Emergent canine visceral leishmaniasis in Argentina: Comparative diagnostics and relevance to proliferation of human disease.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL. METHODOLOGY/PRINCIPAL FINDINGS: Sampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis. CONCLUSIONS/SIGNIFICANCE: Seroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.

Proteomic analysis reveals differentially abundant proteins probably involved in the virulence of amastigote and promastigote forms of Leishmania infantum.

Owing to the importance and clinical diversity of Leishmania infantum, studying its virulence factors is promising for understanding the relationship between parasites and hosts. In the present study, differentially abundant proteins from strains with different degrees of virulence in promastigote and amastigote forms were compared using two quantitative proteomics techniques, differential gel electrophoresis and isobaric mass tag labeling, followed by identification by mass spectrometry. A total of 142 proteins were identified: 96 upregulated and 46 downregulated proteins in the most virulent strain compared to less virulent. The interaction between the proteins identified in each evolutionary form was predicted. The results showed that in the amastigote form of the most virulent strain, there was a large group of proteins related to glycolysis, heat shock, and ribosomal proteins, whereas in the promastigote form, the group consisted of stress response, heat shock, and ribosomal proteins. In addition, biological processes related to metabolic pathways, ribosomes, and oxidative phosphorylation were enriched in the most virulent strain (BH400). Finally, we noted several proteins previously found to play important roles in L. infantum infection, which showed increased abundance in the virulent strain, such as ribosomal proteins, HSP70, enolase, fructose 1,6-biphosphate aldolase, peroxidoxin, and tryparedoxin peroxidase, many of which interact with each other.

Antileishmanial activity of fullerol and its liposomal formulation in experimental models of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a systemic parasitic disease that leads to high rates of morbidity and mortality in humans worldwide. There is a great need to develop new drugs and novel strategies to make chemotherapy for this disease more efficacious and well tolerated. Recent reports on the immunomodulatory effects and the low toxicity of the spherical carbon nanostructure fullerol led us to investigate in vitro and in vivo antileishmanial activity in free and encapsulated forms in liposomes. When assayed against intramacrophagic Leishmania amastigotes, fullerol showed a dose-dependent reduction of the infection index with IC50 of 0.042 mg/mL. When given daily by i.p. route for 20 days (0.05 mg/kg/d) in a murine model of acute VL, fullerol promoted significant reduction in the liver parasite load. To improve the delivery of fullerol to the infection sites, liposomal formulations were prepared by the dehydration-rehydration method. When evaluated in the acute VL model, liposomal fullerol (Lip-Ful) formulations given i.p. at 0.05 and 0.2 mg/kg with 4-days intervals were more effective than the free form, with significant parasite reductions in both liver and spleen. Lip-Ful at 0.2 mg/kg promoted complete parasite elimination in the liver. The antileishmanial activity of Lip-Ful was further confirmed in a chronic model of VL. Lip-Ful was also found to induce secretion of pro-inflammatory TNF-α, IFN-γ and IL-1ß cytokines. In conclusion, this work reports for the first time the antileishmanial activity of fullerol and introduces an innovative approach for treatment of VL based on the association of this nanostructure with liposomes.

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.

Liver infusion tryptose (LIT): the best choice for growth, viability, and infectivity of Leishmania infantum parasites.

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace's insect cell culture medium (Grace's), liver infusion tryptose (LIT), and Schneider's insect medium (Schneider's), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider's with or without FCS when compared to that in Grace's. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace's promoted higher percentage of infected macrophages and parasite load compared with Schneider's media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.

Phenotype evaluation of human and canine isolates of Leishmania infantum.

Human visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in countries of South and Central America are caused by Leishmania infantum and has been endemic in Brazil for several years. The parasite biodiversity as well as the pharmacologic properties of drugs and the host species, are involved in the efficacy or inefficacy of leishmaniasis treatments. Although there are substantial number of reports describing the genetic characterization of the clinical field isolates of L. infantum,the phenotypic parameters have been less studied. In this study isolates from human and canine leishmaniasis (Hum1 and Can1) obtained in Campinas, São Paulo state, Brazil were identified as L. infantum. The Hum1 and Can1 isolates exhibited typical promastigote growth pattern. Regarding morphological features Can1 isolate differed in cell size. The infectivity in vitro of both isolatesis lower compared to the reference strain of L. infantum. Moreover, the in vivo infectivity of the three parasites is similar in Balb/c mice. The Hum1 isolate is more sensitive to leishmanial drugs (amphotericin B, miltefosine and glucantime) than the Can1 isolate when inside human macrophages, but not when inside canine macrophages. These findings indicated that L. infantum isolates differs in some phenotypic characteristics.

