Essential oil of oregano (Origanum vulgare L.) reduces infection and proliferation of Toxoplasma gondii in BeWo cells with induction of autophagy and death of tachyzoites through a mechanism similar to necrosis.

Toxoplasmosis poses a global health threat, ranging from asymptomatic cases to severe, potentially fatal manifestations, especially in immunocompromised individuals and congenital transmission. Prior research suggests that oregano essential oil (OEO) exhibits diverse biological effects, including antiparasitic activity against Toxoplasma gondii. Given concerns about current treatments, exploring new compounds is important. This study was to assess the toxicity of OEO on BeWo cells and T. gondii tachyzoites, as well as to evaluate its effectiveness in in vitro infection models and determine its direct action on free tachyzoites. OEO toxicity on BeWo cells and T. gondii tachyzoites was assessed by MTT and trypan blue methods, determining cytotoxic concentration (CC50), inhibitory concentration (IC50), and selectivity index (SI). Infection and proliferation indices were analyzed. Direct assessments of the parasite included reactive oxygen species (ROS) levels, mitochondrial membrane potential, necrosis, and apoptosis, as well as electron microscopy. Oregano oil exhibited low cytotoxicity on BeWo cells (CC50: 114.8 µg/mL ± 0.01) and reduced parasite viability (IC50 12.5 ± 0.06 µg/mL), demonstrating 9.18 times greater selectivity for parasites than BeWo cells. OEO treatment significantly decreased intracellular proliferation in infected cells by 84% after 24 h with 50 µg/mL. Mechanistic investigations revealed increased ROS levels, mitochondrial depolarization, and lipid droplet formation, linked to autophagy induction and plasma membrane permeabilization. These alterations, observed through electron microscopy, suggested a necrotic process confirmed by propidium iodide labeling. OEO treatment demonstrated anti-T. gondii action through cellular and metabolic change while maintaining low toxicity to trophoblastic cells.

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.

3,3',5,5'-Tetramethoxybiphenyl-4,4'diol triggers oxidative stress, metabolic changes, and apoptosis-like process by reducing the PI3K/AKT/NF-κB pathway in the NCI-H460 lung cancer cell line.

Lung cancer is one of the leading causes of cancer-related deaths in men and women worldwide. Current treatments have limited efficacy, cause significant side effects, and cells can develop drug resistance. New therapeutic strategies are needed to discover alternative anticancer agents with high efficacy and low-toxicity. TMBP, a biphenyl obtained by laccase-biotransformation of 2,6-dimethoxyphenol, possesses antitumor activity against A549 adenocarcinoma cells. Without causing damage to sheep erythrocytes and mouse peritoneal macrophages of BALB/c mice. In addition to being classified as a good oral drug according to in-silico studies. This study evaluated the in-vitro cytotoxic effect of TMBP on lung-cancer cell-line NCI-H460 and reports mechanisms on immunomodulation and cell death. TMBP treatment (12.5-200 µM) inhibited cell proliferation at 24, 48, and 72 h. After 24-h treatment, TMBP at IC50 (154 µM) induced various morphological and ultrastructural changes in NCI-H460, reduced migration and immunofluorescence staining of N-cadherin and ß-catenin, induced increased reactive oxygen species and nitric oxide with reduced superoxide radical-anion, increased superoxide dismutase activity and reduced glutathione reductase. Treatment also caused metabolic stress, reduced glucose-uptake, intracellular lactate dehydrogenase and lactate levels, mitochondrial depolarization, increased lipid droplets, and autophagic vacuoles. TMBP induced cell-cycle arrest in the G2/M phase, death by apoptosis, increased caspase-3/7, and reduced STAT-3 immunofluorescence staining. The anticancer effect was accompanied by decreasing PI3K, AKT, ARG-1, and NF-κB levels, and increasing iNOS. These results suggest its potential as a candidate for use in future lung anticancer drug design studies.

Viremia and Inflammatory Cytokines in Dengue: Interleukin-2 as a Biomarker of Infection, and Interferon-α and -γ as Markers of Primary versus Secondary Infection.

The pathogenesis of Dengue virus (DENV) infection is complex and involves viral replication that may trigger an inflammatory response leading to severe disease. Here, we investigated the correlation between viremia and cytokine levels in the serum of DENV-infected patients. Between 2013 and 2014, 138 patients with a diagnosis of acute-phase DENV infection and 22 patients with a non-dengue acute febrile illness (AFI) were enrolled. Through a focus-forming assay (FFU), we determined the viremia levels in DENV-infected patients and observed a peak in the first two days after the onset of symptoms. A higher level of viremia was observed in primary versus secondary DENV-infected patients. Furthermore, no correlation was observed between viremia and inflammatory cytokine levels in DENV-infected patients. Receiver operating characteristic (ROC) curve analysis revealed that IL-2 has the potential to act as a marker to distinguish dengue from other febrile illnesses and is positively correlated with Th1 cytokines. IFN-α and IFN-γ appear to be potential markers of primary versus secondary infection in DENV-infected patients, respectively. The results also indicate that viremia levels are not the main driving force behind inflammation in dengue and that cytokines could be used as infection biomarkers and for differentiation between primary versus secondary infection.

