Antileishmanial activity of a dillapiole derivative obtained from Piper aduncum L. (Piperaceae).

Cutaneous leishmaniasis (CL) is considered a public health problem. Current treatments have disadvantages because they are invasive and have serious side effects, and thus there is a need for research into new, more effective pharmacological alternatives. Plants are promising sources of bioactive substances, and new analogues can be obtained through chemical reactions. The present study aimed to evaluate the antileishmanial effects of the analog dillapiole n-butyl ether (DBE) extracted from Piper aduncum leaves. The cytotoxic potential of DBE was evaluated at concentrations of 15.62 to 500 µM in peritoneal macrophages for 48 h, and in RAW 264.7 macrophages for 72 h using a dose-response method. The antileishmanial activity in L. amazonensis promastigotes used concentrations of 0.2 to 4.5 µM for 24, 48 and 72 h and the quantification of the cellular infection rate used a concentration of 4.5 µM of DBE against the amastigote forms internalized in macrophages for 24 h and 48 h. Nitric oxide was quantified from macrophages previously treated with DBE for 24 h and 48 h. The dosage of reactive oxygen species used a concentration of 4.5 µM of DBE incubated together with dichlorofluorescein acetate for 1, 3, 6, and 24 h. For the molecular modeling of DBE, the Leishmania protein, available in the "Protein Data Bank" database, was used. The studied molecule was not toxic to cells and presented a CC50 of 413 µM in peritoneal macrophages and 373.5 µM in RAW 264.7. The analogue inhibited promastigote forms of L. amazonensis with an IC50 of 1.6 µM for 72 h. DBE presented an infection rate of 17% and 12%, dillapiole of 24% and 14% and Pentacarinat® of 10% and 9% over 48 h. DBE demonstrated a binding energy of -7.8 for the U53 enzyme. It is concluded that the analogue showed promising antileishmanial activity for future in vivo tests.

Microemulsions strongly promoted the activity of α-bisabolol against different Leishmania species and its skin permeation.

This study aimed to develop microemulsions (MEs) containing α-bisabolol for the topical treatment of cutaneous leishmaniasis (CL). Initially, pseudoternary phase diagrams were developed using α-bisabolol as the oil phase, Eumulgin® CO 40 as the surfactant, Polymol® HE as the co-surfactant, and distilled water as the aqueous phase. Two transparent liquid systems (TLS) containing 5% of α-bisabolol were selected and characterized (F5E25 and F5EP25). Next, skin permeation and retention assays were performed using Franz cells. The interaction of the formulation with the stratum corneum (SC) was evaluated using the FTIR technique. The cytotoxicity was evaluated in murine peritoneal macrophages. Finally, the antileishmanial activity of microemulsions was determined in promastigotes and amastigotes of L. amazonensis (strain MHOM/BR/77/LTB 0016). As a result, the selected formulations showed isotropy, nanometric size (below 25 nm), Newtonian behavior and pH ranging from 6.5 to 6.9. The MEs achieved a 2.5-fold increase in the flux and skin-permeated amount of α-bisabolol. ATR-FTIR results showed that microemulsions promoted fluidization and extraction of lipids and proteins of the stratum corneum, increasing the diffusion coefficient and partition coefficient of the drug in the skin. Additionally, F5E25 and F5EP25 showed higher activity against promastigotes (IC50 13.27 and 18.29, respectively) compared to unencapsulated α-bisabolol (IC50 53.8). Furthermore, F5E25 and F5EP25 also showed antileishmanial activity against intracellular amastigotes of L. amazonensis, with IC50 50 times lower than free α-bisabolol and high selectivity index (up to 15). Therefore, the systems obtained are favorable to topical administration, with significant antileishmanial activity against L. amazonensis promastigotes and amastigotes, being a promising system for future in vivo trials.

Mevalonate kinase of Leishmania donovani promotes its survival and plays a pivotal role in pathogenesis.