Parasitological and immunological evaluation of a novel chemotherapeutic agent against visceral leishmaniasis.

AIMS: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. METHODS AND RESULTS: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4'-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. CONCLUSION: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent against VL.

Experimental infections and co-infections with Leishmania braziliensis and Leishmania infantum in two sand fly species, Lutzomyia migonei and Lutzomyia longipalpis.

Leishmaniases are neglected tropical diseases and Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis are the most important causative agents of leishmaniases in the New World. These two parasite species may co-circulate in a given endemic area but their interactions in the vector have not been studied yet. We conducted experimental infections using both single infections and co-infections to compare the development of L. (L.) infantum (OGVL/mCherry) and L. (V.) braziliensis (XB29/GFP) in Lutzomyia longipalpis and Lutzomyia migonei. Parasite labelling by different fluorescein proteins enabled studying interspecific competition and localization of different parasite species during co-infections. Both Leishmania species completed their life cycle, producing infective forms in both sand fly species studied. The same happens in the co infections, demonstrating that the two parasites conclude their development and do not compete with each other. However, infections produced by L. (L.) infantum reached higher rates and grew more vigorously, as compared to L. (V.) braziliensis. In late-stage infections, L. (L.) infantum was present in all midgut regions, showing typical suprapylarian type of development, whereas L. (V.) braziliensis was concentrated in the hindgut and the abdominal midgut (peripylarian development). We concluded that both Lu. migonei and Lu. longipalpis are equally susceptible vectors for L. (L.) infantum, in laboratory colonies. In relation to L. (V.) braziliensis, Lu. migonei appears to be more susceptible to this parasite than Lu. longipalpis.

Leishmania dual-specificity tyrosine-regulated kinase 1 (DYRK1) is required for sustaining Leishmania stationary phase phenotype.

Although the multiplicative and growth-arrested states play key roles in Leishmania development, the regulators of these transitions are largely unknown. In an attempt to gain a better understanding of these processes, we characterised one member of a family of protein kinases with dual specificity, LinDYRK1, which acts as a stasis regulator in other organisms. LinDYRK1 overexpressing parasites displayed a decrease in proliferation and in cell cycle re-entry of arrested cells. Parasites lacking LinDYRK1 displayed distinct fitness phenotypes in logarithmic and stationary growth phases. In logarithmic growth phase, LinDYRK1-/- parasites proliferated better than control lines, supporting a role of this kinase in stasis, while in stationary growth phase, LinDYRK1-/- parasites had important defects as they rounded up, accumulated vacuoles and lipid bodies and displayed subtle but consistent differences in lipid composition. Moreover, they expressed less metacyclic-enriched transcripts, displayed increased sensitivity to complement lysis and a significant reduction in survival within peritoneal macrophages. The distinct LinDYRK1-/- growth phase phenotypes were mirrored by the distinct LinDYRK1 localisations in logarithmic (mainly in flagellar pocket area and endosomes) and late stationary phase (mitochondrion). Overall, this work provides first evidence for the role of a DYRK family member in sustaining promastigote stationary phase phenotype and infectivity.

Thymic Microenvironment Is Modified by Malnutrition and Leishmania infantum Infection.

Detrimental effects of malnutrition on immune responses to pathogens have long been recognized and it is considered a main risk factor for various infectious diseases, including visceral leishmaniasis (VL). Thymus is a target of both malnutrition and infection, but its role in the immune response to Leishmania infantum in malnourished individuals is barely studied. Because we previously observed thymic atrophy and significant reduction in cellularity and chemokine levels in malnourished mice infected with L. infantum, we postulated that the thymic microenvironment is severely compromised in those animals. To test this, we analyzed the microarchitecture of the organ and measured the protein abundance in its interstitial space in malnourished BALB/c mice infected or not with L. infantum. Malnourished-infected animals exhibited a significant reduction of the thymic cortex:medulla ratio and altered abundance of proteins secreted in the thymic interstitial fluid. Eighty-one percent of identified proteins are secreted by exosomes and malnourished-infected mice showed significant decrease in exosomal proteins, suggesting that exosomal carrier system, and therefore intrathymic communication, is dysregulated in those animals. Malnourished-infected mice also exhibited a significant increase in the abundance of proteins involved in lipid metabolism and tricarboxylic acid cycle, suggestive of a non-proliferative microenvironment. Accordingly, flow cytometry analysis revealed decreased proliferation of single positive and double positive T cells in those animals. Together, the reduced cortical area, decreased proliferation, and altered protein abundance suggest a dysfunctional thymic microenvironment where T cell migration, proliferation, and maturation are compromised, contributing for the thymic atrophy observed in malnourished animals. All these alterations could affect the control of the local and systemic infection, resulting in an impaired response to L. infantum infection.

