Trilobolide-6-O-isobutyrate from Sphagneticola trilobata acts by inducing oxidative stress, metabolic changes and apoptosis-like processes by caspase 3/7 activation of human lung cancer cell lines.

BACKGROUND: Lung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored. PURPOSE: This study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death. METHODS: TBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed. RESULTS: In silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors. CONCLUSION: These findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

Leishmania amazonensis infection regulates oxidate stress in hyperglycemia and diabetes impairing macrophage's function and immune response.

Leishmaniasis is a group of infectious diseases caused by protozoa of the Leishmania genus and its immunopathogenesis results from an unbalanced immune response during the infection. Diabetes is a chronic disease resulting from dysfunction of the body's production of insulin or the ability to use it properly, leading to hyperglycemia causing tissue damage and impairing the immune system. AIMS: The objective of this work was to evaluate the effects of hyperglycemia and diabetes during Leishmania amazonensis infection and how these conditions alter the immune response to the parasite. METHODS: An in vitro hyperglycemic stimulus model using THP-1-derived macrophages and an in vivo experimental diabetes with streptozotocin (STZ) in C57BL/6 mice was employed to investigate the impact of diabetes and hyperglicemia in Leishmania amazonensis infection. RESULTS: We observed that hyperglycemia impair the leishmanicidal capacity of macrophages derived from THP-1 cells and reverse the resistance profile that C57BL/6 mice have against infection by L. amazonensis, inducing more exacerbated lesions compared to non-diabetic animals. In addition, the hyperglycemic stimulus favored the increase of markers related to the phenotype of M2 macrophages. The induction of experimental diabetes in C57BL/6 mice resulted in a failure in the production of nitric oxide (NO) in the face of infection and macrophages from diabetic animals failed to process and present Leishmania antigens, being unable to activate and induce proliferation of antigen-specific lymphocytes. CONCLUSION: Together, these data demonstrate that diabetes and hyperglycemia can impair the cellular immune response, mainly of macrophages, against infection by parasites of the genus Leishmania.

Biogenic silver nanoparticle exhibits schistosomicidal activity in vitro and reduces the parasitic burden in experimental schistosomiasis mansoni.

Schistosomiasis is a neglected tropical parasitic disease that affects millions of people, being the second most prevalent parasitic disease worldwide. The current treatment has limited effectiveness, drug-resistant strains, and is not effective in different stages of the disease. This study investigated the antischistosomal activity of biogenic silver nanoparticles (Bio-AgNp) against Schistosoma mansoni. Bio-AgNp presented direct schistosomicidal activity on newly transformed schistosomula causing plasma membrane permeabilization. In S. mansoni adult worms, reduced the viability and affected the motility, increasing oxidative stress parameters, and inducing plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid bodies accumulation, and autophagic vacuoles formation. During the experimental schistosomiasis mansoni model, Bio AgNp restored body weight, reduced hepatosplenomegaly, and decrease the number of eggs and worms in feces and liver tissue. The treatment also ameliorates liver damage and reduces macrophage and neutrophil infiltrates. A reduction in count and size was evaluated in the granulomas, as well as a change to an exudative-proliferative phase, with a local increase of IFN-γ. Together our results showed that Bio-AgNp is a promising therapeutic candidate for studies of new therapeutic strategies against schistosomiasis.

Biogenic silver nanoparticles reduce Toxoplasma gondii infection and proliferation in RAW 264.7 macrophages by inducing tumor necrosis factor-alpha and reactive oxygen species production in the cells.

Owing to the serious adverse effects caused by pyrimethamine and sulfadiazine, the drugs commonly used to treat toxoplasmosis, there is a need for treatment alternatives for this disease. Nanotechnology has enabled significant advances toward this goal. This study was conducted to evaluate the activity of biogenic silver nanoparticles (AgNp-Bio) in RAW 264.7 murine macrophages infected with the RH strain of Toxoplasma gondii. The macrophages were infected with T. gondii tachyzoites and then treated with various concentrations of AgNp-Bio. The cells were evaluated by microscopy, and culture supernatants were collected for ELISA determination of their cytokine concentration. Treatment with 6 µM AgNp-Bio reduced the infection and parasite load in infected RAW 264.7 macrophages without being toxic to the cells. The treatment also induced the synthesis of reactive oxygen species and tumor necrosis factor-alpha (both pro-inflammatory mediators), which resulted in ultrastructural changes in the tachyzoites and their intramacrophagic destruction. Our findings suggest that AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and pro-inflammatory mechanisms and may be a potential alternative treatment for toxoplasmosis.