The infectivity of Leishmania is determined by its ability to invade and evade host and its thriving capacity within the macrophage. Our study revealed the role of Leishmania donovani mevalonate kinase (MVK), an enzyme of mevalonate pathway in visceral leishmaniasis pathogenesis. Peritoneal exudate cells (PEC)-derived macrophages from BALB/c mice were infected with wild type (WT), MVK over expressing (MVK OE) and knockdown (KD) parasites and MVK OE parasites were found to be more infective than WT and MVK KD parasites. Incubation of macrophages with MVK OE parasites declined inducible nitric oxide synthase (iNOS) expression as well as nitric oxide (NO) production, both by 2 times in comparison to WT parasites. Moreover, ∼3 fold increase in Arginase1 expression indicated that MVK might induce polarization of macrophage towards M2, favouring the survival of parasite within the macrophages. Post 24 h infection of the macrophages with mutant strains, the levels of different cytokines (TNF-α, IL-12, IL-10 and IFN-γ) were measured. Infection of macrophages with MVK OE parasites showed an increase in the level of anti-inflammatory cytokine: IL-10 while infection with MVK KD parasites exhibited an increase in the level of pro-inflammatory cytokines: TNF-α, IL-12, and IFN-γ. Hence, Leishmania donovani mevalonate kinase (LdMVK) modulates macrophage functions and has a significant role in pathogenesis.

Susceptibility of Leishmania to novel pentavalent organometallics: Investigating impact on DNA and membrane integrity in antimony(III)-sensitive and -resistant strains.

The aim the present study was to investigate the impact of novel pentavalent organobismuth and organoantimony complexes on membrane integrity and their interaction with DNA, activity against Sb(III)-sensitive and -resistant Leishmania strains and toxicity in mammalian peritoneal macrophages. Ph3M(L)2 type complexes were synthesized, where M = Sb(V) or Bi(V) and L = deprotonated 3-(dimethylamino)benzoic acid or 2-acetylbenzoic acid. Both organobismuth(V) and organoantimony(V) complexes exhibited efficacy at micromolar concentrations against Leishmania amazonensis and L. infantum but only the later ones demonstrated biocompatibility. Ph3Sb(L1)2 and Ph3Bi(L1)2 demonstrated distinct susceptibility profiles compared to inorganic Sb(III)-resistant strains of MRPA-overexpressing L. amazonensis and AQP1-mutated L. guyanensis. These complexes were able to permeate the cell membrane and interact with the Leishmania DNA, suggesting that this effect may contribute to the parasite growth inhibition via apoptosis. Taken altogether, our data substantiate the notion of a distinct mechanism of uptake pathway and action in Leishmania for these organometallic complexes, distinguishing them from the conventional inorganic antimonial drugs.

N6-methyltubercidin gives sterile cure in a cutaneous Leishmania amazonensis mouse model.

Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-ß-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum. Herein, we investigated its in vitro and in vivo activity against L. amazonensis. CL5564 was 6.5-fold (P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg−1, intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg−1, while the combination (10 + 40 mg kg−1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives.

Comparison of biosynthetic zinc oxide nanoparticle and glucantime cytotoxic effects on Leishmania major (MRHO/IR/75/ER).

Currently, zinc oxide (ZnO) particles are used in nanotechnology to destroy a wide range of microorganisms. Although pentavalent antimony compounds are used as antileishmanial drugs, they are associated with several limitations and side effects. Therefore, it is always desirable to try to find new and effective treatments. The aim of this research is to determine the antileishmanial effect of ZnO particles in comparison to the Antimoan Meglumine compound on promastigotes and amastigotes of Leishmania major (MRHO/IR/75/ER). After the extraction and purification of macrophages from the peritoneal cavity of C57BL/6 mice, L. major parasites were cultured in Roswell Park Memorial Institute-1640 culture medium containing fetal bovine serum (FBS) 10% and antibiotic. In this experimental study, the effect of different concentrations of nanoparticles was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric method, in comparison to the glucantime on promastigotes, amastigotes and healthy macrophages in the culture medium. The amount of light absorption of the obtained color from the regeneration of tetrazolium salt to the product color of formazan by the parasite was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the IC50 value was calculated. IC50 after 24 h of incubation was calculated as IC50 = 358.6 µg/mL. The results showed, that the efficacy of ZnO nanoparticles was favorable and dose-dependent. The concentration of 500 µg/mL of ZnO nanoparticles induced 84.67% apoptosis after 72. Also, the toxicity of nanoparticles was less than the drug. Nanoparticles exert their cytotoxic effects by inducing apoptosis. They can be suitable candidates in the pharmaceutical industry in the future.