Synergistic Antileishmanial Effect of Oregano Essential Oil and Silver Nanoparticles: Mechanisms of Action on Leishmania amazonensis.

American tegumentary leishmaniasis, a zoonotic disease caused by the Leishmania genus, poses significant challenges in treatment, including administration difficulty, low efficacy, and parasite resistance. Novel compounds or associations offer alternative therapies, and natural products such as oregano essential oil (OEO), extracted from Origanum vulgare, have been extensively researched due to biological effects, including antibacterial, antifungal, and antiparasitic properties. Silver nanoparticles (AgNp), a nanomaterial with compelling antimicrobial and antiparasitic activity, have been shown to exhibit potent leishmanicidal properties. We evaluated the in vitro effect of OEO and AgNp-Bio association on L. amazonensis and the death mechanisms of the parasite involved. Our results demonstrated a synergistic antileishmanial effect of OEO + AgNp on promastigote forms and L. amazonensis-infected macrophages, which induced morphological and ultrastructural changes in promastigotes. Subsequently, we investigated the mechanisms underlying parasite death and showed an increase in NO, ROS, mitochondrial depolarization, accumulation of lipid-storage bodies, autophagic vacuoles, phosphatidylserine exposure, and damage to the plasma membrane. Moreover, the association resulted in a reduction in the percentage of infected cells and the number of amastigotes per macrophage. In conclusion, our findings establish that OEO + AgNp elicits a late apoptosis-like mechanism to combat promastigote forms and promotes ROS and NO production in infected macrophages to target intracellular amastigote forms.

In-vitro biological evaluation of 3,3',5,5'-tetramethoxy-biphenyl-4,4'-diol and molecular docking studies on trypanothione reductase and Gp63 from Leishmania amazonensis demonstrated anti-leishmania potential.

Available treatments for leishmaniasis have been widely used since the 1940s but come at a high cost, variable efficacy, high toxicity, and adverse side-effects. 3,3',5,5'-Tetramethoxy-biphenyl-4,4'-diol (TMBP) was synthesized through laccase-catalysis of 2,6-dimethoxyphenol and displayed antioxidant and anticancer activity, and is considered a potential drug candidate. Thus, this study aimed to evaluate the anti-leishmanial effect of TMBP against promastigote and amastigote forms of Leishmania (L.) amazonensis and investigated the mechanisms involved in parasite death. TMBP treatment inhibited the proliferation (IC50 0.62-0.86 µM) and induced the death of promastigote forms by generating reactive oxygen species and mitochondrial dysfunction. In intracellular amastigotes, TMBP reduced the percentage of infected macrophages, being 62.7 times more selective to the parasite (CC50 53.93 µM). TMBP did not hemolyze sheep erythrocytes; indicative of low cytotoxicity. Additionally, molecular docking analysis on two enzyme targets of L. amazonensis: trypanothione reductase (TR) and leishmanolysin (Gp63), suggested that the hydroxyl group could be a pharmacophoric group due to its binding affinity by hydrogen bonds with residues at the active site of both enzymes. TMBP was more selective to the Gp63 target than TR. This is the first report that TMBP is a promising compound to act as an anti-leishmanial agent.

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 (AgNp-Bio) restore testosterone levels and increase TNF-α and IL-6 in Leydig cells infected with Toxoplasma gondii.

Toxoplasma gondii, a protozoan parasite, is responsible for toxoplasmosis. The available therapy for patients with toxoplasmosis involves a combination of pyrimethamine and sulfadiazine, which have several adverse effects, including bone marrow suppression, megaloblastic anemia, leukopenia, and granulocytopenia. The development of therapeutic alternatives is essential for the management of toxoplasmosis, emphasizing the recent advances in nanomedicine. This study aimed to evaluate the in vitro effects of biogenic silver nanoparticles (AgNp-Bio) on tachyzoite forms and Leydig cells infected with T. gondii. We observed that the AgNp-Bio reduced the viability of the tachyzoites and did not exhibit cytotoxicity against Leydig cells at low concentrations. Additionally, treatment with AgNp-Bio reduced the rate of infection and proliferation of the parasite, and lowered the testosterone levels in the infected cells. It increased the levels of IL-6 and TNF-α and reduced the levels of IL- 10. Among the morphological and ultrastructural changes, AgNp-Bio induced a reduction in the number of intracellular tachyzoites and caused changes in the tachyzoites with accumulation of autophagic vacuoles and a decrease in the number of tachyzoites inside the parasitophorous vacuoles. Collectively, our data demonstrate that the AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and inflammatory mechanisms and could be a potential alternative treatment for toxoplasmosis.