Investigation of the antileishmanial activity and mechanisms of action of acetyl-thiohydantoins.

The currently available treatment options for leishmaniasis are associated with high costs, severe side effects, and high toxicity. In previous studies, thiohydantoins demonstrated some pharmacological activities and were shown to be potential hit compounds with antileishmanial properties. The present study further explored the antileishmanial effect of acetyl-thiohydantoins against Leishmania amazonensis and determined the main processes involved in parasite death. We observed that compared to thiohydantoin nuclei, acetyl-thiohydantoin treatment inhibited the proliferation of promastigotes. This treatment caused alterations in cell cycle progression and parasite size and caused morphological and ultrastructural changes. We then investigated the mechanisms involved in the death of the protozoan; there was an increase in ROS production, phosphatidylserine exposure, and plasma membrane permeabilization and a loss of mitochondrial membrane potential, resulting in an accumulation of lipid bodies and the formation of autophagic vacuoles on these parasites and confirming an apoptosis-like process. In intracellular amastigotes, selected acetyl-thiohydantoins reduced the percentage of infected macrophages and the number of amastigotes/macrophages by increasing ROS production and reducing TNF-α levels. Moreover, thiohydantoins did not induce cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), or sheep erythrocytes. In silico and in vitro analyses showed that acetyl-thiohydantoins exerted in vitro antileishmanial effects on L. amazonensis promastigotes in apoptosis-like and amastigote forms by inducing ROS production and reducing TNF-α levels, indicating that they are good candidates for drug discovery studies in leishmaniasis treatment. Additionally, we carried out molecular docking analyses of acetyl-thiohydantoins on two important targets of Leishmania amazonensis: arginase and TNF-alpha converting enzyme. The results suggested that the acetyl groups in the N1-position of the thiohydantoin ring and the ring itself could be pharmacophoric groups due to their affinity for binding amino acid residues at the active site of both enzymes via hydrogen bond interactions. These results demonstrate that thiohydantoins are promising hit compounds that could be used as antileishmanial agents.

Botryosphaeran, [(1 → 3)(1 → 6)-ß-D-glucan], induces apoptosis-like death in promastigotes of Leishmania amazonensis, and exerts a leishmanicidal effect on infected macrophages by activating NF-kB and producing pro-inflammatory molecules.

Leishmaniasis is an infectious-parasitic disease caused by the protozoan Leishmania spp. The available treatments are based upon expensive drugs bearing adverse side-effects. The search for new therapeutic alternatives that present a more effective action without causing adverse effects to the patient is therefore important. The objective of this study was to evaluate the in vitro effect of botryosphaeran, a (1 â†’ 3)(1 â†’ 6)-ß-D-glucan, on the promastigote and intracellular amastigote forms of Leishmania amazonensis. The direct activity of botryosphaeran on promastigote forms was evaluated in vitro and inhibited proliferation, the IC50 7 µg/mL in 48 h was calculated. After 48 h treatment, botryosphaeran induced nitric oxide production (NO), caused mitochondrial membrane hyperpolarization, increased reactive oxygen species (ROS), and accumulation of lipid vesicles in promastigotes, resulting in apoptosis, necrosis and autophagy, and was accompanied by morphological and ultrastructural changes. The range of concentrations used did not alter the viability of peritoneal macrophages from BALB/c mice and erythrocytes of sheep. Botryosphaeran was able to reduce the number of infected macrophages and the number of amastigotes per macrophage at 12.5 µg/mL (50.75% ± 6.48), 25 µg/mL (55.66% ± 3.93) and 50 µg/mL (72.9% ± 6.98), and IC50 9.3 µg/mL (±0.66) for intracellular amastigotes forms. The leishmanicidal effect was due to activation of NF-κB and promoted an increase in pro-inflammatory cytokines (TNF-α and IL-6), iNOS and microbial-derived ROS and NO, in addition to decreasing the levels of SOD. Based upon the data obtained, we infer that botryosphaeran exerted an active leishmanicidal and immunomodulatory effect, acting on promastigotes through autophagic, apoptotic and necrosis processes, and in the intracellular amastigote form, through the action of ROS and NO.

Grandiflorenic acid isolated from Sphagneticola trilobata against Trypanosoma cruzi: Toxicity, mechanisms of action and immunomodulation.

