Nanomedicine in leishmaniasis: A promising tool for diagnosis, treatment and prevention of disease - An update overview.

Leishmaniasis is a neglected tropical disease that has a wide spectrum of clinical manifestations, ranging from visceral to cutaneous, with millions of new cases and thousands of deaths notified every year. The severity of the disease and its various clinical forms are determined by the species of the causative agent, Leishmania, as well as the host's immune response. Major challenges still exist in the diagnosis and treatment of leishmaniasis, and there is no vaccine available to prevent this disease in humans. Nanotechnology has emerged as a promising tool in a variety of fields. In this review, we highlight the main and most recent advances in nanomedicine to improve the diagnosis and treatment, as well as for the development of vaccines, for leishmaniasis. Nanomaterials are nanometric in size and can be produced by a variety of materials, including lipids, polymers, ceramics, and metals, with varying structures and morphologies. Nanotechnology can be used as biosensors to detect antibodies or antigens, thus improving the sensitivity and specificity of such immunological and molecular diagnostic tests. While in treatment, nanomaterials can act as drug carriers or, be used directly, to reduce any toxic effects of drug compounds to the host and to be more selective towards the parasite. Furthermore, preclinical studies show that different nanomaterials can carry different Leishmania antigens, or even act as adjuvants to improve a Th1 immune response in an attempt to produce an effective vaccine.

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.

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.

Impact of Toxoplasma gondii infection on TM3 Leydig cells: Alterations in testosterone and cytokines levels.

Leydig cells play pivotal roles in eliciting male characteristics by producing testosterone and any damage to these cells can compromise male fertility Toxoplasma gondii (T. gondii) is an intracellular parasite capable to invade any nucleated cell, including cells from male reproductive system. Herein, we evaluated the capacity of RH strain of T. gondii to infect TM3 Leydig cells and the impact of this infection on testosterone and inflammatory mediators production. We first, by performing adherence, infection, and intracellular proliferation assays, we found a significant increase in the number of infected Leydig cells, peaking 48 h after the infection with T. gondii. Supernatants of TM3 infected cells exhibited, in a time-dependent manner, increased levels of testosterone as well as monocyte chemoattractant protein-1 (MCP-1) and interferon-γ (IFN-γ), which is correlated with the robust T. gondii infection. In conclusion, our study provides new insights regarding the harmful effects of T. gondii infection on male reproductive system.

IgG reactivity profile to Paracoccidioides spp. antigens in people with asymptomatic Paracoccidioidomycosis.

Introduction. Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. As the disease is known to affect mostly men over 40 years old who previously worked handling soil, some cities of agricultural economy in endemic regions may have more cases of paracoccidioidal infection.Gap statement. The true frequency of PCM cannot be established in Brazil because it is not a disease of mandatory reporting. The detection of paracoccidioidal infection may assist in the planning of health services, in order to provide early detection of the disease and to prevent its worsening or even progression to death. In addition, little is described about sera reactivity with antigens from different species of Paracoccidiodes, especially P. lutzii.Aim. Current research was conducted in an inland municipality of southern Brazil, in order to assess infection rate within this endemic region of PCM disease.Methodology. ELISA was employed to evaluate 359 sera from random volunteers from Guarapuava, Paraná, Brazil, to detect IgG against cell-free antigens (CFA) from P. restrepiensis B339, P. americana LDR3 and P. lutzii LDR2. Confirmatory ELISA employed gp43 from B339. Reduction of cross-reactions was sought by treatment with sodium metaperiodate (SMP-CFA, SMP-gp43). Immunoblot was performed with 37 selected sera among those reactive in ELISA. Epidemiological profile was assessed by questionnaire.Results. ELISA reactivity was: CFA/SMP-CFA in general 37.3/17.8 %, B339 25.3/14.5 %, LDR3 24.5/1.4 %, LDR2 8.3/5.8 %; gp43/SMP-gp43 7.2/4.7 %. There were sera reactive with multiple CFAs. In immunoblot, five sera showed the same reaction profile with P. lutzii's antigens as PCM disease sera. Rural residence and soil-related professions were risk factors for paracoccidioidal infection.Conclusion. The low prevalence is in accordance with previous reports of lower PCM disease endemicity in Guarapuava than in other areas of Paraná. Although P. brasiliensis seems to be the prevalent strain of the region, 21 sera from people who only lived in Guarapuava reacted with P. lutzii LDR2. CFA-ELISA with whole antigens seems a good option for serological screening in epidemiological surveys.

