Milena Menegazzo Miranda-Sapla

Brazilian propolis promotes immunomodulation on human cells from American Tegumentar Leishmaniasis patients and healthy donors infected with L. braziliensis

American Tegumentar Leishmaniasis (ATL) is an infectious disease caused by Leishmania parasites with ineffective treatment. The properties of propolis have been studied in different experimental studies, however, few works have investigated the effects of propolis on human-derived peripheral blood mononuclear cells (PBMC) in leishmaniasis models. Thus, we investigate the immunomodulatory effects of propolis treatment on PBMC from ATL patients and on PBMC from healthy donors infected with Leishmania braziliensis. Our data demonstrate that propolis pretreatment shows immunomodulatory effects on both healthy donors and ATL patients adherent cells, increasing IL-4 and IL-17 and decreasing IL-10, in either the presence or absence of the L. braziliensis infection, demonstrating that propolis contributes with the decrease of the inflammation and could also contribute with parasite control.

Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×105) were infected with T. gondii tachyzoites (5×105) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×105) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms of T. gondii, giving these drugs new therapeutic uses.

Biogenic silver nanoparticles inducing Leishmania amazonensis promastigote and amastigote death in vitro

American Cutaneous Leishmaniasis (ACL) is a zoonosis caused by Leishmania protozoa. The ACL chemotherapy available is unsatisfactory motivating researches to seek alternative treatments. In this study, we investigated the action of biogenic silver nanoparticle (AgNp-bio) obtained from Fusarium oxysporium, against Leishmania amazonensis promastigote and amastigote forms. The AgNp-bio promastigote treatment results in promastigote death leading to apoptosis-like events due an increased production of reactive oxygen species (ROS), loss of mitochondrial integrity, phosphatidylserine exposure and damage on promastigotes membrane. In L. amazonensis infected macrophages, AgNp-bio treatment was still able to reduce the percentage of infected macrophages and the amount of amastigotes per macrophage, consequently, the amount of promastigotes recovered. This leishmanicidal effect was also accompanied by a decrease in the levels of ROS and nitric oxide. By observing the ultrastructural integrity of the intracellular amastigotes, we found that the AgNp-bio treatment made a significant damage, suggesting that the compound has a direct effect on intracellular amastigotes. These results demonstrated that AgNp-bio had a direct effect against L. amazonensis forms and acted on immunomodulatory ability of infected macrophages, reducing the infection without inducing the synthesis of inflammatory mediators, which continuous stimulation can generate and aggravate leishmaniotic lesions. Overall, our findings suggest that the use of AgNp-bio stands out as a new therapeutic option to be considered for further in vivo investigations representing a possible treatment for ACL.

Caryocar coriaceum extracts exert leishmanicidal effect acting in promastigote forms by apoptosis-like mechanism and intracellular amastigotes by Nrf2/HO-1/ferritin dependent response and iron depletion: Leishmanicidal effect of Caryocar coriaceum leaf exracts

Leishmania (L.) amazonensis is the American Cutaneous Leishmaniasis-causing agents, and the available drugs for this disease present toxicity, low efficiency and difficulty of administration. Plants belong23ing to the Caryocar genus are found in Brazilian Cerrado, where fruits are used as food and in folk medicine, and previous studies showed several biological effects of extracts of this plant. The present work evaluated the leishmanicidal and immunomodulatory activity of ethyl acetate (EAC) and methanol (MET) C. coriaceum leaf extracts EAC and MET showed an antipromastigote effect after 24, 48 and 72 h. The extracts also induced loss of mitochondrial membrane potential, reactive oxygen species (ROS) production, damage to the plasma membrane, and phosphatidylserine exposure on promastigote forms, and most parasites were going through a late apoptosis-like process. The range of concentrations used did not alter the viability of peritoneal macrophages of BALB/c mice; therefore, we observed that the treatment with extracts was able to reduce the infection of this cells. Thereafter, the extracts were able to significantly improve the levels of TNFα, IL-6, MCP-1, and IL-10, and reduced the levels of MDA and ROS without interfering on NO levels released by infected macrophages. In addition, both EAC and MET up-regulated Nrf2/HO-1/Ferritin expression and reduced the labile iron pool in infected macrophages. Based on the data obtained, it is possible to infer that different solvent extracts of the C. coriaceum leaves exert leishmanicidal effect, acting on promastigote forms through apoptosis-like mechanisms and intracellular amastigote forms involving a Nrf2/HO-1 dependent antioxidant response, which culminates in a depletion of available iron for L. amazonensis replication.