Grandiflorenic acid (GFA) is one of the main kaurane diterpenes found in different parts of Sphagneticola trilobata. It has several biological activities, especially antiprotozoal action. In turn, Chagas disease is a complex systemic disease caused by the protozoan Trypanosoma cruzi, and the drugs available to treat it involve significant side effects and impose an urgent need to search for therapeutic alternatives. In this context, our goal was to determine the effect of GFA on trypomastigote and intracellular amastigote forms. Our results showed that GFA treatment led to significantly less viability of trypomastigote forms, with morphological and ultrastructural changes in the parasites treated with IC50 of GFA (24.60 nM), and larger levels of reactive oxygen species (ROS), mitochondrial depolarization, lipid droplets accumulation, presence of autophagic vacuoles, phosphatidylserine exposure, and plasma membrane damage. In addition, the GFA treatment was able to reduce the percentage of infected cells and the number of amastigotes per macrophage (J774A.1) without showing cytotoxicity in mammalian cell lines (J774A.1, LLCMK2, THP-1, AMJ2-C11), in addition to increasing TNF-α and reducing IL-6 levels in infected macrophages. In conclusion, the GFA treatment exerted influence on trypomastigote forms through an apoptosis-like mechanism and by eliminating intracellular parasites via TNF-α/ROS pathway, without generating cellular cytotoxicity.

Exploring the antileishmanial activity of N1,N2-disubstituted-benzoylguanidines: synthesis and molecular modeling studies.

In this report, we describe the synthesis and evaluation of nine N1,N2-disubstituted-benzoylguanidines against promastigotes and amastigotes forms of Leishmania amazonensis. The derivatives 2g and 2i showed low IC50 values against promastigote form (90.8 ± 0.05 µM and 68.4 ± 0.03 µM, respectively), low cytotoxicity profile (CC50 396 ± 0.02 µM and 857.9 ± 0.06 µM) for peritoneal macrophages cells and SI of 5.5 and 12.5, respectively. Investigations about the mechanism of action of 2g and 2i showed that both compounds cause mitochondrial depolarization, increase in ROS levels, and generation of autophagic vacuoles on free promastigotes forms. These compounds were also capable of reducing the number of infected macrophages with amastigotes forms (59.5% ± 0.08% and 98.1% ± 0.46%) and the number of amastigotes/macrophages (79.80% ± 0.05% and 96.0% ± 0.16%), through increasing induction of microbicide molecule NO. Additionally, ADMET-Tox in silico predictions showed drug-like features and free of toxicological risks. The molecular docking studies with arginase and gp63 showed that relevant intermolecular interactions could explain the experimental results. Therefore, these results reinforce that benzoylguanidines could be a starting scaffold for the search for new antileishmanial drugs.Communicated by Ramaswamy H. Sarma.

Murine Susceptibility to Leishmania amazonensis Infection Is Influenced by Arginase-1 and Macrophages at the Lesion Site.

Cutaneous leishmaniasis is a zoonotic infectious disease broadly distributed worldwide, causing a range of diseases with clinical outcomes ranging from self-healing infections to chronic disfiguring disease. The effective immune response to this infection is yet to be more comprehensively understood and is fundamental for developing drugs and vaccines. Thus, we used experimental models of susceptibility (BALB/c) and partial resistance (C57BL/6) to Leishmania amazonensis infection to investigate the local profile of mediators involved in the development of cutaneous leishmaniasis. We found worse disease outcome in BALB/c mice than in C57BL/6 mice, with almost 15 times higher parasitic load, ulcerated lesion formation, and higher levels of IL-6 in infected paws. In contrast, C57BL/6 presented higher levels of IFN-γ and superoxide anion (•O2-) after 11 weeks of infection and no lesion ulcerations. A peak of local macrophages appeared after 24 h of infection in both of the studied mice strains, followed by another increase after 240 h, detected only in C57BL/6 mice. Regarding M1 and M2 macrophage phenotype markers [iNOS, MHC-II, CD206, and arginase-1 (Arg-1)], we found a pronounced increase in Arg-1 levels in BALB/c after 11 weeks of infection, whereas C57BL/6 showed an initial predomination of markers from both profiles, followed by an M2 predominance, coinciding with the second peak of macrophage infiltration, 240 h after the infection. Greater deposition of type III collagen and lesion resolution was also observed in C57BL/6 mice. The adoptive transfer of macrophages from C57BL/6 to infected BALB/c at the 11th week showed a reduction in both edema and the number of parasites at the lesion site, in addition to lower levels of Arg-1. Thus, C57BL/6 mice have a more effective response against L. amazonensis, based on a balance between inflammation and tissue repair, while BALB/c mice have an excessive Arg-1 production at late infection. The worst evolution seems to be influenced by recruitment of Arg-1 related macrophages, since the adoptive transfer of macrophages from C57BL/6 mice to BALB/c resulted in better outcomes, with lower levels of Arg-1.