Leishmania infantum arginase: biochemical characterization and inhibition by naturally occurring phenolic substances.

Inhibition of Leishmania arginase leads to a decrease in parasite growth and infectivity and thus represents an attractive therapeutic strategy. We evaluated the inhibitory potential of selected naturally occurring phenolic substances on Leishmania infantum arginase (ARGLi) and investigated their antileishmanial activity in vivo. ARGLi exhibited a Vmax of 0.28 ± 0.016 mM/min and a Km of 5.1 ± 1.1 mM for L-arginine. The phenylpropanoids rosmarinic acid and caffeic acid (100 µM) showed percentages of inhibition of 71.48 ± 0.85% and 56.98 ± 5.51%, respectively. Moreover, rosmarinic acid and caffeic acid displayed the greatest effects against L. infantum with IC50 values of 57.3 ± 2.65 and 60.8 ± 11 µM for promastigotes, and 7.9 ± 1.7 and 21.9 ± 5.0 µM for intracellular amastigotes, respectively. Only caffeic acid significantly increased nitric oxide production by infected macrophages. Altogether, our results broaden the current spectrum of known arginase inhibitors and revealed promising drug candidates for the therapy of visceral leishmaniasis.

Lutzomyia longipalpis TGF-ß Has a Role in Leishmania infantum chagasi Survival in the Vector.

Despite the increasing number of studies concerning insect immunity, Lutzomyia longipalpis immune responses in the presence of Leishmania infantum chagasi infection has not been widely investigated. The few available studies analyzed the role of the Toll and IMD pathways involved in response against Leishmania and microbial infections. Nevertheless, effector molecules responsible for controlling sand fly infections have not been identified. In the present study we investigated the role a signal transduction pathway, the Transforming Growth Factor-beta (TGF-ß) pathway, on the interrelation between L. longipalpis and L. i. chagasi. We identified an L. longipalpis homolog belonging to the multifunctional cytokine TGF-ß gene family (LlTGF-ß), which is closely related to the activin/inhibin subfamily and potentially involved in responses to infections. We investigated this gene expression through the insect development and in adult flies infected with L. i. chagasi. Our results showed that LlTGF-ß was expressed in all L. longipalpis developmental stages and was upregulated at the third day post L. i. chagasi infection, when protein levels were also higher as compared to uninfected insects. At this point blood digestion is finished and parasites are in close contact with the insect gut. In addition, we investigated the role of LlTGF-ß on L. longipalpis infection by L. i. chagasi using either gene silencing by RNAi or pathway inactivation by addition of the TGF-ß receptor inhibitor SB431542. The blockage of the LlTGF-ß pathway increased significantly antimicrobial peptides expression and nitric oxide levels in the insect gut, as expected. Both methods led to a decreased L. i. chagasi infection. Our results show that inactivation of the L. longipalpis TGF-ß signal transduction pathway reduce L. i. chagasi survival, therefore suggesting that under natural conditions the parasite benefits from the insect LlTGF-ß pathway, as already seen in Plamodium infection of mosquitoes.

Evaluation of the in vitro and in vivo antileishmanial activity of a chloroquinolin derivative against Leishmania species capable of causing tegumentary and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, since the currently used drugs are toxic and/or have high costs. In addition, parasite resistance has increased. As a consequence, in this study, a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine or GF1059, was in vitro and in vivo tested against Leishmania parasites. Experiments were performed to evaluate in vitro antileishmanial activity and cytotoxicity, as well as the treatment of infected macrophages and the inhibition of infection using pre-treated parasites. This study also investigated the GF1059 mechanism of action in L. amazonensis. Results showed that the compound was highly effective against L. infantum and L. amazonensis, presenting a selectivity index of 154.6 and 86.4, respectively, against promastigotes and of 137.6 and 74.3, respectively, against amastigotes. GF1059 was also effective in the treatment of infected macrophages and inhibited the infection of these cells when parasites were pre-incubated with it. The molecule also induced changes in the parasites' mitochondrial membrane potential and cell integrity, and caused an increase in the reactive oxygen species production in L. amazonensis. Experiments performed in BALB/c mice, which had been previously infected with L. amazonensis promastigotes, and thus treated with GF1059, showed that these animals presented significant reductions in the parasite load when the infected tissue, spleen, liver, and draining lymph node were evaluated. GF1059-treated mice presented both lower parasitism and low levels of enzymatic markers, as compared to those receiving amphotericin B, which was used as control. In conclusion, data suggested that GF1059 can be considered a possible therapeutic target to be tested against leishmaniasis.

