Edilene O. Silva

Kojic acid, a secondary metabolite from Aspergillus sp., acts as an inducer of macrophage activation.

KA (kojic acid) is a secondary metabolite isolated from Aspergillus fungi that has demonstrated skin whitening, antioxidant and antitumour properties among others. However, limited information is available regarding its effects on macrophages, the major cell involved in cell defence. The aim of the present study was to analyse whether KA affects functional properties related to macrophage activation, such as phagocytosis and spreading ability over a substrate. Treatment of resident macrophages with 50 μg/ml KA for 1 h induced both morphological and physiological alterations in cells. Immunofluorescence microscopy revealed enhanced cell spreading and an increase in cell surface exposure, associated with a rearrangement of microtubules, actin filaments and intermediate filaments. KA also potentiated phagocytosis by macrophages, as demonstrated by the increase in phagocytic activity towards yeast, when compared to untreated cells. KA increased the production of ROS (reactive oxygen species), but not NO (nitric oxide) production. Three tests were used to assess cell viability; MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide], NR (neutral red) uptake and PI (propidium iodide) exclusion test, which showed that macrophages maintain their viability following KA treatment. Results indicate that KA can modulate macrophage activation through cytoskeleton rearrangement, increase cell surface exposure, enhance the phagocytic process and ROS production. The study demonstrates a new role for KA as a macrophage activator.

A novel function for kojic acid, a secondary metabolite from Aspergillus fungi, as antileishmanial agent.

Kojic acid (KA) is a fungal metabolite used as a topical treatment skin-whitening cosmetic agent for melasma in humans; however its potential as an anti-leishmanial agent is unknown. Chemotherapy is one of the most effective treatments for Leishmaniasis. However, the drugs available are expensive, invasive, require long-term treatment and have severe side effects. Thus, the development of new effective leishmanicidal agents is a necessity. In this study we investigated the anti-leishmanial effect of KA on L. amazonensis, following in vitro and in vivo infections. KA (50 μg/mL) was found to decrease the growth by 62% (IC50 34 μg/mL) and 79% (IC50 27.84 μg/mL) of promastigotes and amastigotes in vitro, respectively. Ultrastructural analysis of KA-treated amastigotes showed the presence of vesicles bodies into the flagellar pocket, and an intense intracellular vacuolization and swelling of the mitochondrion. During the in vitro interaction of parasites and the host cell, KA reverses the superoxide anions (O2 -) inhibitory mechanism promoted by parasite. In addition, 4 weeks after KA-topical formulation treatment of infected animals, a healing process was observed with a high production of collagen fibers and a decrease in parasite burden. Thus, these results demonstrated the great potential of KA as an anti-leishmanial compound.

Combined Kinetic Studies and Computational Analysis on Kojic Acid Analogs as Tyrosinase Inhibitors

Tyrosinase is a key enzyme in melanin synthesis and widely distributed in plants and animals tissues. In mammals, this enzyme is related to pigment production, involved in wound healing, primary immune response and it can also contribute to catecholamines synthesis in the brain. Consequently, tyrosinase enzyme represents an attractive and selective target in the field of the medicine, cosmetics and bio-insecticides. In this paper, experimental kinetics and computational analysis were used to study the inhibition of tyrosinase by analogs of Kojic acid. The main interactions occurring between inhibitors-tyrosinase complexes and the influence of divalent cation (Cu2+) in enzymatic inhibition were investigated by using molecular docking, molecular dynamic simulations and electrostatic binding free energy by using the Linear Interaction Energy (LIE) method. The results showed that the electrostatic binding free energy are correlated with values of constant inhibition (r2 = 0.97).Thus, the model obtained here could contribute to future studies of this important system and, therefore, eventually facilitate development of tyrosinase inhibitors.

Phosphatidylserine Exposure and Surface Sugars in Two Leishmania (Viannia) braziliensis Strains Involved in Cutaneous and Mucocutaneous Leishmaniasis

BACKGROUND Phosphatidylserine (PS) and surface carbohydrates (SC) are known as virulence factors that may contribute to the different clinical symptoms ranging from self-healing cutaneous leishmaniasis lesions to fatal visceral disease. Leishmania (Viannia) braziliensis causes localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). METHODS We analyzed PS exposure and SC expression associated with 2 primary L. braziliensis isolates from patients with LCL or MCL. The role of PS exposure was also addressed during promastigotes phagocytosis by macrophages. RESULTS We observed higher PS exposure on the surface of late stationary growth phase promastigotes from patients with LCL, compared with those from patients with MCL, and both strains were alive during PS display. Reduction in the infectivity index was observed during macrophage interaction with late stationary growth phase promastigotes in which PS was blocked by annexin V. The major surface carbohydrates detected on LCL and MCL promastigotes were α-Man, α-Glc, and α-Gal. However, α-β-GalNAc, although observed on the surface of the LCL strain during the late stationary growth phase was highly expressed on the surface of early stationary growth phase promastigotes. CONCLUSIONS Our results suggest that PS and SC can modulate interactions between Leishmania organisms and host cells and may be important for the outcome of the clinical course of diseases caused by L. braziliensis.