Impairment of effector molecules response in diabetes induces susceptibility to Leishmania amazonensis infection.

Type 2 Diabetes is a chronic disease resulting from insulin dysfunction that triggers a low-grade inflammatory state and immune impairment. Leishmaniasis is an infectious disease characterized by chronic inflammation resulted from the parasite's immunomodulation ability. Thus, due to the delicate immune balance required in the combat and resistance to Leishmania infection and the chronic deregulation of the inflammatory response observed in type 2 diabetes, we evaluated the response of PBMC from diabetic patients to in vitro Leishmania amazonensis infection. For that, peripheral blood was collected from 25 diabetic patients and 25 healthy controls matched for age for cells extraction and subsequent experimental infection for 2 or 24 h and analyzed for phagocytic and leishmanicidal capacity by optical microscopy, oxidative stress by GSSG generation, labeling of intracellular mediators by enzyme-Linked immunosorbent assay, and cytokines measurement with cytometric beads array technique. We found that the diabetic group had a higher percentage of infected cells and a greater number of amastigotes per cell. Also, even inducing NF-kB phosphorylation and increasing TNF production after infection, cells from diabetic patients were unable to downregulate NRF2 and generate oxidative stress, which may be associated with the exacerbated levels of IL-6 observed. PBMC of diabetic individuals are more susceptible to infection by L. amazonensis and fail to control the infection over time due to the inability to generate effector microbicidal molecules.

Solidagenone acts on promastigotes of L. amazonensis by inducing apoptosis-like processes on intracellular amastigotes by IL-12p70/ROS/NO pathway activation.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the Leishmania genus. Currently, the treatment has limited effectiveness and high toxicity, is expensive, requires long-term treatment, induces significant side effects, and promotes drug resistance. Thus, new therapeutic strategies must be developed to find alternative compounds with high efficiency and low cost. Solidagenone (SOL), one of the main constituents of Solidago chilensis, has shown gastroprotective, anti-inflammatory and immunomodulatory effects. PURPOSE: This study assessed the in vitro effect of SOL on promastigotes and Leishmania amazonensis-infected macrophages, as well its microbicide and immunomodulatory mechanisms. METHODS: SOL was isolated from the roots of S. chilensis, 98% purity, and identified by chromatographic methods, and the effect of SOL on leishmanicidal activity against promastigotes in vitro, SOL-induced cytotoxicity in THP-1, J774 cells, sheep erythrocytes, and L. amazonensis-infected J774 macrophages, and the mechanisms of death involved in this action were evaluated. RESULTS: In silico predictions showed good drug-likeness potential for SOL with high oral bioavailability and intestinal absorption. SOL treatment (10-160 µM) inhibited promastigote proliferation 24, 48, and 72 h after treatment. After 24 h of treatment, SOL at the IC50 (34.5 µM) and 2 × the IC50 (69 µM) induced several morphological and ultrastructural changes in promastigotes, altered the cell cycle and cellular volume, increased phosphatidylserine exposure on the cell surface, induced the loss of plasma membrane integrity, increased the reactive oxygen species (ROS) level, induced loss of mitochondrial integrity (characterized by an apoptosis-like process), and increased the number of lipid droplets and autophagic vacuoles. Additionally, SOL induced low cytotoxicity in J774 murine macrophages (CC50 of 1587 µM), THP-1 human monocytes (CC50 of 1321 µM), and sheep erythrocytes. SOL treatment reduced the percentage of L. amazonensis-infected macrophages and the number of amastigotes per macrophage (IC50 9.5 µM), reduced TNF-α production and increased IL-12p70, ROS and nitric oxide (NO) levels. CONCLUSION: SOL showed in vitro leishmanicidal effects against the promastigotes by apoptosis-like mechanism and amastigotes by reducing TNF-α and increasing IL-12p70, ROS, and NO levels, suggesting their potential as a candidate for use in further studies on the design of antileishmanial drugs.

Trans-chalcone induces death by autophagy mediated by p53 up-regulation and ß-catenin down-regulation on human hepatocellular carcinoma HuH7.5 cell line.