Flavonoid Composition and Biological Activities of Ethanol Extracts of Caryocar coriaceum Wittm., a Native Plant from Caatinga Biome

Caryocar coriaceum fruits, found in Brazilian Cerrado and Caatinga, are commonly used as food and in folk medicine, as anti-inflammatory, bactericide, fungicide, leishmanicide, and nematicide. Due to the biological potential of this plant, this study focuses on the evaluation of antifungal and antileishmanial activities, including anticholinesterase and antioxidant tests, correlating with total phenols and flavonoids content. Peel extracts contain higher yield of phenols and flavonoids as analyzed by spectrophotometric methods. HPLC analysis of flavonoids revealed that isoquercitrin is the main flavonoid in both parts of the fruit, and peel extract showed the best antioxidant activity. In the inhibition of the acetylcholinesterase assay, both extracts demonstrate action comparable to physostigmine. The antimicrobial activity of extracts was evaluated against strains of Malassezia sp. and Microsporum canis, using the broth microdilution technique, in which the extracts showed similar MIC and MFC. The extracts present antileishmanial activity and low toxicity on murine macrophages and erythrocytes. Therefore, these results suggest a potential for the application of C. coriaceum fruits ethanol extracts in the treatment against dermatophyte fungi and leishmaniasis, probably due to the presence of active flavonoids. Further in vivo studies are recommended aiming at the development of possible new pharmaceutical compounds.

Nanotechnology as a potential therapeutic alternative for schistosomiasis

Schistosomiasis is a neglected disease that affects millions of people worldwide, recognized as the most important human helminth infection in terms of morbidity and mortality. The treatment of choice presents low bioavailability and water solubility, in addition to the induction of parasite resistance. In this context, researchers have been conducting studies seeking to develop new drugs to ensure safety, quality, and efficacy against this parasitosis. In this scenario, nanotechnology arises including the drug delivery systems in nanoscale: nanoemulsions, liposomes and nanoparticles. These drug delivery systems have been extensively applied for in vitro and in vivo studies against Schistosoma spp. with promising results. This review pointed out the most relevant development scenarios regarding the treatment of schistosomiasis as well as the application of nanotechnology as a vaccine, highlighting the use of nanotechnology as an alternative therapy for both the repositioning of drugs and the use of new pharmaceutical products, with promising results regarding the aforementioned disease.

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.

Leishmanicidal and fungicidal activity of lipases obtained from endophytic fungi extracts

This work describes the production of lipases from endophytic fungi: Vermisporium-like, Emericella nidulans, Dichotomophtora portulacae and D. boerhaaviae and the biological activity against the dermatophyte fungi Malassezia sp and Microsporum canis and the parasite Leishmania amazonensis. All fungal enzymes extract showed lipolysis action in the media that contains long carbon chain lipids. The proteomic analysis of lipases exhibits several molecules mostly ranging in size from 220 to 20 kDa, with clear differences in protein profile’s yield. All fungal enzymes were competent to eliminate promastigote forms of Leishmania amazonensis at 5 mg.mL-1. The antileishmanial activity of lipases from Vermisporium-like, E. nidulans, D. portulacae and D. boerhaaviae in amastigote forms, promoted the reduction in viability of 78.88, 39.65, 63.17 and 98.13%, with selectivity index of 19.56, 30.68, 18.09 and 20.99. In relation to antifungal activity, Dichothomophtora enzymes demonstrate best action with MFC of 14.65 μg.mL-1 against Malassezia sp and Microsporum canis, respectively. These results allow us to infer that lipases from entophytic fungi displays activity against dermatophyte fungi (Malassezia sp. and Microsporum canis) as well as Leishmania.

Concanavalin-A displays leishmanicidal activity by inducing ROS production in human peripheral blood mononuclear cells

Abstract The context of the article: Leishmania amazonensis has a wide geographical distribution throughout South American countries and can cause self-healing to severe cases as mucocutaneous or visceral forms. Leishmaniasis presents a balance of inflammatory and anti-inflammatory cytokines which is responsible for promoting the activation of phagocytes, essential to control the infection and lead to tissue repair/resolution of the disease, respectively. Results and discussion: Our model revealed that the treatment with Con-A was capable to stimulate human PBMC cells by increasing the phagocytic capacity and promoting parasite elimination. The pretreatment with Con-A promoted inflammatory (IFN-γ, TNF-α, IL-2 and IL-6) and anti-inflammatory (IL-4 and IL-10) cytokines production, increased the reactive oxygen species (ROS) sinthesys as well as the expression and presence of iNOS enzyme, but not nitric oxide production. Conclusion: Based on the data obtained, it was possible to infer that Con-A induces the ROS production, responsible for eliminating parasites in addition to regulatory cytokines synthesis which are important for disease resolution.