Solidagenone in vivo leishmanicidal activity acting in tissue repair response, and immunomodulatory capacity in Leishmania amazonensis.

Leishmaniasis is a group of chronic parasitic diseases in humans caused by species of the Leishmania genus. Current treatments have high toxicity, cost, duration, limited effectiveness, significantly complex administration, and drug-resistant strains. These factors highlight the importance of research into new therapies that use drugs without toxic effects. Solidagenone (SOL), the main labdane diterpene isolated from the plant Solidago chilensis, has anti-inflammatory, gastroprotective, antioxidant, tissue repair-inducing effects, suggesting a role in novel drug development. This study investigates in vivo mechanism action of SOL treatment in L. amazonensis-infected BALB/c mice. SOL was isolated from the roots of S. chilensis, and L. amazonensis-infected mice were treated daily with SOL (10, 50, 100 mg/kg) by gavage for 30 days. Gastric (NAG, MPO), hepatic (AST, ALT), systemic (body weight, NO) toxicity, leishmanicidal activity (lesion size, parasite burden), cell profile (macrophage, neutrophil infiltration), antioxidant (ABTS, NBT, NO), oxidant parameters (FRAP, ABTS), Th1, Th2, Th17 cytokines (CBA), collagen deposition (picrosirius), arginase, iNOS, NF-kB, and NRF2 (immunofluorescence) were evaluated. In vivo results showed SOL-treatment did not induce gastric, hepatic, or systemic toxicity in L. amazonensis-infected mice. SOL was able to reduce the lesion size and parasite load at the site of infection, increasing macrophage infiltration and neutrophil migration, exerting a balance in antioxidant (increased ABTS, NBT reduction, and NO), oxidative (increased FRAP and ABTS), and anti-inflammatory responses (reduced TNF-α, IFN-γ and increased IL-6, IL-17 production), and inducing arginase, iNOS, NF-kB, NRF2 and collagen deposition (type III), favoring wound healing and accelerating tissue repair at the site injury.

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.

Caryocar coriaceum Wittm. fruit extracts as Leishmania inhibitors: in-vitro and in-silico approaches.

Leishmaniasis is a group of neglected diseases caused by parasites of the Leishmania genus. The treatment of Leishmaniasis represents a great challenge, because the available drugs present high toxicity and none of them is fully effective. Caryocar is a botanical genus rich in phenolic compounds, which leaves extracts have already been described by its antileishmanial action. Thus, we investigated the effect of pulp and peel extracts of the Caryocar coriaceum fruit on promastigote and amastigote forms of Leishmania amazonensis. Both extracts had antipromastigote effect after 24, 48, and 72 h, and this effect was by apoptosis-like process induction, with reactive oxygen species (ROS) production, damage to the mitochondria and plasma membrane, and phosphatidylserine exposure. Knowing that the fruit extracts did not alter the viability of macrophages, we observed that the treatment reduced the infection of these cells. Thereafter, in the in vitro infection context, the extracts showed antioxidant proprieties, by reducing NO, ROS, and MDA levels. Besides, both peel and pulp extracts up-regulated Nrf2/HO-1/Ferritin expression and increase the total iron-bound in infected macrophages, which culminates in a depletion of available iron for L. amazonensis replication. In silico, the molecular modeling experiments showed that the three flavonoids presented in the C. coriaceum extracts can act as synergistic inhibitors of Leishmania proteins, and compete for the active site. Also, there is a preference for rutin at the active site due to its greater interaction binding strength.Communicated by Ramaswamy H. Sarma.

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.

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.

Patterns of Cell Death Induced by Thiohydantoins in Human MCF-7 Breast Cancer Cells.