The Impact of the CTHRSSVVC Peptide Upon Experimental Models of Trypanosoma cruzi Infection.

Chagas disease (CD), caused by the hemoflagellate protozoan Trypanosoma cruzi, affects more than six million people worldwide and presents an unsatisfactory therapy, based on two nitroderivatives, introduced in clinical medicine for decades. The synthetic peptide, with CTHRSSVVC sequence (PepA), mimics the CD163 and TNF-α tripeptide "RSS" motif and binds to atheromatous plaques in carotid biopsies of human patients, spleen tissues, and a low-density lipoprotein receptor knockout (LDLr-/-) mouse model of atherosclerosis. CD163 receptor is present on monocytes, macrophages, and neutrophils, acting as a regulator of acute-phase processes and modulating aspects of the inflammatory response and the establishment of infections. Due to the potential theranostic role of PepA, our aim was to investigate its effect upon T. cruzi infection in vitro and in vivo. PepA and two other peptides with shuffled sequences were assayed upon different binomials of host cell/parasite, including professional [as peritoneal mouse macrophages (PMM)] and non-professional phagocytes [primary cultures of cardiac cells (CM)], under different protocols. Also, their impact was further addressed in vivo using a mouse model of acute experimental Chagas disease. Our in-vitro findings demonstrate that PepA and PepB (the peptide with random sequence retaining the "RS" sequence) reduced the intracellular parasitism of the PMM but were inactive during the infection of cardiac cells. Another set of in-vitro and in-vivo studies showed that they do not display a trypanocidal effect on bloodstream trypomastigotes nor exhibit in-vivo efficacy when administered after the parasite inoculation. Our data report the in-vitro activity of PepA and PepB upon the infection of PMM by T. cruzi, possibly triggering the microbicidal arsenal of the host professional phagocytes, capable of controlling parasitic invasion and proliferation.

The Ecto-5'nucleotidase/CD73 Mediates Leishmania amazonensis Survival in Macrophages.

Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals in vivo. Extracellularly released nucleotides such as ATP are rapidly hydrolyzed to adenosine by the coordinated ectonucleotidase activities of CD39 and CD73. Leishmania is an obligate intracellular parasite of macrophages and capable of modulating host immune response in order to survive and multiply within host cells. In this study, the activity of CD73 induced by Leishmania amazonensis in infected macrophages has been investigated and correlated with parasite survival and infection in vitro. For this, the expression of CD39 and CD73, by flow cytometry, in murine peritoneal macrophages infected with metacyclic promastigotes of L. amazonensis has been analyzed. Our results showed that L. amazonensis-infected macrophages, unlike LPS-treated macrophages, increased CD73 expression. It was also noted that when CD73 enzymatic activity was blocked by α, ß-methyleneadenosine 5'-diphosphate sodium salt (APCP), macrophage parasitism was significantly decreased. Interestingly, these effects were not associated with the production of TNF-α, IL-10, or nitric oxide (NO). Together, these data demonstrate that L. amazonensis induces a regulatory phenotype in macrophages, which by activating the CD39/CD73 pathway allows parasite survival through the action of immunomodulatory adenosine receptors.

Design, synthesis and biological evaluation of N-oxide derivatives with potent in vivo antileishmanial activity.

Leishmaniasis is a neglected disease that affects 12 million people living mainly in developing countries. Herein, 24 new N-oxide-containing compounds were synthesized followed by in vitro and in vivo evaluation of their antileishmanial activity. Compound 4f, a furoxan derivative, was particularly remarkable in this regard, with EC50 value of 3.6 µM against L. infantum amastigote forms and CC50 value superior to 500 µM against murine peritoneal macrophages. In vitro studies suggested that 4f may act by a dual effect, by releasing nitric oxide after biotransformation and by inhibiting cysteine protease CPB (IC50: 4.5 µM). In vivo studies using an acute model of infection showed that compound 4f at 7.7 mg/Kg reduced ~90% of parasite burden in the liver and spleen of L. infantum-infected BALB/c mice. Altogether, these outcomes highlight furoxan 4f as a promising compound for further evaluation as an antileishmanial agent.

