Marcos Santos

DETC Induces Leishmania Parasite Killing in Human In Vitro and Murine In Vivo Models: A Promising Therapeutic Alternative in Leishmaniasis

Background Chemotherapy remains the primary tool for treatment and control of human leishmaniasis. However, currently available drugs present serious problems regarding side-effects, variable efficacy, and cost. Affordable and less toxic drugs are urgently needed for leishmaniasis. Methodology/Principal Findings We demonstrate, by microscopy and viability assays, that superoxide dismutase inhibitor diethyldithiocarbamate (DETC) dose-dependently induces parasite killing (p<0.001) and is able to “sterilize” Leishmania amazonensis infection at 2 mM in human macrophages in vitro. We also show that DETC-induced superoxide production (p<0.001) and parasite destruction (p<0.05) were reverted by the addition of the antioxidant N-acetylcysteine, indicating that DETC-induced killing occurs through oxidative damage. Furthermore, ultrastructural analysis by electron microscopy demonstrates a rapid and highly selective destruction of amastigotes in the phagosome upon DETC treatment, without any apparent damage to the host cell, including its mitochondria. In addition, DETC significantly induced parasite killing in Leishmania promastigotes in axenic culture. In murine macrophages infected with Leishmania braziliensis, DETC significantly induced in vitro superoxide production (p = 0.0049) and parasite killing (p = 0.0043). In vivo treatment with DETC in BALB/C mice infected with Leishmania braziliensis caused a significant decrease in lesion size (p<0.0001), paralleled by a 100-fold decrease (p = 0.0087) in parasite burden. Conclusions/Significance Due to its strong leishmanicidal effect in human macrophages in vitro, its in vivo effectiveness in a murine model, and its previously demonstrated in vivo safety profile in HIV treatment, DETC treatment might be considered as a valuable therapeutic option in human leishmaniasis, including HIV/Leishmania co-infection.

Photodynamic antimicrobial chemotherapy (PACT) using phenothiazines derivatives associated with the red-orange LED against staphylococcus aureus

The objective of this study was to evaluate the bactericidal effect of photodynamic antimicrobial chemotherapy (PACT) using phenothiazinium dye (Toluidine blue O and methylene blue) at a low concentration of 1μg/mL irradiated with the red laser at doses of 2.4 e 4.8 J/cm² on strain of Staphylococcus aureus (ATCC 23529) in vitro. For this research, tests were performed in triplicate and the samples were distributed into six test groups: (L-P-) Negative control (L1+P-) and (L2+P-) bacterial suspensions were irradiated with laser energy 2.4 and 4.8 J/cm2 respectively in the absence of photosensitizer; (L1+P+) and (L2+P+) bacterial suspensions were irradiated with laser in the presence of 1μg/ml of photosensitizer and finally (L-P+) bacterial suspensions only in the presence of phenothiazinium dye. Therefore, were analyzed the potential bactericidal PACT by counting of colony-forming units and analyzed statistically (ANOVA, Tukey test, p<0.05). The results showed that the negative control group when compared with laser group (L2+P-) it was observed a statistically significant increase (p<0.01) which L2+P- showed a higher number of CFU, on the other hand when compared to L1+P- no statistically significant difference was found, relation to the groups submitted to PACT, only showed a statistically significant reduction relative to the group irradiated L2+P+ (p<0.01) that showed a decrease in the number of CFU. There was no statistically significant difference between the groups submitted to PDT (L1+P+ and L2+P+). Although the results of this study have shown a reduction in average number of colony forming units by the appropriate laser-dye treatment combination, it needs further investigation.

Influence of Laser Therapy and Muscle Relaxant on the Masseter Muscle under Occlusal Wear: An Ultrastructural Study