BACKGROUND: Hepatocellular Carcinoma (HCC) is extremely aggressive and presents low rates of response to the available chemotherapeutic agents. Many studies have focused on the search for alternative low-cost natural compounds with antiproliferative potential that selectively respond to liver cancer cells. PURPOSE: This study assessed the in vitro direct action of trans-chalcone (TC) on cells of the human HCC HuH7.5 cell line. METHODS: We subjected the HuH7.5 tumor cells to TC treatment at increasing concentrations (12.5-100 µM) for 24 and 48 h. Cell viability was verified through MTT and 50% inhibitory concentration of cells (IC50 23.66 µM) was determined within 48 h. We quantified trypan blue proliferation and light microscopy, ROS production, mitochondrial depolarization and autophagy, cell cycle analysis, and apoptosis using Muse® cell analyzer and immunocytochemical markings of p53 and ß-catenin. RESULTS: Data showed an effective dose- and time-dependent TC-cytotoxic action at low micromolar concentrations without causing toxicity to non-cancerous cells, such as erythrocytes. TC-treatment caused mitochondrial membrane damage and cell cycle G0/G1 phase arrest, increasing the presence of the p53 protein and decreasing ß-catenin, in addition, to inducing cell death by autophagy. Additionally, TC decreased the metastatic capacity of HuH7.5, which affected the migration/invasion of this type of cell. CONCLUSION: In vitro TC activity in the human HCC HuH7.5 tumor cell line is shown to be a potential molecule to develop new therapies to repair the p53 pathway and prevent the overexpression of Wnt/ß-catenin tumor development inducing autophagy cell death and decreasing metastatic capacity of HuH7.5 cell line.

3,3',5,5'-tetramethoxybiphenyl-4,4'diol induces cell cycle arrest in G2/M phase and apoptosis in human non-small cell lung cancer A549 cells.

Lung cancer is one of the leading causes of cancer-related death worldwide. It has aggressive manifestation, high ability to promote metastasis and late diagnosis. In the present study, we investigated the cytotoxic effect of 3,3',5,5'-tetramethoxybiphenyl-4,4'diol (TMBP), against the A549 human non-small cell lung carcinoma lineage. The A549 cell line was treated for 72h with TMBP (12.5-200 µM) with and subsequently defined the 50% inhibitory concentration (148 µM ± 0.05), from which tests were performed to determine the viability, volume, and regulation of the cell cycle. Finally, we investigated the death mechanisms involved in the action of the treatments by flow cytometry and fluorimetry. The TMBP-treatment of primary cells, peritoneal macrophages, and sheep erythrocytes did not reduce the viability of these cells. On the other hand, TMBP was able to reduce the viability of the investigated cell line, by cytotoxic action and to promote the reduction of cell size. Subsequently, we found that TMBP treatment was able to increase the production of reactive oxygen species, cause mitochondrial depolarization, induce cell cycle arrest in G2/M phase and lead to death by direct apoptosis. Thus, this study revealed that TMBP could be a promising candidate for the development of antitumor drugs targeting lung cancer.

trans-Chalcone modulates Leishmania amazonensis infection in vitro by Nrf2 overexpression affecting iron availability.

Leishmania parasites infect macrophages causing a wide spectrum of human diseases encompassing from cutaneous to visceral forms. The drugs currently used in leishmaniasis treatment are highly toxic and associated with acquired resistance. Seeking novel therapeutic targets, we conducted a comprehensive in vitro study to investigate the action of trans-chalcone (TC) against Leishmania amazonensis promastigote and amastigote forms. TC is a common precursor of flavonoids, however, no extensive research has been developed regarding its pharmacological properties. In silico predictions showed good drug-likeness potential for TC with high oral bioavailability and intestinal absorption. The TC-treatment had a direct action on promastigote forms leading to death by late apoptosis-like process resulting from an increased production of reactive oxygen species (ROS), loss of mitochondrial integrity, phosphatidylserine exposure, and damage on the membrane. Similar results were found for L. amazonensis-axenic amastigotes. The TC-treatment of L.amazonensis-infected macrophages proved to reduce the percentage of infected cells as well as the number of amastigotes per macrophage, consequently, the number of promastigotes recovered without cytotoxic effects on macrophages, having indicated a selectivity index (SI) of 53.8 for the parasite. Such leishmanicidal effect was followed by a decrease in the levels of TNF-α, TGF-ß, IL-10, ROS and NO, in addition to upregulation mRNA expression of Nrf2, heme oxygenase 1, and ferritin, modulating iron metabolism, depleting available iron for parasite replication, and survival within macrophages. These results suggested trans-chalcone as a satisfactory support for further studies as well as a possible further lead molecule for the design of new prototypes of antileishmanial drugs.

