracellular amastigote form of L. infantum. Entry of promastigotes through massive actin accumulation was associated with a subsequent delay in fusion of the parasitophorous vacuole (PV) with the lysosomal markers LAMP-1 and Cathepsin D. Actin accumulation was also associated with entry through CR3, since macrophages from CD11b knockout (KO) mice did not form massive aggregates of actin during phagocytosis of metacyclic promastigotes. Furthermore, intracellular survival of L. infantum was significantly decreased in CD11b KO compared to wild type macrophages, although entry rates were similar. We conclude that both promastigote virulence and host cell CR3 are needed for the formation of ruffle-like membrane structures at the site of metacyclic promastigote phagocytosis, and that formation of actin-rich aggregates during entry correlates with the intracellular survival of virulent promastigotes.

Vernonia brasiliana (L.) Druce induces ultrastructural changes and apoptosis-like death of Leishmania infantum promastigotes.

The present study aimed to evaluate the antileishmanial effect, the mechanisms of action and the association with miltefosine of Vernonia brasiliana essential oil against Leishmania infantum promastigotes. This essential oil was obtained by hydrodistillation and its chemical composition was determined by gas chromatography-mass spectrometry (GC-MS). The antileishmanial activity against L. infantum promastigotes and cytotoxicity on DH82 cells were evaluated by MTT colorimetric assay. Ultrastructural alterations were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential, in the production of reactive oxygen species, and analysis of apoptotic events were determined by flow cytometry. The association between the essential oil and miltefosine was evaluated using the modified isobologram method. The most abundant component of the essential oil was ß-caryophyllene (21.47 %). Anti-Leishmania assays indicated an IC50 of 39.01 ±â€¯1.080 µg/mL for promastigote forms after 72 h of treatment. The cytotoxic concentration for DH82 cells was 63.13 ±â€¯1.211 µg/mL after 24 h of treatment. The effect against L. infantum was proven through the ultrastructural changes caused by the oil, such as kinetoplast and mitochondrial swelling, vesicles in the flagellar pocket, discontinuity of the nuclear membrane, nuclear fragmentation and condensation, and loss of organelles. It was observed that the oil leads to a decrease in the mitochondrial membrane potential (35.10 %, p = 0.0031), increased reactive oxygen species production, and cell death by late apoptosis (17.60 %, p = 0.020). The combination of the essential oil and miltefosine exhibited an antagonistic effect. This study evidences the antileishmanial action of V. brasiliana essential oil against L. infantum promastigotes.

Boosting immunity to treat parasitic infections: Asaia bacteria expressing a protein from Wolbachia determine M1 macrophage activation and killing of Leishmania protozoans.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of AsaiaWSP, a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with AsaiaWSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, AsaiaWSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to AsaiaWSP. In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation.

Assessing the composition of the plasma membrane of Leishmania (Leishmania) infantum and L. (L.) amazonensis using label-free proteomics.

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.

Leishmanicidal activity of green tea leaves and pomegranate peel extracts on L. infantum.

Leishmania infantum is responsible for the cutaneous and visceral form of this zoonotic disease, which is potentially lethal for humans and has dogs as natural reservoir. In the light of the antiparasitic properties displayed by several natural products, L. infantum promastigotes were exposed to green tea (Camellia sinensis) leaves extract (GTE) and pomegranate (Punica granatum) peel extract (PPE). Both extracts, characterized by NMR and HPLC analysis, inhibited parasite proliferation in a dose-dependent manner, as proved by IC50 evaluation determined by MTT assay.Moreover, the reversibility assay showed that GTE and PPE have an aptotosis-mediated leishmanicidal effect, as evidenced by DNA degradation and confirmed by DNA fragmentation and real-time PCR analyses. Finally, for the first time morphological and ultrastructural alterations induced by a P. granatum extract on Leishmania were shown by the use of light, transmission and scanning electron microscopy.

Effects of nanoemulsions prepared with essential oils of copaiba- and andiroba against Leishmania infantum and Leishmania amazonensis infections.

Plant products are an important source of bioactive agents against parasitic diseases, including leishmaniasis. Among these products, vegetable oils have gained ground in the pharmaceutical field. Here we report the development of nanoemulsions as a delivery system for copaiba and andiroba oils (nanocopa and nanoandi) in order to test their effects on Leishmania infantum and L. amazonensis. The nanocopa and nanoandi had an average particle size of 76.1 and 88.1, respectively with polydispersity index 0.14 to 0.16 and potential zeta -2.54 to -3.9. The data indicated toxic activity of nanocopa and nanoandi against promastigotes of both Leishmania species ultrastructural analyses by scanning electron microscopy revealed that exposition to nanoemulsions induced oval cell shape and retracted flagella. The treatment with nanocopa and nanoandi led to a reduction in L. infantum and L. amazonensis infection levels in macrophage cultures. The nanoemulsions treatment have significant beneficial effects on all the parameters evaluated in lesions induced by L. amazonensis (lesion size, parasite burden and histopathology) on BALB/c mice. The treatment of L. infantum-infected BALB/c mice with nanoemulsions also showed promising results reducing parasite burden in spleen and liver and improving histopathological features.