Wolbachia introduction into Lutzomyia longipalpis (Diptera: Psychodidae) cell lines and its effects on immune-related gene expression and interaction with Leishmania infantum.

BACKGROUND: The leishmaniases are important neglected diseases caused by Leishmania spp. which are transmitted by sand flies, Lutzomyia longipalpis being the main vector of visceral leishmaniasis in the Americas. The methodologies for leishmaniasis control are not efficient, causing 1.5 million reported cases annually worldwide, therefore showing the need for development of novel strategies and interventions to control transmission of the disease. The bacterium Wolbachia pipientis is being used to control viruses transmitted by mosquitoes, such as dengue and Zika, and its introduction in disease vectors has been effective against parasites such as Plasmodium. Here we show the first successful establishment of Wolbachia into two different embryonic cell lines from L. longipalpis, LL-5 and Lulo, and analysed its effects on the sand fly innate immune system, followed by in vitro Leishmania infantum interaction. RESULTS: Our results show that LL-5 cells respond to wMel and wMelPop-CLA strains within the first 72 h post-infection, through the expression of antimicrobial peptides and inducible nitric oxide synthase resulting in a decrease of Wolbachia detection in the early stages of infection. In subsequent passages, the wMel strain was not able to infect any of the sand fly cell lines while the wMelPop-CLA strain was able to stably infect Lulo cells and LL-5 at lower levels. In Wolbachia stably infected cells, the expression of immune-related genes involved with downregulation of the IMD, Toll and Jak-Stat innate immune pathways was significantly decreased, in comparison with the uninfected control, suggesting immune activation upon Wolbachia transinfection. Furthermore, Wolbachia transinfection did not promote a negative effect on parasite load in those cells. CONCLUSIONS: Initial strong immune responses of LL5 cells might explain the inefficiency of stable infections in these cells while we found that Lulo cells are more permissive to infection with Wolbachia causing an effect on the cell immune system, but not against in vitro L. infantum interaction. This establishes Lulo cells as a good system for the adaptation of Wolbachia in L. longipalpis.

In vitro antileishmanial activity of leaf and stem extracts of seven Brazilian plant species.

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis is a parasitic disease that affects people all over the world. The number of cases of leishmaniasis is increasing and the drugs used for its treatment are toxic and not always effective. The recognition of the global nature of this disease and its direct or indirect effects on health economics and actions focuses attention on the development of new therapeutic options. In Brazil, this parasitic disease is endemic in many regions. The plants used by the population against leishmaniasis can be good starting points in the search of new lead compounds for antileishmanial drugs. AIM OF THE STUDY: The aim of the present study was to investigate the antileishmanial activity of extracts from leaves and stems of seven Brazilian plant species used by the population to treat leishmaniasis, and symptoms that might be related to Leishmania infections. MATERIALS AND METHODS: Twenty two extracts from seven plants belonging to five different botanical families were prepared by different methods and evaluated for their effect on the viability of promastigote forms of Leishmania infantum (MHOM/BR/1967/BH46) using the resazurin-based colorimetric assay. The extracts were considered active when they inhibited the growth of promastigotes in a percentage greater than or equal to 50% at 100 and 200 µg/mL. The active samples were further investigated to determine IC50, CC50 and SI values against promastigote forms of L. infantum. The active and non-cytotoxic extracts (SI> 10) were evaluated against amastigote forms of L. infantum. In addition, the active extracts against the amastigote forms were analyzed by TLC and HPLC, while the EtOAc extract of stems from Aspidosperma tomentosum was also evaluated by GC/MS. RESULTS: Among the twenty two extracts evaluated, two were considered active against L. infantum. The EtOH extract of leaves from Dyospiros hispida (IC50 55.48 ±â€¯2.77 µg/mL and IC50 80.63 ±â€¯13.17 µg/mL, respectively) and the EtOAc extract of stems from Aspidosperma tomentosum (IC50 9.70 ±â€¯2.82 µg/mL and IC50 15.88 ±â€¯1.53 µg/mL, respectively) inhibited significantly the growth of promastigote and amastigote forms of L. infantum. Some extracts, although active in the initial screening, were considered toxic since the SI was lower than 10. In TLC and HPLC analysis the leaf extract of Dyospiros hispida showed the presence of anthraquinones, terpenes and saponins, and in the EtOAc extract of stems from Aspidosperma tomentosum alkaloids and flavonoids were detected. In addition, in the latter extract the indole alkaloids uleine and dasycarpidone could be identified by GC/MS. CONCLUSIONS: The ethnopharmacological data of Aspidosperma tomentosum and Dyospiros hispida in part support the results found in the biological models used. Extracts of Aspidosperma tomentosum and Dyospiros hispida presented promising results against L. infantum.

