Noemi Nosomi Taniwaki

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis.

Antiparasitic, antineuroinflammatory, and cytotoxic polyketides from the marine sponge Plakortis angulospiculatus collected in Brazil.

Investigation of the bioactive crude extract from the sponge Plakortis angulospiculatus from Brazil led to the isolation of plakortenone ( 1) as a new polyketide, along with five known polyketides ( 2- 6) previously isolated from other Plakortis sponges. The known polyketides were tested in antileishmanial, antitrypanosomal, antineuroinflammatory, and cytotoxicity assays. The results show that plakortide P ( 3) is a potent antiparasitic compound, against both Leishmania chagasi and Trypanosona cruzi, and exhibited antineuroinflammatory activity. The known polyketides 2- 6 were tested for cytotoxicity against four human cancer cell lines, but displayed only moderate cytotoxic activity.

Intracellular localization of myeloperoxidase in murine peritoneal B-lymphocytes and macrophages.

Generation of hypochlorous acid (HOCl), an important microbicidal agent, is considered to be the main function of myeloperoxidase (MPO), an enzyme present in phagocytes. High amounts of MPO are present in neutrophil azurophilic granules, which are mobilized into the phagolysosome vacuole during phagocytosis. MPO is also present in monocytes and macrophages, although to a lesser degree than in neutrophils. In the present study, we investigated the distribution of MPO in murine peritoneal cells using flow cytometry, confocal microscopy (CM) and transmission electron microscopy (TEM). MPO was observed in macrophages, and surprisingly, we detected MPO in B lymphocytes, specifically in B1-a. MPO was present in cytoplasmic granules, vesicles, mitochondria and the nucleus of murine peritoneal cells. Together, these findings suggest that, in addition to its known microbicidal activity, MPO has a myriad of other unanticipated cellular functions.

In Vitro Antileishmanial Activity of Essential Oil of Vanillosmopsis arborea (Asteraceae) Baker

The search for new immunopharmacological chemical agents to treat various diseases caused by bacteria, fungi, and protozoa, such as leishmaniasis, for example, has led to the exploration of potential products from plant species and their main active ingredients. Antimonial drugs are the current treatment for leishmaniasis. These drugs cause major side effects and frequent discontinuation of treatment. In this study, we evaluated the in vitro leishmanicidal activity of essential oil of Vanillosmopsis arborea (VAEO) and its major compound α-bisabolol against Leishmania amazonensis. The essential oil and α-bisabolol showed activity against promastigotes (IC50 7.35 and 4.95 μg/mL resp.) and intracellular amastigotes (IC50 12.58 and 10.70 μg/mL, resp.). Neither product showed any cytotoxicity on treated macrophages. The ultrastructural analysis of promastigotes incubated with VAEO or α-bisabolol at 30 μg/mL, showed morphological changes with the accumulation of vesicles electrodense lipid inclusions. The results give evidence that both VAEO and α-bisabolol have potential as new therapeutic agents against leishmaniasis.

In vitro evaluation of (-)α-bisabolol as a promising agent against Leishmania amazonensis.

Current treatments for leishmaniasis present some difficulties due to their toxicity, the use of the intravenous route for administration and therapy duration, which may lead to treatment discontinuation. The aim of this study is to investigate new treatment alternatives to improve patients well being. Therefore, we evaluated the inhibitory effect of (-)α-bisabolol, a sesquiterpene alcohol found in various essential oils of different plant species, against the promastigotes and intracellular amastigotes forms of Leishmania amazonensis, as well as the cytotoxic, morphological and ultrastructural alterations of treated cells. Promastigotes forms of L. amazonensis were incubated with (-)α-bisabolol to determine the antileishmanial activity of this compound. The cytotoxicity effect was evaluated by testing against J774.G8 cells. After these tests, the infected and uninfected cells with L. amazonensis were used to determine if the (-)α-bisabolol was able to kill intracellular parasites and to cause some morphological changes in the cells. The (-)α-bisabolol compound showed significant antileishmanial activity against promastigotes with a 50% effective concentration of 8.07 µg/ml (24 h) and 4.26 µg/ml (48 h). Against intracellular amastigotes the IC50 (inhibitory concentration) of (-)α-bisabolol (24 h) was 4.15 µg/ml. The (-)α-bisabolol also showed a cytotoxic effect against the macrophage strain J774.G8. The value of 50% cytotoxic concentration was 14.82 µg/ml showing that (-)α-bisabolol is less toxic to macrophages than to the parasite. Ultrastructural studies of treated promastigotes and amastigotes showed several alterations, such as loss of cytoplasmic organelles, including the nucleus, and the presence of lipid inclusions. This study showed that (-)α-bisabolol has promising antileishmanial properties, as it can act against the promastigote forms and is able to penetrate the cell, and is also active against the amastigote forms. About 69% of the promastigotes forms suffered mitochondrial membrane damage after treatment with IC50 of (-)α-bisabolol, suggesting inhibition of the metabolic activity of parasites. These results open new prospects for research that can contribute to the development of products based on essential oils or isolated compounds from plants for the treatment of cutaneous leishmaniasis.

Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes

Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. The host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. In parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca2+ fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.

Disruption of myofibrillar proteins in cardiac muscle of Calomys callosus chronically infected with Trypanosoma cruzi and treated with immunosuppressive agent.

Calomys callosus (Rodentia: Cricetidae) chronically infected with CL strain of Trypanosoma cruzi undergo recrudescence of the acute phase when treated with the immunosuppressor cyclophosphamide. The distribution of cytoskeletal proteins in cardiac tissue of immunosuppressed animals was mapped by immunofluorescence and electron microscopy to evaluate myofibrillar distribution during the intracellular life cycle of T. cruzi. Cardiac muscle sections showed enhancement of myocarditis and parasite proliferation after immunosuppression. Immunofluorescence using monoclonal antibodies against myosin, actin, desmin, titin, tropomyosin, and troponin T demonstrated disruption and loss of contractile proteins, such as myosin and actin. Desmin and titin were irregularly distributed in close proximity to parasite nests. Ultrastructural observations confirmed alterations of cardiac cells with Z-line fragmentation, indistinguishable I-bands and A-bands, and loss of myofibrillar elements. The disruption of the muscle cell architecture was greater as infection progressed, probably as a result of increased myocarditis and physical displacement due to the activity of flagellated parasites.

Antiviral Action of Hydromethanolic Extract of Geopropolis from Scaptotrigona postica against Antiherpes Simplex Virus (HSV-1)

The studies on chemical composition and biological activity of propolis had focused mainly on species Apis mellifera L. (Hymenoptera: Apidae). There are few studies about the uncommon propolis collected by stingless bees of the Meliponini tribe known as geopropolis. The geopropolis from Scaptotrigona postica was collected in the region of Barra do Corda, Maranhão state, Brazil. The chemical analysis of hydromethanolic extract of this geopropolis (HMG) was carried out through HPLC-DAD-ESI-MS/MS and the main constituents found were pyrrolizidine alkaloids and C-glycosyl flavones. The presence of alkaloids in extracts of propolis is detected for the first time in this sample. The antiviral activity of HMG was evaluated through viral DNA quantification experiments and electron microscopy experiments. Quantification of viral DNA from herpes virus showed reduction of about 98% in all conditions and concentration tested of the HMG extract. The results obtained were corroborated by transmission electron microscopy, in which the images did not show particle or viral replication complex. The antiviral activity of C-glycosyl flavones was reported for a variety of viruses, being observed at different points in the viral replication. This work is the first report about the antiviral activity of geopropolis from Scaptotrigona postica, in vitro, against antiherpes simplex virus (HSV).

Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum.

Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZs activity against L. infantum was confirmed, producing IC50 values of 42.71μg/mL against promastigotes and 6.78μg/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX(®) Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets.

Ultrastructural Changes and Death of Leishmania infantum Promastigotes Induced by Morinda citrifolia Linn. Fruit (Noni) Juice Treatment

The search for new treatments against leishmaniasis has increased due to high frequency of drug resistance registered in endemics areas, side effects, and complications caused by coinfection with HIV. Morinda citrifolia Linn., commonly known as Noni, has a rich chemical composition and various therapeutic effects have been described in the literature. Studies have shown the leishmanicidal activity of M. citrifolia; however, its action on the parasite has not yet been elucidated. In this work, we analyzed leishmanicidal activity and ultrastructural changes in Leishmania infantum promastigotes caused by M. citrifolia fruit juice treatment. M. citrifolia fruit extract showed a yield of 6.31% and high performance liquid chromatography identified phenolic and aromatic compounds as the major constituents. IC50 values were 260.5 µg/mL for promastigotes and 201.3 µg/mL for intracellular amastigotes of L. infantum treated with M. citrifolia. Cytotoxicity assay with J774.G8 macrophages showed that M. citrifolia fruit juice was not toxic up to 2 mg/mL. Transmission electron microscopy showed cytoplasmic vacuolization, lipid inclusion, increased exocytosis activity, and autophagosome-like vesicles in L. infantum promastigotes treated with M. citrifolia fruit juice. M. citrifolia fruit juice was active against L. infantum in the in vitro model used here causing ultrastructural changes and has a future potential for treatment against leishmaniasis.