BACKGROUND: Conventional therapies for breast cancer are still a challenge due to cytotoxic drugs not being highly effective with significant adverse effects. Thiohydantoins are biologically active heterocyclic compounds reported for several biological activities, including anticarcinogenic properties, etc. This work aims to assess the use of thiohydantoin as a potential antitumor agent against MCF-7 breast cancer cells. METHODS: MTT and neutral red assays were used to assess the possible cytotoxic activity of compounds against MCF-7 cells. Cell volume measurement and analysis were performed by flow cytometry. Fluorescence analysis was carried out to determine patterns of cell death induced by thiohydantoins. RESULTS: The treatment with micromolar doses of thiohydantoins promoted a decrease in the viability of MCF-7 breast tumor cells. An increase in the ROS and NO production, reduction in cell volume, loss of membrane integrity, mitochondrial depolarization, and increased fluorescence for annexin-V and caspase-3 were also observed. These findings indicate cell death by apoptosis and increased autophagic vacuoles, stopping the cell cycle in the G1/ G0 phase. CONCLUSION: Our results indicate that thiohydantoins are cytotoxic to breast tumor cells, and this effect is linked to the increase in ROS production. This phenomenon changes tumorigenic pathways, which halt the cell cycle in G1/G0. This is an essential checkpoint for DNA errors, which may have altered how cells produce energy, causing a decrease in mitochondrial viability and thus leading to the apoptotic process. Furthermore, the results indicate increased autophagy, a vital process linked to a decrease in lysosomal viability and thus considered a cell death and tumor suppression mechanism.

Thiohydantoins as anti-leishmanial agents: n vitro biological evaluation and multi-target investigation by molecular docking studies.

Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. The first-line treatment of this disease is still based on pentavalent antimonial drugs that have a high toxicity profile, which could induce parasitic resistance. Therefore, there is a critical need to discover more effective and selective novel anti-leishmanial agents. In this context, thiohydantoins are a versatile class of substances due to their simple synthesis and several biological activities. In this work, thiohydantoins 1a-l were evaluated in vitro for antileishmania activity. Among them, four derivatives (1c, 1e, 1h and 1l) showed promising IC50 values around 10 µM against promastigotes forms of Leishmania amazonensis and low cytotoxicity profile for peritoneal macrophages cells. Besides, these compounds induce oxidative stress through an increase in ROS production and the labeling of annexin-V and propidium iodide, indicating that promastigotes were undergoing a late apoptosis-like process. Additionally, molecular consensual docking analysis was carried out against two important targets to L. amazonensis: arginase and trypanothione reductase enzymes. Docking results suggest that thiohydantoin ring could be a pharmacophoric group due to its binding affinity by hydrogens bond interactions with important amino acid residues at the active site of both enzymes. These results demonstrate that compounds 1c, 1e, 1h and 1l may are promising in future advance studies.Communicated by Ramaswamy H. Sarma.

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.

Recent Advances in Biotransformation by Cunninghamella Species.

The goal of the biotransformation process is to develop structural changes and generate new chemical compounds, which can occur naturally in mammalian and microbial organisms, such as filamentous fungi, and represent a tool to achieve enhanced bioactive compounds. Cunninghamella spp. is among the fungal models most widely used in biotransformation processes at phase I and II reactions, mimicking the metabolism of drugs and xenobiotics in mammals and generating new molecules based on substances of natural and synthetic origin. Therefore, the goal of this review is to highlight the studies involving the biotransformation of Cunninghamella species between January 2015 and March 2021, in addition to updating existing studies to identify the similarities between the human metabolite and Cunninghamella patterns of active compounds, with related advantages and challenges, and providing new tools for further studies in this scope.

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.

Larval excretion/secretion of dipters of Lucilia cuprina species induces death in promastigote and amastigote forms of Leishmania amazonensis.

Leishmaniasis is a neglected tropical disease that affects millions of people around the world. Larval excretion/secretion (ES) of the larvae of flies of the Calliphoridae family has microbicidal activity against Gram-positive and Gram-negative bacteria, in addition to some species of Leishmania. Our study aimed at assessing the in vitro efficacy of Lucilia cuprina larval ES against the promastigote and amastigote forms of Leishmania amazonensis, elucidating possible microbicidal mechanisms and routes of death involved. Larval ES was able to inhibit the viability of L. amazonensis at all concentrations, induce morphological and ultrastructural changes in the parasite, retraction of the cell body, roughness of the cytoplasmic membrane, leakage of intracellular content, ROS production increase, induction of membrane depolarization and mitochondrial swelling, the formation of cytoplasmic lipid droplets and phosphatidylserine exposure, thus indicating the possibility of apoptosis-like death. To verify the efficacy of larval ES on amastigote forms, we performed a phagocytic assay, measurement of total ROS and NO. Treatment using larval ES reduced the percentage of infection and the number of amastigotes per macrophage of lineage J774A.1 at all concentrations, increasing the production of ROS and TNF-α, thus indicating possible pro-inflammatory immunomodulation and oxidative damage. Therefore, treatment using larval ES is effective at inducing the death of promastigotes and amastigotes of L. amazonensis even at low concentrations.