In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum.

OBJECTIVE: In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root (AEPa) on Leishmania infantum proliferation, morphology, and the driving mechanism in leishmanicidal activity and modulatory action on macrophages. METHODS: L. infantum promastigotes were treated with 50 and 100 µg/mL AEPa for 72 h and then antipromastigote assay was performed by counts in a Newbauer chamber, morphological changes were analyzed by transmission electron microscopy and the mechanism of the leishmanicidal activity was detected. In addition, macrophages were infected with L. infantum and were used to evaluate anti-amastigote activity of AEPa and effects of AEPa on cytokine secretion after 72-hour treatment. RESULTS: Treatment with AEPa reduced the numbers of L. infantum promastigotes (50% inhibitory concentration (IC50) = 65.9 µg/mL; selectivity index (SI) = 22.1) and amastigotes (IC50 = 37.9 µg/mL; SI = 38.5) compared with the untreated control. Amphotericin B reduced 100% of the promastigote numbers after 72 h of treatment (IC50 = 0.2 µg/mL). AEPa induced several morphological changes and increased the production of reactive oxygen species and apoptotic death in promastigotes after treating for 72 h. AEPa (100 µg/mL) promoted tumor necrosis factor-α secretion in macrophages infected with L. infantum after 72 h of treatment, but did not induce an increase in this cytokine in noninfected macrophages. In addition, AEPa showed no cytotoxic effect on J774-A1 cells (50% cytotoxic concentration >1000 µg/mL). CONCLUSION: AEPa presented antileishmanial activity against the promastigotes and amastigotes of L. infantum without macrophage cytotoxicity; these results show that natural products such as P. angulata have leishmanicidal potential and in the future may be an alternative treatment for leishmaniasis.

Growth arrested live-attenuated Leishmania infantum KHARON1 null mutants display cytokinesis defect and protective immunity in mice.

There is no safe and efficacious vaccine against human leishmaniasis available and live attenuated vaccines have been used as a prophylactic alternative against the disease. In order to obtain an attenuated Leishmania parasite for vaccine purposes, we generated L. infantum KHARON1 (KH1) null mutants (ΔLikh1). This gene was previously associated with growth defects in L. mexicana. ΔLikh1 was obtained and confirmed by PCR, qPCR and Southern blot. We also generate a KH1 complemented line with the introduction of episomal copies of KH1. Although ΔLikh1 promastigote forms exhibited a growth pattern similar to the wild-type line, they differ in morphology without affecting parasite viability. L. infantum KH1-deficient amastigotes were unable to sustain experimental infection in macrophages, forming multinucleate cells which was confirmed by in vivo attenuation phenotype. The cell cycle analysis of ΔLikh1 amastigotes showed arrested cells at G2/M phase. ΔLikh1-immunized mice presented reduced parasite burden upon challenging with virulent L. infantum, when compared to naïve mice. An effect associated with increased Li SLA-specific IgG serum levels and IL-17 production. Thus, ΔLikh1 parasites present an infective-attenuated phenotype due to a cytokinesis defect, whereas it induces immunity against visceral leishmaniasis in mouse model, being a candidate for antileishmanial vaccine purposes.

New heterobimetallic ferrocenyl derivatives: Evaluation of their potential as prospective agents against trypanosomatid parasites and Mycobacterium tuberculosis.