Unraveling the susceptibility of paracoccidioidomycosis: Insights towards the pathogen-immune interplay and immunogenetics.

Paracoccidioidomycosis (PCM) is a life-threatening systemic mycosis caused by Paracoccidioides spp. This disease comprises three clinical forms: symptomatic acute and chronic forms (PCM disease) and PCM infection, a latent form without clinical symptoms. PCM disease differs markedly according to severity, clinical manifestations, and host immune response. Fungal virulence factors and adhesion molecules are determinants for entry, latency, immune escape and invasion, and dissemination in the host. Neutrophils and macrophages play a paramount role in first-line defense against the fungus through the recognition of antigens by pattern recognition receptors (PRRs), activating their microbicidal machinery. Furthermore, the clinical outcome of the PCM is strongly associated with the variability of cytokines and immunoglobulins produced by T and B cells. While the mechanisms that mediate susceptibility or resistance to infection are dictated by the immune system, some genetic factors may alter gene expression and its final products and, hence, modulate how the organism responds to infection and injury. This review outlines the main findings relative to this topic, addressing the complexity of the immune response triggered by Paracoccidioides spp. infection from preclinical investigations to studies in humans. Here, we focus on mechanisms of fungal pathogenesis, the patterns of innate and adaptive immunity, and the genetic and molecular basis related to immune response and susceptibility to the development of the PCM and its clinical forms. Immunogenetic features such as HLA system, cytokines/cytokines receptors genes and other immune-related genes, and miRNAs are likewise discussed. Finally, we point out the occurrence of PCM in patients with primary immunodeficiencies and call attention to the research gaps and challenges faced by the PCM field.

4-nitrochalcone exerts leishmanicidal effect on L. amazonensis promastigotes and intracellular amastigotes, and the 4-nitrochalcone encapsulation in beeswax copaiba oil nanoparticles reduces macrophages cytotoxicity.

The Leishmaniasis treatment currently available involves some difficulties, such as high toxicity, variable efficacy, high cost, therefore, it is crucial to search for new therapeutic alternatives. Over the past few years, research on new drugs has focused on the use of natural compounds such as chalcones and nanotechnology. In this context, this research aimed at assessing the in vitro leishmanicidal activity of free 4-nitrochalcone (4NC) on promastigotes and encapsulated 4NC on L. amazonensis-infected macrophages, as well as their action mechanisms. Free 4NC was able to reduce the viability of promastigotes, induce reactive oxygen species production, decrease mitochondrial membrane potential, increase plasma membrane permeability, and expose phosphatidylserine, in addition to altering the morphology and lowering parasite cellular volume. Treatment containing encapsulated 4NC in beeswax-copaiba oil nanoparticles (4NC-beeswax-CO Nps) did not alter the viability of macrophages. Furthermore, 4NC-beeswax-CO Nps reduced the percentage of infected macrophages and the number of amastigotes per macrophages, increasing the production of reactive oxygen species, NO, TNF-α, and IL-10. Therefore, free 4NC proved to exert anti-promastigote effect, while 4NC-beeswax-CO Nps showed a leishmanicidal effect on L. amazonensis-infected macrophages by activating the macrophage microbicidal machinery.

Diethyldithiocarbamate encapsulation reduces toxicity and promotes leishmanicidal effect through apoptosis-like mechanism in promastigote and ROS production by macrophage.

The use of compounds from natural or synthetic sources and nanotechnology may represent an alternative to develop new drugs for the leishmaniasis treatment. DETC is an inhibitor of the SOD1 enzyme, which leads to increased ROS production, important for the elimination of Leishmania. Thus, our objective was to assess the leishmanicidal in vitro effect of free Diethydithiocarbamate (DETC) and DETC loaded in beeswax-copaiba oil nanoparticles (DETC-Beeswax-CO Nps) on L. amazonensis forms and elucidate the possible mechanisms involved in the parasite death. DETC-Beeswax-CO Nps presented size below 200 nm, spherical morphology, negative zeta potential, and high encapsulation efficiency. Free DETC reduced the viability of promastigotes and increase ROS production, lower the mitochondrial membrane potential, cause phosphatidylserine exposure, and enhance plasma membrane permeability, in addition to promoting morphological changes in the parasite. Free DETC proved toxic in the assessment of toxicity to murine macrophages, however, the encapsulation of this compound was able to reduce these toxic effects on macrophages. DETC-Beeswax-CO Nps exerted anti-amastigote effect by enhancing the production of ROS, superoxide anion, TNF-α, IL-6, and reduced IL-10 in macrophages. Therefore, free DETC induces antipromastigote effect by apoptosis-like; and DETC-Beeswax-CO Nps exerted anti-leishmanial effect due to pro-oxidant and pro-inflammatory response.

