Mônica Pereira Garcia

Characterisation of a Trypanosoma cruzi acidic 30 kDa cysteine protease.

A novel proteolytic activity was identified in epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Epimastigotes showed enzyme activity to be 2-fold higher than amastigotes and trypomastigotes. The protease that displays this activity was purified from epimastigote forms by a four step chromatographic procedure: Diethylaminoethyl-Sephacel, Phenyl-Sepharose, Phenyl-Superose, and Concanavalin A Sepharose columns. The purified enzyme is a glycoprotein that migrates as a 30 kDa protein in 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), under reducing conditions. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. The inhibition pattern of the purified 30 kDa protease showed that it belongs to the cysteine-protease class. In addition to the synthetic substrate, the purified protease hydrolysed bovine serum albumin (BSA) and human type I collagen. The N-terminal amino acid sequence of the protease shows similarity to the mammalian cathepsin B protease.

Biodistribution and biocompatibility of DMSA-stabilized maghemite magnetic nanoparticles in nonhuman primates (Cebus spp.)

AIM This work represents the first reported investigation on the effects of magnetic nanoparticles (MNPs) in nonhuman primates. Biodistribution, biocompatibility and nanotoxicity of maghemite nanoparticles stabilized with dimercaptosuccinic acid (DMSA) were accessed. MATERIALS & METHODS A control animal was used and three other animals were intravenously injected with DMSA-MNPs and euthanized 12 h, 30 and 90 days following administration. Extracted organs were processed by histological techniques. An additional animal was used to collect blood samples to complementarily assess biocompatibility 12 h, 7, 15, 30, 60 and 90 days after DMSA-MNP injection. RESULTS DMSA-MNPs were preferentially addressed to the lungs, liver and kidneys. Hematological and serum biochemical results corroborated histological findings, supporting DMSA-MNP biocompatibility while preserving both hepatic and renal normal activity. CONCLUSION DMSA-MNPs were preferentially distributed to the lung, liver and kidneys. Furthermore, DMSA-MNPs were considered biocompatible, supporting their application as a promising nanomaterial platform for future biomedical use.

Light Microscopical and Ultrastructural Characterization of Black Howler Monkey (Alouatta caraya) Ovarian Follicles

The present study describes the morphological characteristics of black howler monkey (Alouatta caraya) ovarian follicles. One ovary of an adult healthy black howler monkey was collected and processed for light and electron microscopy. Primordial, primary, secondary, tertiary and pre‐ovulatory follicles were evaluated for their morphometrical aspects. The ovary of black howler monkey presented a distinct conformation with a uniform distribution of the follicles mostly in the peripheric cortex. This black howler monkey ovary presented a total of 59 921 ovarian follicles. From this amount, 71.1% were classified as primordial, 18.9% as primary, 8.1% as secondary, 1.4% as tertiary and 0.5% as pre‐ovulatory follicles. From all these developmental stages, the mean diameters of follicles, oocytes, oocytes nuclei and the mean number of granulosa cells are described. Moreover, primordial, primary and secondary follicles have been observed by electron microscopy.

Photodynamic Therapy Based on Arrabidaea chica (Crajiru) ExtractNanoemulsion: In vitro Activity against Monolayers and Spheroids ofHuman Mammary Adenocarcinoma MCF-7 Cells

Natural products have been sources of numerous drugs over the history. However, little is known about the therapeutic potential of Amazon forest species. This work aimed at testing the potential of the hydrophobic extract of Arrabidaea chica (ACE), an Amazon plant, as a source of photosensitizers for anticancer photodynamic therapy. ACE was tested as a nanoemulsion (ACE-NE) produced by phase inversion temperature. It was found that ACE-NE intensely absorbs red light and, under photoactivation (λ 660 nm, 4.5 J/cm2), produces reactive oxygen species. Photoactivated ACE-NE presented, in vitro, a CC50 of 1.3 μg ACE/mL against human breast adenocarcinoma MCF-7 cells, and was effective in lysing MCF-7 spheroids. In the dark, ACE was toxic neither to human mammary epithelial MCF-10A cells nor to MCF-7 cells. On that ground, this work is the first to show that A. chica is a source of photosensitizers potentially useful for anticancer photodynamic therapy.

Morphological Analysis of Reticuloendothelial System in Capuchin Monkeys (Sapajus spp.) after Meso-2,3-Dimercaptosuccinic Acid (DMSA) Coated Magnetic Nanoparticles Administration.

Magnetic nanoparticles can be used for numerous in vitro and in vivo applications. However, since uptake by the reticuloendothelial system represents an obstacle for the achievement of nanoparticle diagnostic and therapeutic goals, the aim of the present study was to evaluate the uptake of dimercaptosuccinic acid coated magnetic nanoparticles by reticuloendothelial system phagocytic cells present in lymph nodes, spleen, and liver tissue and how the presence of these particles could have an impact on the morphology of these organs in capuchin monkeys (Sapajus spp.). Animals were intravenously injected with dimercaptosuccinic acid coated magnetic nanoparticles and euthanized 12 hours and 90 days post-injection. Organs were processed by transmission electron microscopy and histological techniques. Samples of spleen and lymph nodes showed no morphological changes. Nevertheless, liver samples collected 90 days post-administration showed slight morphological alteration in space of Disse. Moreover, morphometrical analysis of hepatic mitochondria was performed, suggesting a clear positive correlation between mitochondrial area and dimercaptosuccinic acid coated magnetic nanoparticles administration time. The present results are directly relevant to current safety considerations in clinical diagnostic and therapeutic uses of magnetic nanoparticles.