El objetivo del estudio fue analizar la influencia de la terapia laser de baja intensidad y del relajante muscular sobre las caracteristicas ultraestructurales del musculo masetero en el desgaste oclusal. 40 ratas macho Wistar, se dividieron al azar en cuatro grupos: grupo de control (GI), desgaste oclusal (G-II), laserterapia desgaste oclusal (G-III), y relajante muscular desgaste oclusal (G-IV). Bajo anestesia general por via intraperitoneal, los animales de los grupos II, III y IV sufrieron amputacion unilateral de las cuspides de los molares superiores e inferiores para simular una situacion de desgaste oclusal. El musculo masetero del G-III recibio la terapia con laser (830nm, 4J/cm2, 40mW, f ~ 2mm) despues del procedimiento el cual se repitio durante 14/30 dias. Los animales del G-IV fueron tratados con una inyeccion diaria de Dantroleno® (2,5 mg/Kg en 0,5 ml de H2O). Los animales fueron sacrificados con una sobredosis de anestesia general en los dias 14 y 30. Despues de la remocion de las cuspides el musculo masetero ipsilateral se extirpo y se dividio en dos, una mitad fue procesada para microscopia de luz y la otra para microscopia electronica. No hubo diferencias estadisticamente significativas entre cada grupo experimental y el control, asi como, entre los periodos en cada grupo experimental. Sin embargo, las fibras musculares en el G-II mostraron los cambios mas pronunciados. En conclusion no existe relacion causal entre las lesiones de las fibras musculares y la oclusion, a pesar que los signos de lesion de los tejidos musculares fue mas evidente en el grupo con desgaste oclusal. Los resultados indican una accion moderada ejercida por la terapia laser y de los relajantes musculares sobre el musculo esqueletico.

The biochemical characterization, stabilization studies and the antiproliferative effect of bromelain against B16F10 murine melanoma cells

Abstract The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50 °C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell proliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

Antifungal mechanism of [RuIII(NH3)4catechol]+ complex on fluconazole-resistant Candida tropicalis

Abstract Candidiasis, a major opportunistic mycosis caused by Candida sp., may comprise life‐threatening systemic infections. The incidence of non‐albicans species is rising, particularly in South America and they are frequently drug resistant, causing unresponsive cases. Thus, novel antimycotic agents are required. Here we tested the antifungal activity of [RuIII(NH3)4catechol]+ complex (RuCat), approaching possible action mechanisms on fluconazole‐resistant Candida tropicalis. RuCat significantly (P < 0.05) inhibited the growth and viability of C. tropicalis dose‐dependently (IC50 20.3 &mgr;M). Cytotoxicity of RuCat upon murine splenocytes was lower (Selectivity Index = 16). Scanning electron microscopy analysis showed pseudohyphae formation, yeast aggregation and surface damage. RuCat‐treated samples investigated by transmission electron microscopy showed melanin granule trafficking to cell surfaces and extracellular milieu. Surface‐adherent membrane fragments and extracellular debris were also observed. RuCat treatment produced intense H2DCFDA labeling, indicating reactive oxygen species (ROS) production which caused increased lipoperoxidation. ROS are involved in the fungicidal effect as N‐acetyl‐L‐cysteine completely restored cell viability. Calcofluor White chitin staining suggests that 70 or 140 &mgr;M RuCat treatment for 2 h affected cell‐wall structure. PI labeling indicated necrotic cell death. The present data indicate that RuCat triggers ROS production, lipoperoxidation and cell surface damage, culminating in selective necrotic death of drug‐resistant C. tropicalis.

Green LED Associated to 20%Hydrogen Peroxide for Dental Bleaching: Nanomorfologic Study of Enamel by Scanning Electron Microscopy

Dental bleaching is a much requested procedure in clinical dental practice and widely related to dental esthetics. The literature is contradictory regarding the effects of bleaching agents on the morphology and demineralization of enamel after bleaching. The aim of this study was to analyze in vitro by scanning electron microscopy (SEM) the effect of hydrogen peroxide at 20% at neutral pH, cured by the green LED, to evaluate the action of these substances on dental enamel. We selected 15 pre-molars, lingual surfaces were sectioned and previously marked with a central groove to take the experimental and control groups on the same specimen. The groups were divided as follows. The mesial hemi-faces were the experimental group and distal ones as controls. For morphological analysis were performed 75 electron micrographs SEM with an increase of X 43, X 220 and X 1000 and its images were evaluated by tree observers. Was also performed quantitative analysis of the determination of the surface atomic composition of the samples through microanalysis with the aid of scanning electron microscopy. The use of hydrogen peroxide at a concentration of 20% at photoactivated green LED showed no significant changes in mineral composition of the samples or the dental morphological structure of the same when compared to their controls, according to the study protocol.