Increased thiol levels in antimony-resistant Leishmania infantum isolated from treatment-refractory visceral leishmaniasis in Brazil.

BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.

Investigation of the Anti-Leishmania (Leishmania) infantum Activity of Some Natural Sesquiterpene Lactones.

Leishmaniases are neglected infectious diseases caused by parasites of the 'protozoan' genus Leishmania. Depending on the parasite species, different clinical forms are known as cutaneous, muco-cutaneous, and the visceral leishmaniasis (VL). VL is particularly fatal and the therapy presents limitations. In the search for new anti-leishmanial hit compounds, seven natural sesquiterpene lactones were evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum, a pathogen causing VL. The pseudoguaianolides mexicanin I and helenalin acetate demonstrated the highest selectivity and potency against intracellular amastigotes. In addition, promastigotes treated with helenalin acetate were subject to an ultrastructural and biochemical investigation. The lethal action of the compound was investigated by fluorescence-activated cell sorting and related techniques to detect alterations in reactive oxygen species (ROS) content, plasma membrane permeability, and mitochondrial membrane potential. Helenalin acetate significantly reduced the mitochondrial membrane potential and the mitochondrial structural damage was also confirmed by transmission electron microscopy, displaying an intense organelle swelling. No alteration of plasma membrane permeability or ROS content could be detected. Additionally, helenalin acetate significantly increased the production of nitric oxide in peritoneal macrophages, probably potentiating the activity against the intracellular amastigotes. Helenalin acetate could hence be a useful anti-leishmanial scaffold for further optimization studies.

Antileishmanial activity of meroditerpenoids from the macroalgae Cystoseira baccata.

The development of novel drugs for the treatment of leishmaniases continues to be crucial to overcome the severe impacts of these diseases on human and animal health. Several bioactivities have been described in extracts from macroalgae belonging to the Cystoseira genus. However, none of the studies has reported the chemical compounds responsible for the antileishmanial activity observed upon incubation of the parasite with the aforementioned extracts. Thus, this work aimed to isolate and characterize the molecules present in a hexane extract of Cystoseira baccata that was found to be bioactive against Leishmania infantum in a previous screening effort. A bioactivity-guided fractionation of the C. baccata extract was carried out and the inhibitory potential of the isolated compounds was evaluated via the MTT assay against promastigotes and murine macrophages as well as direct counting against intracellular amastigotes. Moreover, the promastigote ultrastructure, DNA fragmentation and changes in the mitochondrial potential were assessed to unravel their mechanism of action. In this process, two antileishmanial meroditerpenoids, (3R)- and (3S)-tetraprenyltoluquinol (1a/1b) and (3R)- and (3S)-tetraprenyltoluquinone (2a/2b), were isolated. Compounds 1 and 2 inhibited the growth of the L. infantum promastigotes (IC50 = 44.9 ± 4.3 and 94.4 ± 10.1 µM, respectively), inducing cytoplasmic vacuolization and the presence of coiled multilamellar structures in mitochondria as well as an intense disruption of the mitochondrial membrane potential. Compound 1 decreased the intracellular infection index (IC50 = 25.0 ± 4.1 µM), while compound 2 eliminated 50% of the intracellular amastigotes at a concentration > 88.0 µM. This work identified compound 2 as a novel metabolite and compound 1 as a biochemical isolated from Cystoseira algae displaying antileishmanial activity. Compound 1 can thus be an interesting scaffold for the development of novel chemotherapeutic molecules for canine and human visceral leishmaniases studies. This work reinforces the evidence of the marine environment as source of novel molecules.

Antiproliferative and ultrastructural effects of phenethylamine derivatives on promastigotes and amastigotes of Leishmania (Leishmania) infantum chagasi.

Leishmania (Leishmania) infantum chagasi is one of the agents that cause visceral leishmaniasis. This disease occurs more frequently in third world countries, such as Brazil. The treatment is arduous, and is dependent on just a few drugs like the antimonial derivatives and amphotericin B. Moreover, these drugs are not only expensive, but they can also cause severe side effects and require long-term treatment. Therefore, it is very important to find new compounds that are effective against leishmaniasis. In the present work we evaluated a new group of synthetic amides against the promastigote and amastigote forms of L. infantum chagasi. The results showed that one of these amides in particular, presented very effective activity against the promastigotes and amastigotes of L. infantum chagasi at low concentrations and it also presented low toxicity for mammal cells, which makes this synthetic amide a promising drug for combating leishmaniasis.