The fine structure of Garnia gonadati and its association with the host cell

Abstract Most of the studies on the fine structure of protozoa of the Apicomplexa group have been carried out with members of the Toxoplasma, Eimeria, and Plasmodium genera. In the present study we analyzed the fine structure of Garnia gonadoti parasitizing the red blood cells of the Amazonian reptile Gonatodes humeralis (Reptilia: Lacertilia). Transmission electron microscopy of thin sections showed that G. gonadoti presented all structures characteristic of the group, including the apicoplast. However, four special features were observed: (1) absence of the hemozoin (malarial) pigment; (2) a group of microtubules associated with the mitochondrion; (3) a vacuole containing electron-dense material, which resembled the acidocalcisome described in trypanosomatids; and (4) a special array of the host-cell endoplasmic reticulum around the parasitophorous vacuole.

Hematological parameters of Ameiva ameiva (Reptilia: Teiidae) naturally infected with hemogregarine: Confirmation of monocytosis

Little is known on how hematozoan infection changes reptile hematology. The lizard Ameiva ameiva is widely distributed in the Americas and is infected by hematozoan parasites. Previous studies on this lizard have shown that the parasite of monocytes causes a variety of ultrastructural changes in infected host cells. The present study reports that this infection does not cause any change to the erythrocytic values. However, a marked increase in the number of leukocytes (especially monocytes) was detected. This indicates that the hemogregarine not only modulates the infected monocyte, but also increases the blood pool of this leukocyte. A Plasmodium sp was also found infecting erythrocytes of one lizard.

Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages

BackgroundThe bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells.ResultsIncreased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa.ConclusionResults demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent.

Blood monocyte alteration caused by a hematozoan infection in the lizard Ameiva ameiva (Reptilia: Teiidae).

Although hematozoa have been described from many different host species, little is known about the infection and its relationship to the physiology of the host. We studied a hematozoan, regarded as a species of Lainsonia Landau, 1973 (Lankestereliidae), which infects the monocytes of the lizard Ameiva ameiva. The infected animals show a huge monocytosis and morphological changes in the monocytes. Ultrastructurally, the parasite has an apical complex, dense bodies, electron lucent structures, plasma membrane projections and folding which may be involved with nutrition. The parasite occupies a parasitophorous vacuole (PV) exhibiting high electron density at its membrane. Mitochondria and the Golgi complex of the monocytes were concentrated around the PV, and the cytoplasm was totally occupied by a vimentin type of intermediate filament radiating from (or to) the cytosolic surface of the PV. Vimentin was identified by diameter measurement, immunofluorescence and immunoelectron microscopy. These observations indicate that this infection alters the physiological state of the host and suggest that this parasite has the ability to modify monocyte vimentin assembly.

Leishmanicidal Activity of (+)-Phyllanthidine and the Phytochemical Profile of Margaritaria nobilis (Phyllanthaceae)

The effects of the Securinega alkaloid (+)-phyllanthidine on Leishmania (L.) amazonensis and the first chemical investigation of Margaritaria nobilis L.f. (Phyllanthaceae) are described. Treating the parasites with this alkaloid caused a dose-dependent reduction in promastigote growth of 67.68% (IC50 82.37 μg/mL or 353 µM) and in amastigote growth of 83.96% (IC50 49.11 μg/mL or 210 µM), together with ultrastructural alterations in the promastigotes. No cytotoxic effect was detected in mammalian cells (CC50 1727.48 µg/mL or CC50 5268 µM). Classical chromatographic techniques and spectral methods led to the isolation and identification of betulinic acid, kaempferol, corilagin, gallic acid and its methyl ester, besides (+)-phyllanthidine from M. nobilis leaves and stems. Margaritaria nobilis is another source of the small group of Securinega alkaloids, together with other Phyllanthaceae (Euphorbiaceae s.l.) species. The low toxicity to macrophages and the effects against promastigotes and amastigotes are suggestive that (+)-phyllanthidine could be a promising antileishmanial agent for treating cutaneous leishmaniasis.

Some observations on the fine structure of trophozoites of the haemogregarine Cyrilia lignieresi (Adeleina: Haemogregarinidae) in erythrocytes of the fish Synbranchus marmoratus (Synbranchidae)

Abstract. This communication describes the fine structure of trophozoites of the haemogregarine Cyrilia lignieresi (Laveran, 1906) found in erythrocytes of the fresh-water fish Synbranchus marmoratus from Belém, Pará, North Brazil. The parasite possesses the usual structures, such as conoid, rhoptries and micronemes, seen in members of the phylum Apicomplexa. Three structures, however, appear to be characteristic features of this parasite. The parasitophorous vacuole is unusual in containing a large number of spherical bodies. Secondly, some of the dense bodies, which are usually spherical organelles, may appear as elongated structures. Thirdly, peculiar invaginations of the inner membrane appear to divide the parasite into compartments.