Fernanda Tomiotto-Pellissier

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.

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.

Galleria mellonella hemocytes: A novel phagocytic assay for Leishmania (Viannia) braziliensis.

Galleria mellonella is an excellent invertebrate model for the study of diseases that involve interactions with cells from the innate immune system, since they have an innate immune system capable of recognizing the pathogens. Here we present for the first time, an alternative model for an in vitro phagocytic assay using hemocytes of G. mellonella larvae to study infection by Leishmania (Viannia) braziliensis. We showed that the insect phagocytic cells were able to engulf promastigotes. Furthermore, this infective form differentiated into the amastigote form inside those cells. However, the cells in this model seem resistant to the parasite, since amastigotes were depleted after 24h and NO levels were maintained after infection. Our model opens an avenue of possibilities for new investigations regarding other Leishmania species, mechanisms of invasion and evasion, receptors involved, release of signaling molecules and, above all, it is a novel infection model using invertebrate animals.

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.

Antileishmanial Activity and Inducible Nitric Oxide Synthase Activation by RuNO Complex

Parasites of the genus Leishmania are capable of inhibiting effector functions of macrophages. These parasites have developed the adaptive ability to escape host defenses; for example, they inactivate the NF-κB complex and suppress iNOS expression in infected macrophages, which are responsible for the production of the major antileishmanial substance nitric oxide (NO), favoring then its replication and successful infection. Metal complexes with NO have been studied as potential compounds for the treatment of certain tropical diseases, such as ruthenium compounds, known to be exogenous NO donors. In the present work, the compound cis-[Ru(bpy)2SO3(NO)]PF6, or RuNO, showed leishmanicidal activity directly and indirectly on promastigote forms of Leishmania (Leishmania) amazonensis. In addition, treatment with RuNO increased NO production by reversing the depletion of NO caused by Leishmania. We also found increased expression of Akt, iNOS, and NF-κB in infected and treated macrophages. These results demonstrated that RuNO was able to kill the parasite by NO release and modulate the transcriptional capacity of the cell.

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.

Sodium nitroprusside has leishmanicidal activity independent of iNOS

INTRODUCTION Leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania . Cutaneous leishmaniasis is the most common form, with millions of new cases worldwide each year. Treatments are ineffective due to the toxicity of existing drugs and the resistance acquired by certain strains of the parasite. METHODS We evaluated the activity of sodium nitroprusside in macrophages infected with Leishmania (Leishmania) amazonensis . Phagocytic and microbicidal activity were evaluated by phagocytosis assay and promastigote recovery, respectively, while cytokine production and nitrite levels were determined by ELISA and by the Griess method. Levels of iNOS and 3-nitrotyrosine were measured by immunocytochemistry. RESULTS Sodium nitroprusside exhibited in vitro antileishmanial activity at both concentrations tested, reducing the number of amastigotes and recovered promastigotes in macrophages infected with L. amazonensis . At 1.5µg/mL, sodium nitroprusside stimulated levels of TNF-α and nitric oxide, but not IFN-γ. The compound also increased levels of 3-nitrotyrosine, but not expression of iNOS, suggesting that the drug acts as an exogenous source of nitric oxide. CONCLUSIONS Sodium nitroprusside enhances microbicidal activity in Leishmania -infected macrophages by boosting nitric oxide and 3-nitrotyrosine.

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.