Effective anti-leishmanial activity of minimalist squaramide-based compounds.

In order to evaluate the in vitro leishmanicidal activity of N,N'-Squaramides derivatives, compounds that feature both hydrogen bond donor and acceptor groups and are capable of multiple interactions with complementary sites, against Leishmania infantum, Leishmania braziliensis and Leishmania donovani a series of 18compounds was prepared and assayed on extracellular and intracellular parasite forms. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. Changes in metabolite excretion by 1H-NMR and the ultrastructural alterations occurring in the parasites treated using transmission electron microscopy (TEM), was analyzed. Compounds 1, 7, 11, 14 and 17 were the more active and less toxic. Infection rates showed that the order of effectiveness was 17 > 11 > 14 > 7 for both L. infantum and L. braziliensis and in the same way, the compound 1 for L. donovani. All these compounds have altered the typical structure of the promastigotes, glycosomes and mitochondria. These severe modifications by the compounds are the ultimate reasons for the alterations observed in the excretion products. The Squaramide 17 (3-(butylamino)-4-((3-(dimetilamino)propyl)(methyl)amino)cyclobut-3-en-1,2-dione) was clearly the most efficient of all compounds. The data appear to confirm that the severe modifications generated in organelles such as glycosomes or mitochondria by the compounds are the ultimate reasons for the alterations observed in the excretion products of all species. The activity, stability, low cost of starting materials, and straightforward synthesis make amino squaramides appropriate molecules for the development of an affordable anti-leishmanial agent.

In vitro leishmanicidal activity of 1,3-disubstituted 5-nitroindazoles.

The antiprotozoal activity of some indazole-derived amines (2, 3, 5-8) as well as that of some simple structurally related 3-alkoxy-1-alkyl-5-nitroindazoles (1, 4) against promastigote and amastigote forms of Leishmania infantum and Leishmania braziliensis is reported. In some cases, these compounds showed in vitro activities against the different morphological forms of Leishmania similar to or higher than those of the reference drug glucantime; this fact, along with low unspecific cytotoxicities against macrophages shown by some of them, led to good selectivity indexes (SI). The high efficiency of some 5-nitroindazoles against the mentioned protozoa was confirmed by further in vitro studies on infection rates. Complementary analyses by (1)H NMR of the changes on the metabolites excreted by parasites after treatment with the more active indazole derivatives in many cases showed the decreased excretion of succinate and increased levels of acetate, lactate and alanine, as well as, in some cases, the appearance of glycine and pyruvate as new metabolites. Damage caused by indazoles at the glycosomal or mitochondrial level are consistent with these metabolic changes as well as with the huge ultrastructural alterations observed by transmission electron microscopy (TEM), especially affecting the mitochondria and other cytoplasmic organelles.

Immucillins Impair Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis Multiplication In Vitro.

Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05-500 µM and, when needed, 0.01-50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 µM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 µM for IA and 0.019 µM for IH). IA, IH and SMIH at 10 µM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.

Ultrastructural analysis of Leishmania infantum chagasi promastigotes forms treated in vitro with usnic acid.

Leishmaniasis is considered by the World Health Organization as one of the infectious parasitic diseases endemic of great relevance and a global public health problem. Pentavalent antimonials used for treatment of this disease are limited and new phytochemicals emerge as an alternative to existing treatments, due to the low toxicity and cost reduction. Usnic acid is uniquely found in lichens and is especially abundant in genera such as Alectoria, Cladonia, Evernia, Lecanora, Ramalina, and Usnea. Usnic acid has been shown to exhibit antiviral, antiprotozoal, antiproliferative, anti-inflammatory, and analgesic activity. The aim of this study was to evaluate the antileishmanial activity of usnic acid on Leishmania infantum chagasi promastigotes and the occurrence of drug-induced ultrastructural damage in the parasite. Usnic acid was effective against the promastigote forms (IC50=18.30±2.00 µg/mL). Structural and ultrastructural aspects of parasite were analyzed. Morphological alterations were observed as blebs in cell membrane and shapes given off, increasing the number of cytoplasmic vacuoles, and cellular and mitochondrial swelling, with loss of cell polarity. We concluded that the usnic acid presented antileishmanial activity against promastigote forms of Leishmania infantum chagasi and structural and ultrastructural analysis reinforces its cytotoxicity. Further, in vitro studies are warranted to further evaluate this potential.

In vitro leishmanicidal activity of pyrazole-containing polyamine macrocycles which inhibit the Fe-SOD enzyme of Leishmania infantum and Leishmania braziliensis species.