Cutting Edge: CD36 Mediates Phagocyte Tropism and Avirulence of Toxoplasma gondii.

Resistance and tolerance are vital for survivability of the host-pathogen relationship. Virulence during Toxoplasma infection in mice is mediated by parasite kinase-dependent antagonism of IFN-γ-induced host resistance. Whether avirulence requires expression of parasite factors that induce host tolerance mechanisms or is a default status reflecting the absence of resistance-interfering factors is not known. In this study, we present evidence that avirulence in Toxoplasma requires parasite engagement of the scavenger receptor CD36. CD36 promotes macrophage tropism but is dispensable for the development of resistance mechanisms. Instead CD36 is critical for re-establishing tissue homeostasis and survival following the acute phase of infection. The CD36-binding capacity of T. gondii strains is negatively controlled by the virulence factor, ROP18. Thus, the absence of resistance-interfering virulence factors and the presence of tolerance-inducing avirulence factors are both required for long-term host-pathogen survival.

Nitric Oxide Induction in Peritoneal Macrophages by a 1,2,3-Triazole Derivative Improves Its Efficacy upon Leishmania amazonensis In Vitro Infection.

1,2,3-Triazole is one of the most flexible chemical scaffolds broadly used in various fields. Here, we report the antileishmanial activity of 1,2,3-triazole derivatives, the ultrastructural alterations induced by their treatment, and the nitric oxide (NO) modulation effect on their efficacy against Leishmania amazonensis in vitro infection. After the screening of eleven compounds, compound 4 exhibited better results against L. amazonensis promastigotes (IC50 = 15.52 ± 3.782 µM) and intracellular amastigotes (IC50 = 4.10 ± 1.136 µM), 50% cytotoxicity concentration at 84.01 ± 3.064 µM against BALB/c peritoneal macrophages, and 20.49-fold selectivity for the parasite over the cells. Compound 4 induced ultrastructural mitochondrial alterations and lipid inclusions in L. amazonensis promastigotes, upregulated tumor necrosis factor α, interleukin (IL)-1ß, IL-6, IL-12, and IL-10 messenger RNA expressions, and enhanced the NO production, verified by nitrite (p = 0.0095) and inducible nitric oxide synthase expression (p = 0.0049) quantification, which played an important role in its activity against intramacrophagic L. amazonensis. In silico prediction in association with antileishmanial activity results showed compound 4 as a hit compound with promising potential for further studies of new leishmaniasis treatment options.

Excretory/secretory proteins of adult Toxocara canis induce changes in the expression of proteins involved in the NOD1-RIP2-NF-κB pathway and modulate cytokine production in mouse macrophages.

Dog roundworm (Toxocara canis) is the major causative agent of toxocarosis, a parasitic disease of both veterinary and medical importance. Knowledge gaps in fundamental and applied aspects hinder the control of this important zoonotic disease. To have a better understanding of Toxocara infection and host immune responses, mouse macrophages were exposed to excretory/secretory (ES) proteins released by adult worms of T. canis in vitro. The messenger RNA transcription and protein expression of nucleotide-binding oligomerization domain-containing protein 1 (NOD1), receptor interacting protein 2 (RIP2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in macrophages were analysed using quantitative real-time PCR (qRT-PCR) and Western blot. The levels of tumour necrosis factor alpha (TNF-ɑ), interleukin-1 beta (IL-1ß) and IL-6 released by the stimulated macrophages were analysed using enzyme-linked immunosorbent assay. It was found that 20 µg/mL ES proteins of adult T. canis induced the expression of NOD1, RIP2 and NF-κB in mouse macrophages at both transcriptional and translational levels after 9 h of incubation in vitro. Incubation with 20 µg/mL ES proteins also modulated the production of pro-inflammatory cytokines TNF-ɑ, IL-1ß and IL-6 by the macrophages. Taken together, ES proteins of adult T. canis appeared to be able to affect the macrophage NOD1-RIP2-NF-κB signalling pathway, which might play a role in regulating the production of proinflammatory cytokines. Further investigation of these aspects should lead to a better understanding of immune recognition of and modulation by Toxocara canis in host animals.