Biogenic silver nanoparticles reduce adherence, infection, and proliferation of toxoplasma gondii RH strain in HeLa cells without inflammatory mediators induction.

The highlights of biogenic silver nanoparticles (AgNp-Bio) include low toxicity - depending on size and concentration - and efficient antiparasitic activity. Therefore, the objective of this study was to assess the action of the AgNp-Bio on HeLa cells in an infection with strain of RH Toxoplasma gondii. Firstly, we performed a cellular viability test and characterized the AgNp-Bio to proceed with the infection of HeLa cells with T. gondii to be treated using AgNp-Bio or conventional drugs. Subsequently, we determined the level of standard cytokines Th1/Th2 as well as the content of nitric oxide (NO) and reactive oxygen species (ROS). Results indicated a mean size of 69 nm in diameter for the AgNp-Bio and obtained a dose-dependent toxicity. In addition, the concentrations of 3 and 6 µM promoted a significant decrease in adherence, infection, and intracellular proliferation. We also found lower IL-8 and production of inflammatory mediators. Thus, the nanoparticles reduced the adherence, infection, and proliferation of ROS and NO, in addition to immunomodulating the IL-8. Therefore, our data proved relevant to introduce a promising therapeutic alternative to toxoplasmosis.

Investigating the role of circulating microRNAs in human immunodeficiency virus infection: friends or foes

Introduction: Human immunodeficiency virus (HIV), which causes Acquired Immunodeficiency Syndrome, still affects millions of people worldwide. Despite recent advances in the understanding of biological mechanisms of viral replication, there are relevant gaps regarding the virus-host relationship. Unraveling these complexities may lead to the development of new therapeutic strategies and the establishment of new biomarkers useful for the diagnosis and prognosis of infection and its comorbidities. Therefore, in this study we discuss the main biological characteristics of microRNAs and the potential use of these nucleic acids in their free circulating form as indicators of risk or protection against HIV infection. Methods: A narrative review of the literature was carried out in the following databases through keyword and/or health descriptor searches: i) Google Scholar; ii) CAPES periodicals portal; iii) United States National Library of Medicine (PubMed) and iv) Elsevier's Science Direct library. The keywords "microRNA; HIV infection; circulating microRNA; biomarkers" were used to search the databases as mentioned above.Results: Circulating microRNAs (ci-miRNA) are closely related to numerous processes in the HIV infection pathophysiology. They are involved in viral latency, increased viremia, hepatic injury, heart dysfunction, pulmonary hypertension, immune response impairment, and participate in Kaposi's sarcoma pathology. Additionally, these molecules may indicate protection in elite controllers, reduce viral replication and load, and be useful markers of the infection's eclipse phase. Conclusion: Ci-miRNA levels are altered levels in individuals with HIV, playing a dual role in infection. Advances in research have shown that ci-miRNAs could differentiate stages of HIV infection and diseases associated with a viral infection and serve as biomarkers for antiretroviral therapy's effectiveness through changes in their expression. (AU)

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.

Quercetin promotes antipromastigote effect by increasing the ROS production and anti-amastigote by upregulating Nrf2/HO-1 expression, affecting iron availability.

American cutaneous leishmaniasis is a zoonotic disease caused by protozoans of the genus Leishmania. The treatment of cutaneous leishmaniasis is unsatisfactory, thus, much research effort has been focused on investigating new compounds with lower collateral effects to the patients and derived from low-cost sources, such as natural products. In the present study, we evaluated the in vitro directly effect of the flavonoid quercetin against Leishmania (Viannia) braziliensis. Quercetin inhibited the proliferation of promastigote forms at all tested concentrations, these effect were due to increasing the reactive oxygen species (ROS) production, phosphatidylserine exposure and loss of plasma membrane integrity. Moreover, quercetin reduced the number of parasites in L. braziliensis-infected macrophages, reducing the levels of TNF-α and increasing IL-10 synthesis without modulate nitric oxide (NO) production. In addition, quercetin upregulated Nrf2/HO-1 expression and modulated the labile iron pool in infected macrophages, culminating in a depletion of available iron for L. braziliensis replication.