The in vitro leishmanicidal activity and cytotoxicity of pyrazole-containing macrocyclic polyamines 1-4 was assayed on Leishmania infantum and Leishmania braziliensis species. Compounds 1-4 were more active and less toxic than glucantime and both infection rates and ultrastructural alterations confirmed that 1 and 2 were highly leishmanicidal and induced extensive parasite cell damage. Modifications in the excretion products of parasites treated with 1-3 were also consistent with substantial cytoplasm alterations. Compound 2 was highlighted as a potent inhibitor of Fe-SOD in both species, whereas its effect on human CuZn-SOD was poor. Molecular modelling suggested that 2 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the enzyme`s antioxidant features.

Essential oil from Chenopodium ambrosioides and main components: activity against Leishmania, their mitochondria and other microorganisms.

Chenopodium ambrosioides is an aromatic herb used by native people to treat parasitic diseases. The aim of this work is to compare the in vitro anti-leishmanial activity of the essential oil (EO) from C. ambrosioides and its major components (ascaridole, carvacrol and caryophyllene oxide) and study their mechanism of action and activity against a panel of microorganism. Antileishmanial activity and cytotoxicity of the EO and major components was study. In addition, experiments to elucidate the mechanism of action were perform and activities against other microorganisms (bacteria, fungi and protozoa) were evaluate. All products were active against promastigote and amastigote forms of Leishmania. Ascaridole exhibited the better antileishmanial activity and the EO the highest selectivity index. The exploration of the mechanism suggests that the products cause a breakdown of mitochondrial membrane potential and a modification of redox indexes. Only EO showed antiprotozoal effect against Plasmodium falciparum and Trypanosoma brucei; while no activity against bacteria and fungi was observed. Our results demonstrate the potentialities of EO in cellular and molecular system, which could be consider in future studies to develop new antileishmanial drugs with a wide anti-parasitic spectrum.

Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum.

Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ's activity against L. infantum was confirmed, producing IC50 values of 42.71µg/mL against promastigotes and 6.78µg/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX(®) Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets.

Effect of allicin on promastigotes and intracellular amastigotes of Leishmania donovani and L. infantum.

Anti-leishmanial activity of allicin (=diallyl thiosulphinate) has been tested in vitro against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania infantum. Macrophage infections have been carried out in vitro in the murine cell line J774 and ex vivo with peritoneal macrophages from BALB/c mice with a modified method to isolate metacyclic promastigotes. The compound has shown a significant in vitro effect on the multiplication of promastigotes of L. donovani and L. infantum in a time- and dose-dependent manner. It has been shown for the first time the inhibition of multiplication of intracellular amastigotes of Leishmania by allicin. Inhibitory concentrations of the compound were in the micromolar range (10-30 µM) for both Leishmania species. Antileishmanial effect of allicin apparently was not related to products of degradation of the molecule as assessed by mass spectrometry analysis. Inhibitory activity of allicin against promastigotes and intracellular amastigotes increased when the compound was added to the cultures every 24 h. Two administrations of 5 µM allicin inhibited by ca. 50% the proliferation of Leishmania amastigotes. Low toxicity for mammalian cells of this compound suggests the interest of exploring the value of allicin in combined therapy against leishmanial infections.

Nuclease activity and ultrastructural effects of new sulfonamides with anti-leishmanial and trypanocidal activities.

Our aim was to evaluate the in vitro efficacy of a series of N-benzenesulfonamides of amine substituted aromatic rings, sulfonamides 1-6, against Trypanosoma cruzi and Leishmania spp. and to compare their trypanocidal and leishmanicidal profile. In order to elucidate the probable mechanism of action, the interaction of selected sulfonamides with pUC18 plasmid DNA was investigated by nuclease activity assays. In addition, the cellular targets of these sulfonamides in treated parasites were also searched by transmission and scanning electron microscopy. The most active compounds 4-nitro-N-pyrimidin-2-ylbenzenesulfonamide 1a and 4-chloro-N-5-methyl-thiazol-2-yl-benzenesulfonamide 2d displayed significant in vitro activity against Leishmania spp. promastigotes, without toxicity to J774 macrophages. Selected sulfonamides 1a, 4-nitro-N-pyrazin-2-yl-benzenesulfonamide 1n and 2d were also active against Leishmania infantum intracellular amastigotes. Compounds 1n and 2d showed nuclease activity in the presence of copper salt analogous to our previous results with sulfonamide 1a. Mechanistic data reveal the involvement of a redox process. Evidence for the formation of reactive oxygen species (ROS) responsible for DNA strand scission is provided for sulfonamides 1a, 1n and 2d. Transmission electron microscopic (TEM) analysis of L. infantum promastigotes treated with compounds 1a, 1n and 2d shows an overall cellular disorganization effects which are mainly addressed to DNA bearing structures such as the nucleus, mitochondria and kinetoplast. Disruption of double nuclear membrane and loss of cellular integrity along with accumulation of cytoplasmic electrodense bodies were also frequently observed.