Antiprotozoal and Antibacterial Activity of Ravenelin, a Xanthone Isolated from the Endophytic Fungus Exserohilum rostratum.

The natural compound ravenelin was isolated from the biomass extracts of Exserohilum rostratum fungus, and its antimicrobial, antiplasmodial, and trypanocidal activities were evaluated. Ravenelin was isolated by column chromatography and HPLC and identified by NMR and MS. The susceptibility of Gram-positive and Gram-negative bacteria strains to ravenelin was determined by microbroth dilution assay. Cytotoxicity was evaluated in hepatocarcinoma cells (HepG2) and BALB/c peritoneal macrophages by using MTT. SYBR Green I-based assay was used in the asexual stages of Plasmodium falciparum. Trypanocidal activity was tested against the epimastigote and intracellular amastigote forms of Trypanosoma cruzi. Ravenelin was active against Gram-positive bacteria strains, with emphasis on Bacillus subtilis (MIC value of 7.5 µM). Ravenelin's antiparasitic activities were assessed against both the epimastigote (IC50 value of 5 ± 1 µM) and the intracellular amastigote forms of T. cruzi (IC50 value of 9 ± 2 µM), as well as against P. falciparum (IC50 value of 3.4 ± 0.4 µM). Ravenelin showed low cytotoxic effects on both HepG2 (CC50 > 50 µM) and peritoneal macrophage (CC50 = 185 ± 1 µM) cells with attractive selectivity for the parasites (SI values > 15). These findings indicate that ravenelin is a natural compound with both antibacterial and antiparasitic activities, and considerable selectivity indexes. Therefore, ravenelin is an attractive candidate for hit-to-lead development.

α-Gal immunization positively impacts Trypanosoma cruzi colonization of heart tissue in a mouse model.

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qß-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue.

Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection.

Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8+ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim+/-, Bim-/- mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim-/- mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim+/- mice. At the peak of parasitemia, peritoneal macrophages of Bim-/- mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim-/- splenocytes. Moreover, an impaired anti-T. cruzi CD8+ T-cell response was found in Bim-/- mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim-/- mice and place Bim as an important protein in the control of T. cruzi infections.

Toll-like receptor-7/8 agonist kill Leishmania amazonensis by acting as pro-oxidant and pro-inflammatory agent.

OBJECTIVES: Evaluation of the anti-Leishmanial activity of imidazoquinoline-based TLR7/8 agonists. METHODS: TLR7/8-active imidazoquinolines (2 and 3) were synthesized and assessed for activity against Leishmania amazonensis-intracellular amastigotes using mouse peritoneal macrophages. The production of reactive oxygen species (ROS), nitric oxide (NO) and cytokines was determined in infected and non-infected macrophages. KEY FINDINGS: The imidazoquinolines, 2 and 3, were primarily agonists of TLR7 with compound 3 also showing modest TLR8 activity. Docking studies showed them to occupy the same binding pocket on TLR7 and 8 as the known agonists, imiquimod and resiquimod. Compounds 2 and 3 inhibited the growth of L. amazonensis-intracellular amastigotes with the most potent compound (3, IC50 = 5.93 µM) having an IC50 value close to miltefosine (IC50 = 4.05 µM), a known anti-Leishmanial drug. Compound 3 induced macrophages to produce ROS, NO and inflammatory cytokines that likely explain the anti-Leishmanial effects. CONCLUSIONS: This study shows that activating TLR7 using compounds 2 or 3 induces anti-Leishmanial activity associated with induction of free radicals and inflammatory cytokines able to kill the parasites. While 2 and 3 had a very narrow cytotoxicity window for macrophages, this identifies the possibility to further develop this chemical scaffold to less cytotoxic TLR7/8 agonist for potential use as anti-Leishmanial drug.

Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain.

BACKGROUND: Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-ß signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES: To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS: Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS: Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS: These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Anti-Trypanosoma cruzi activity, cytotoxicity, and chemical characterization of extracts from seeds of Lonchocarpus cultratus.