Searching for prospective agents against infectious diseases, four new ferrocenyl derivatives, [M(L)(dppf)4](PF6), with M = Pd(II) or Pt(II), dppf = 1,1'-bis(dipheny1phosphino) ferrocene and HL = tropolone (HTrop) or hinokitiol (HHino), were synthesized and characterized. Complexes and ligands were evaluated against the bloodstream form of T. brucei, L. infantum amastigotes, M. tuberculosis (MTB) sensitive strain and MTB clinical isolates. Complexes showed a significant increase of the anti-T. brucei activity with respect to the free ligands (>28- and >46-fold for Trop and 6- and 22-fold for Hino coordinated to Pt-dppf and Pd-dppf, respectively), yielding IC50 values < 5 µM. The complexes proved to be more potent than the antitrypanosomal drug Nifurtimox. The new ferrocenyl derivatives were more selective towards the parasite than the free ligands. The Pt compounds were less toxic on J774 murine macrophages (mammalian cell model), than the Pd ones, showing selectivity index values (SI = IC50 murine macrophage/IC50T. brucei) up to 23. Generation of the {M-dppf} compounds lead to a slightly positive impact on the anti-leishmanial potency. Although the ferrocenyl derivatives were more active on sensitive MTB than the free ligands (MIC90 = 9.88-14.73 µM), they showed low selectivity towards the pathogen. Related to the mechanism of action, the antiparasitic effect cannot be ascribed to an interference of the compounds with the thiol-redox homeostasis of the pathogen. Fluorescence measurements pointed at DNA as a probable target of the new compounds. [Pt(Trop)(dppf)](PF6) and [Pt(Hino)(dppf)](PF6) could be considered prospective anti-T. brucei agents that deserve further research.

Antiparasitic activity of new gibbilimbol analogues and SAR analysis through efficiency and statistical methods.

Chagas' disease and leishmaniasis are parasitic infections enrolled among the neglected tropical diseases, which urge for new treatments. In the search for new chemical entities as prototypes, gibbilimbols A/B have shown antiparasitic activity against Trypanosoma cruzi and Leishmania infantum, and then a set of analogues (LINS03 series) of this natural product were synthesized and evaluated in vitro against the parasites. In the present paper we reported five new compounds with activity against these protozoan parasites, and quite low cytotoxicity. Moreover, the interference of plasma membrane permeability of these analogues were also evaluated. We found that [(4-methoxyphenyl)methyl]octylamine (4) was noteworthy due to its high activity against the amastigote form of both parasites (IC50 1.3-5.8 µM) and good selectivity index. In order to unveil the SAR for this chemotype, we also presented a group efficiency analysis and PCA and HCA study, which indicated that the methoxyl provides good activity with lower cytotoxicity to mammalian cells. The results from SAR analyses suggest different mechanisms of action between the neutral and basic compounds. In summary, the analogues represent important activity against these parasites and must be prototypes for further exploitation.

Selective effects of Euterpe oleracea (açai) on Leishmania (Leishmania) amazonensis and Leishmania infantum.

Leishmania (Leishmania) amazonensis and Leishmania infantum (=Leishmania chagasi) are protozoa that cause American cutaneous and visceral leishmaniasis, respectively. These diseases show a high incidence in developing countries such as Brazil. The treatments used for leishmaniasis are still limited due to their high cost and toxicity. Currently, some natural products are considered an important alternative source of new leishmanicidal agents. Euterpe oleracea Martius, a palm producing black fruits, is frequently consumed in the Amazon region, as a juice, known as açai, with potent antioxidant, anti-inflammatory and anticonvulsant properties. Interestingly, the biological activity of clarified açai juice (EO) on L. (L.) amazonensis and L. infantum (=L. chagasi) is unknown. Therefore, the mechanism of anti-leishmanial action of EO has been evaluated on L. (L.) amazonensis and L. infantum (=L. chagasi). EO reduced the number of promastigotes and caused morphological alterations, increased the production of reactive oxygen species (ROS) and induced cell death phenotypes probably seems by apoptosis in the promastigotes of L. (L.) amazonensis (IC50 = 1:40) and L. infantum (=L. chagasi) (IC50 = 1:38). EO also presented activity against Leishmania amastigotes. Treatment with EO for 72 h strongly reduced IL-17 cytokine levels at all tested concentrations and decreased the number of intracellular amastigotes in macrophages infected with L. (L.) amazonensis (IC50 = 1:30) and L. infantum (=L. chagasi) (IC50 = 1:38). Additionally, no cytotoxic effect was observed in murine macrophages treated with EO (72 h - CC50 > 1:1). Our results demonstrated that EO has leishmanicidal activity against two different species that cause American visceral and cutaneous leishmaniasis without cytotoxic effects for the host cell.