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.

Diethyldithiocarbamate loaded in beeswax-copaiba oil nanoparticles obtained by solventless double emulsion technique promote promastigote death in vitro.

Leishmaniasis is considered a neglected tropical disease that represents a Public Health problem due to its high incidence. In the search of new alternatives for Leishmaniasis treatment diethyldithiocarbamate (DETC) has shown an excellent leishmanicidal activity and the incorporation into drug carrier systems, such as solid lipid nanoparticles (SLNs), is very promising. In the present work DETC loaded in beeswax nanoparticles containing copaiba oil were obtained by the double emulsion/melt technique. The nanoparticles were characterized and leishmanicidal activity against L. amazonensis promastigotes forms and cytotoxicity in murine macrophages were evaluated. SLNs presented size below 200 nm, spherical morphology, negative charge surface, high encapsulation efficiency, above 80%, and excellent stability. Moreover, Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) analyses were performed to evaluate the chemical structure and possible interactions between DETC and SLNs. SLNs provided a protection for DETC, decreasing its cytotoxic effects in macrophages, which led to an improvement in the selectivity against the parasites, which almost doubled from free DETC (11.4) to DETC incorporated in SLNs (18.2). These results demonstrated that SLNs had a direct effect on L. amazonensis promastigotes without affect the viability of macrophage cell, can be a promising alternative therapy for the cutaneous treatment of L. amazonensis.

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.

Trypanocidal activity of copaiba oil and kaurenoic acid does not depend on macrophage killing machinery.

Activity, mechanisms of action, and toxicity of natural compounds have been investigated in a context in which knowledge on which pathway is activated remains crucial to understand the action mechanism of these bioactive substances when treating an infected host. Herein, we showed an ability of copaiba oil and kaurenoic acid to eliminate Trypanosoma cruzi forms by infected macrophages through other mechanisms in addition to nitric oxide, reactive oxygen species, iron metabolism, and antioxidant defense. Both compounds induced an anti-inflammatory response with an increase in IL-10 and TGF-ß as well as a decrease in IL-12 production. Despite being able to modulate the immune response in host cells, the antimicrobial activity of copaiba oil and kaurenoic acid seems to be a direct action of the compounds on the parasites, causing their death.

Dehydroabietic acid isolated from Pinus elliottii exerts in vitro antileishmanial action by pro-oxidant effect, inducing ROS production in promastigote and downregulating Nrf2/ferritin expression in amastigote forms of Leishmania amazonensis.

Dehydroabietic acid (DHA) is one of the main constituents of the resin that have antiprotozoal activity against Leishmania spp., but the leishmanicidal mechanism is unknown. The objective of the study was to investigate in vitro the leishmanicidal activity of the natural compound DHA against intracellular and extracellular forms of L. amazonensis and the mechanism of action involved. The antileishmanial activity of DHA was evaluated in vitro against promastigote forms of L. amazonensis by counting in Neubauer chamber. The morphological changes were observed by scanning electron microscopy and cell death mechanism by fluorescence assay using 2',7'-dichlorofluorescein diacetate probe (H2DCFDA), tetramethylrhodamine ethyl ester (TMRE), annexin-V and propidium iodide (PI). The antiamastigote effect was observed by counting the number of amastigotes per macrophage and percentage of infected cells. In addition, reactive oxygen species (ROS) production, nitric oxide (NO), cytokines, free iron and total iron-binding capacity (TIBC), expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and ferritin were evaluated. DHA inhibited the proliferation of promastigotes at all times tested. The compound (IC50, 40 ±â€¯0.1458 µg/mL) altered the morphology of the promastigote forms, caused mitochondrial depolarization, induced ROS production, increased phosphatidylserine exposure and caused loss of plasma membrane integrity. DHA also reduced the number of amastigotes and the percentage of infected macrophages by increasing ROS production, free iron and TIBC, and also caused downregulation of Nrf2 and ferritin expression. DHA was effective in the elimination of L. amazonensis both in its promastigote forms by apoptosis-like mechanisms and intracellular form by ROS production.