INTRODUCTION: Trypanosoma cruzi is the agent of Chagas' disease and affects approximately 6-8 million people worldwide. The search for new anti-T. cruzi drugs are relevant because only two drugs exist actually. The objective of this study was to investigate the effect of the extracts from the seeds of Lonchocarpus cultratus on T. cruzi, its cytotoxicity as well as to elucidate its chemical profile. METHODOLOGY: The characterization of the extracts was done using 1H-RMN. T. cruzi forms were treated with increasing concentrations of the extracts and after, the percentage of inhibition and IC50 or LC50 were calculated. Murine peritoneal macrophages were treated with different concentrations of the extracts to evaluate the cellular viability. The hemotoxicity was accessed by verifying the levels of hemolysis caused by the extracts on human red blood cells. RESULTS: Chalcones isocordoin and lonchocarpin were detected in the dichloromethane extract, and chalcone lonchocarpin was detected in the hexane extract. The dichloromethane extract showed higher activity against all the forms of T. cruzi compared to the other two extracts, but the hexane showed the best selectivity index. The cytotoxicity observed in murine macrophages was confirmed in human erythrocytes, with dichloromethane extract having the highest toxicity. The methanolic extract showed the lowest anti-T. cruzi activity but was nontoxic to peritoneal murine macrophages and red blood cells. CONCLUSIONS: L. cultratus extracts have the potential to be explored for the development of new anti-trypanosomal drugs. This study was the first to demonstrate the action of extracts from the genus Lonchocarpus on infecting forms of T. cruzi.

Gene expression patterns associated with Leishmania panamensis infection in macrophages from BALB/c and C57BL/6 mice.

Leishmania parasites can trigger different host immune responses that result in varying levels of disease severity. The C57BL/6 and BALB/c mouse strains are among the host models commonly used for characterizing the immunopathogenesis of Leishmania species and the possible antileishmanial effect of novel drug candidates. C57BL/6 mice tend to be resistant to Leishmania infections, whereas BALB/c mice display a susceptible phenotype. Studying species-specific interactions between Leishmania parasites and different host systems is a key step to characterize and validate these models for in vivo studies. Here, we use RNA-Seq and differential expression analysis to characterize the transcriptomic profiles of C57BL/6 and BALB/c peritoneal-derived macrophages in response to Leishmania panamensis infection. We observed differences between BALB/c and C57BL/6 macrophages regarding pathways associated with lysosomal degradation, arginine metabolism and the regulation of cell cycle. We also observed differences in the expression of chemokine and cytokine genes associated with regulation of immune responses. In conclusion, infection with L. panamensis induced an inflammatory gene expression pattern in C57BL/6 macrophages that is more consistently associated with a classic macrophage M1 activation, whereas in BALB/c macrophages a gene expression pattern consistent with an intermediate inflammatory response was observed.

Oral administration of eugenol oleate cures experimental visceral leishmaniasis through cytokines abundance.

Visceral leishmaniasis (VL) is an endemic fatal infectious disease in tropical and subtropical nations. The limited treatment options, long treatment regimens, invasive mode of administration of drugs, and lack of effective vaccination are the main reasons for the search of new alternative therapeutics against VL. On this quest, from a series of eugenol derivatives, we had demonstrated eugenol oleate as a lead immunomodulatory anti-VL molecule earlier. In this report, the oral efficacy and mechanism of eugenol oleate in inducing immunomodulatory anti-VL activity has been studied in BALB/c mice model. The plasma pharmacokinetic and acute toxicity studies suggested that the eugenol oleate is safe with an appreciable pharmacokinetic profile. Eugenol oleate (30 mg/kg B.W.) showed 86.5% of hepatic and 84.1% of splenic parasite clearance. The increased Th1 cytokine profile and decreased Th2 cytokine profile observed from ELISA and qRTPCR suggested that the eugenol oleate induced the parasite clearance through the activation of the host immune system. Subsequently, the mechanistic insights behind the anti-leishmanial activity of eugenol oleate were studied in peritoneal macrophages in vitro by inhibitor response study and immunoblotting. The results inferred that eugenol oleate activated the PKC-ßII-p38 MAPK and produced IL-12 and IFN-γ which intern activated the iNOS2 to produce NO free radicals that cleared the intracellular parasite.