Leukocyte transepithelial migration in lung induced by DMSA functionalized magnetic nanoparticles

Magnetic nanoparticles surface-covered with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute a promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. However, they can also induce a transient transendothelial migration of leukocytes in the organ as a side effect after endovenous administration of MNPs-DMSA. We demonstrated that monocytes/macrophages constitute the main subpopulation of leukocytes involved in this process. Our recent research found that MNPs-DMSA up-regulated the mRNA expression of E-, L- and P-selectin and macrophage-1 antigen, and increased concentration of tumor necrosis factor-α in lung, in a time dependent manner. The critical relevance of the β2 integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identifies β2 integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.

Antifungal Activity of Amphotericin B Conjugated to Nanosized Magnetite in the Treatment of Paracoccidioidomycosis

This study reports on in vitro and in vivo tests that sought to assess the antifungal activity of a newly developed magnetic carrier system comprising amphotericin B loaded onto the surface of pre-coated (with a double-layer of lauric acid) magnetite nanoparticles. The in vitro tests compared two drugs; i.e., this newly developed form and free amphotericin B. We found that this nanocomplex exhibited antifungal activity without cytotoxicity to human urinary cells and with low cytotoxicity to peritoneal macrophages. We also evaluated the efficacy of the nanocomplex in experimental paracoccidioidomycosis. BALB/c mice were intratracheally infected with Paracoccidioides brasiliensis and treated with the compound for 30 or 60 days beginning the day after infection. The newly developed amphotericin B coupled with magnetic nanoparticles was effective against experimental paracoccidioidomycosis, and it did not induce clinical, biochemical or histopathological alterations. The nanocomplex also did not induce genotoxic effects in bone marrow cells. Therefore, it is reasonable to believe that amphotericin B coupled to magnetic nanoparticles and stabilized with bilayer lauric acid is a promising nanotool for the treatment of the experimental paracoccidioidomycosis because it exhibited antifungal activity that was similar to that of free amphotericin B, did not induce adverse effects in therapeutic doses and allowed for a reduction in the number of applications.

Emprego de quatro exames imunológicos na determinaçäo da prevalência da doença de Chagas nos garis do Serviço de Limpeza Urbana do Distrito Federal

Seropositivity for Trypanosoma cruzi infection was studied in 308 street-sweepers of the SLU. Federal District. Brazil, with the aid of haemaglutination. immunofluorescence and, also, a delayed-type skin test to the parasite T12E antigen. It showed 32,1%, 42.7% and 38.6% positive results, respectively for cach assay. Among these, however, only 47% were positive with each of three exams performed. In addition, 19.7% were positive with two out of three exams performed. The remaining 33-3% sera yielded one positive result out of three exams employed and were submitted to the immunoblot assay. This analysis confirmed 3 cases (37.5%) positive by hemmaglutination. 3 (11.5%) positive by skin test, and 1 (3-7%) positive by immunofluorescence. At the end of the analysis, it was shown that 129 (35%) individuals yielded at least two positive assays and therefore, they should be cousidered as T. cruzi-infected individuals.

Itraconazole encapsulated PLGA-nanoparticles covered with mannose as potential candidates against leishmaniasis: ITRACONAZOLE/MANNOSE PLGA-NANOPARTICLES AGAINST LEISHMANIASIS

Leishmaniasis is a neglected disease threatening over 350 million people. Antimonials are first-line drugs due to resistance and side effects there is a demand for alternative chemotherapy. Itraconazole (ITZ) is an antimycotic. It was encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and covered with mannose. The NPs were 250 nm and -1.1 mV ± 0.7. PLGA-ITZ-mannose NPs presented a toxicity of 20.7% for J774 cells, and no toxicity for THP 1. The J774 cells were infected with three Leishmania promastigotes strains and treated with ITZ loaded PLGA NPs with/without mannose. The parasite percentage of L.(V.) panamensis intracellular amastigotes significantly (p < 0.01) decreased from 34.4% to 13.7% and 5.7% for PLGA-ITZ-mannose NPs and PLGA-ITZ NPs, respectively. For L.(L.) infantum there was a reduction (p < 0.001) from 18.1% to 4.8% and 8.3% for PLGA-ITZ-mannose NPs and PLGA-ITZ NPs, respectively. Further with L.(L.) braziliensis amastigotes there was a significant reduction (p < 0.001) from 54.9% to 28% and 21.1% for PLGA-ITZ-mannose NPs and PLGA-ITZ NPs, respectively. Adding mannose increased the efficacy PLGA-ITZ NPs against L.(L.) infantum, while it had no effect against L(V.) panamensis and L.(L.) braziliensis amastigotes. We recommend further investigation of PLGA-ITZ-mannose NPs in animal models to evaluate their potential.