Caffeic acid has antipromastigote activity by apoptosis-like process; and anti-amastigote by TNF-α/ROS/NO production and decreased of iron availability.

BACKGROUND: Leishmaniasis is a disease caused by protozoan parasites of the Leishmania genus whose current treatment has high cost, highly toxic, and difficult administration, which makes it very important to find alternative natural compounds of high efficiency and low cost. PURPOSE: This study assessed the in vitro effect of caffeic acid (CA) on promastigotes and L. amazonensis-infected macrophages. METHODS: Evaluation of the in vitro leishmanicidal activity of CA against promastigotes and L. amazonensis infected peritoneal macrophages, as well its microbicide mechanisms. RESULTS: CA 12.5-100 µg/ml were able to inhibit promastigotes proliferation at all tested periods. The IC50, 12.5 µg/ml, also altered promastigote cell morphology and cell volume accompanied by loss of mitochondrial integrity, increase in reactive oxygen species (ROS) production, phosphatidylserine exposure, and loss of plasma membrane integrity - characterizing the apoptosis-like process. Moreover, CA reduced the percentage of infected macrophages and the number of amastigotes per macrophages increasing TNF-α, ROS, NO and reducing IL-10 levels as well as iron availability. CONCLUSION: CA showed in vitro antipromastigote and antiamostigote by increasing oxidant and inflammatory response important to eliminate the parasite.

Macrophage Polarization in Leishmaniasis: Broadening Horizons.

Leishmaniasis is a vector-borne neglected tropical disease that affects more than 700,000 people annually. Leishmania parasites cause the disease, and different species trigger a distinct immune response and clinical manifestations. Macrophages are the final host cells for the proliferation of Leishmania parasites, and these cells are the key to a controlled or exacerbated response that culminates in clinical manifestations. M1 and M2 are the two main macrophage phenotypes. M1 is a pro-inflammatory subtype with microbicidal properties, and M2, or alternatively activated, is an anti-inflammatory/regulatory subtype that is related to inflammation resolution and tissue repair. The present review elucidates the roles of M1 and M2 polarization in leishmaniasis and highlights the role of the salivary components of the vector and the action of the parasite in the macrophage plasticity.

Grandiflorenic acid promotes death of promastigotes via apoptosis-like mechanism and affects amastigotes by increasing total iron bound capacity.

BACKGROUND: American tegumentary leishmaniasis (ATL) is a zoonotic disease caused by protozoa of the genus Leishmania. The high toxicity, high costs and resistance of some strains to current drugs has prompted the search for therapeutic alternatives for the management of this disease. Sphagneticola trilobata is a plant that has diterpenes as main constituents, including grandiflorenic acid (GFA) that has antiinflammatory, antiprotozoal, antibacterial and antinociceptive activity. PURPOSE: The aim of our study was to determine the effect of GFA on both the promastigotes and the amastigotes of Leishmania amazonensis. METHODS: Isolation by chromatographic methods and chemical identification of GFA, then evaluation of the in vitro leishmanicidal activity of this compound against Leishmania amazonensis promastigotes and L. amazonensis infected peritoneal Balb/c macrophages, as well its action and microbicide mechanisms. RESULTS: GFA treatment significantly inhibited the proliferation of promastigotes. This antiproliferative effect was accompanied by morphological changes in the parasite with 25 nM GFA. Afterwards, we investigated the mechanisms involved in the death of the protozoan; there was an increase in the production of reactive oxygen species (ROS), phosphatidylserine exposure, permeabilization of the plasma membrane and decreased mitochondrial depolarization. In addition, we observed that the treatment caused a reduction in the percentage of infected cells and the number of amastigotes per macrophage, without showing cytotoxicity in low doses to peritoneal macrophages and sheep erythrocytes. GFA increased IL-10 and total iron bound to transferrin in infected macrophages. Our results showed that GFA treatment acts on promastigote forms through an apoptosis-like mechanism and on intracellular amastigote forms, dependent of regulatory cytokine IL-10 modulation with increase in total iron bound to transferrin. CONCLUSION: GFA showed in vitro antileishmanial activity on L. amazonensis promastigotes forms and on L. amazonensis-infected macrophages.