Ana Paula dos Santos

Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures

Chronic exposure to excessive levels of Mn results in a movement disorder termed manganism, which resembles Parkinsons disease (PD). The pathogenic mechanisms underlying this disorder are not fully understood. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity. In the present study, we investigated the effects of Mn on mitochondrial function. Primary astrocyte cultures were prepared from cerebral cortices of one-day-old Sprague-Dawley rats. We have examined the cellular toxicity of Mn and its effects on the phosphorylation of extracellular signal-regulated kinase (ERK) and activation of the precursor protein of caspase-3. The potentiometric dye, tetramethyl rhodamine ethyl ester (TMRE), was used to assess the effect of Mn on astrocytic mitochondrial inner membrane potential (DeltaPsi(m)). Our studies show that, in a concentration-dependent manner, Mn induces significant (p<0.05) activation of astrocyte caspase-3 and phosphorylated extracellular signal-regulated kinase (p-ERK). Mn treatment (1 and 6 h) also significantly (p<0.01) dissipates the DeltaPsi(m) in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. These results suggest that activations of astrocytic caspase-3 and ERK are involved in Mn-induced neurotoxicity via mitochondrial-dependent pathways.

Detection of TH1/TH2 cytokine signatures in yellow fever 17DD first-time vaccinees through ELISpot assay

Immunity to yellow fever (YF) is conferred by the interplay of humoral and cellular immune response. Despite the extensive literature on the humoral immune response to the YF vaccine virus, little is known about its cellular immune response to vaccination. The analysis of cytokine production by ex-vivo antigen-stimulated T cells has been considered as a valuable tool for understanding cellular immune response. Thus, we have analyzed two T(H)1/T(H)2 signature cytokines (IFN-gamma and IL-4) from 12 healthy first-time adults vaccinated with YF17DD virus. The cells, harvested on day 0 (before vaccination) and 7, 15 and 30 days after immunization were antigen-stimulated and analyzed by ELISpot. A significant increase in the number of spot-forming cells during the response to YF 17DD live virus stimulation by ELISpot assay was observed. IFN-gamma-and IL-4-producing cells were significantly increased on the 15th day after vaccination in all volunteers. These results presented herein are important for understanding the role of cytokines in the immune response to YF 17DD virus.

Lymphocyte subset analyses in healthy adults vaccinated with yellow fever 17DD virus

In this study the kinetics of humoral and cellular immune responses in first-time vaccinees and re-vaccinees with the yellow fever 17DD vaccine virus was analyzed. Flow cytometric analyses were used to determine percentual values of T and B cells in parallel to the yellow fever neutralizing antibody production. All lymphocyte subsets analyzed were augmented around the 30th post vaccination day, both for first-time vaccinees and re-vaccinees. CD3+ T cells increased from 30.8% (SE +/- 4%) to 61.15% (SE +/- 4.2%), CD4+ T cells from 22.4% (SE +/- 3.6%) to 39.17% (SE +/- 2%) with 43% of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2% (SE +/- 2.9%) to 27% (SE +/- 3%) with 70% corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7% (SE +/- 3%) to 80% (SE +/- 2.3%), CD4+ T cells from 24.9% (SE +/- 1.4%) to 40% (SE +/- 3%) presenting a percentage of 95% CD4+CD45RO+ T cells, CD8+ T cells from 19.7% (SE +/- 1.8%) to 25% (SE +/- 2%). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6% in first-time vaccinees and 20.7 to 62.6% in re-vaccinees. Regarding neutralizing antibodies, the re-vaccinees presented high titers even before re-vaccination. The levels of neutralizing antibodies of first-time vaccinees were similar to those presented by re-vaccinees at day 30 after vaccination, indicating the success of primary vaccination. Our data provide a basis for further studies on immunological behavior of the YF 17DD vaccine.

Ecological aspects and molecular detection of Leishmania DNA Ross (Kinetoplastida: Trypanosomatidae) in phlebotomine sandflies (Diptera: Psychodidae) in terra firme and várzea environments in the Middle Solimões Region, Amazonas State, Brazil

BackgroundPhlebotomine sand flies (Diptera: Psychodidae) are insects of medical importance due to the role that some species play in the transmission of leishmaniasis. This work aimed to study some ecological aspects among sand flies fauna inhabiting two different environments: the várzea (lowland Amazonian forest) and terra firme (upland Amazonian forest), both located in Tefé Municipality, Amazonas State, Braziland to detect Leishmania infection in those phlebotomine populations.MethodsSand flies were collected using HP light traps. Collection took place over the course of six months: January, February, April, August, September, and October of 2013. To detect natural infection by Leishmania, DNA samples were extracted from female sand flies and submitted to Polymerase Chain Reaction (PCR) targeting the kDNA gene; Leishmania species were identified by PCR-RFLP targeting the hsp70 gene and genetic sequencing.ResultsIn all, 5,716 individuals were collected, and 46 species were identified. Trichophoromyia ubiquitalis (3,330 – 58.26%) and Nyssomyia antunesi (661 – 11.26%) were the most abundant species. Species richness was greater in terra firme environments (42 species) than in the várzea environments (22 species), and forests ecotopes (43 species) were richer than peridomiciles (28 species). DNA of Leishmania was found in Th. ubiquitalis and Psychodopygus davisi, both of which inhabit the terra firme environment and sequencing analysis confirmed the presence of Leishmania (Viannia) lainsoni DNA in Th. ubiquitalis in Tefé Municipality.ConclusionsThe high abundance of Th. ubiquitalis and Ps. davisi and detection of DNA of Leishmania sp. may indicate that both species could be putative vectors for American Cutaneous Leishmaniasis (ACL) in the terra firme environment of Tefé. The sand fly fauna found in várzea is rich and diverse, exhibiting several species, nevertheless the seasonal hydric stress during part of the year that could influence the local diversity, if compared with other studies. This is the first report in Amazonas State of Th. ubiquitalis with presence of L. (V.) lainsoni DNA.

MOLECULAR CHARACTERIZATION OF AMERICAN CUTANEOUS LEISHMANIASIS IN THE TRI‑BORDER AREA OF ASSIS BRASIL, ACRE STATE, BRAZIL

SUMMARY In this study, Leishmaniaspecies were identified by Polymerase Chain Reaction (PCR). The epidemiology of patients suspected of having American Cutaneous Leishmaniasis in the municipality of Assis Brasil, Acre State, located in the Brazil/Peru/Bolivia triborder was also investigated. By PCR, the DNA of Leishmaniawas detected in 100% of the cases (37 samples) and a PCR-Restriction Fragment Length Polymorphism (RFLP) of the hsp 70gene identified the species in 32 samples: Leishmania (Viannia) braziliensis (65.6%) , L. (V.) shawi (28.1%) , L. (V.) guyanensis (3.1%) and mixed infection L. (V.) guyanensis and L. (Leishmania) amazonensis (3.1%)This is the first report of L. (V.) shawiand L. (L.) amazonensis in Acre. The two predominant species were found in patients living in urban and rural areas. Most cases were found in males living in rural areas for at least three years and involved in rural work. This suggests, in most cases, a possible transmission of the disease from a rural/forest source, although some patients had not engaged in activities associated with permanence in forestall areas, which indicate a possible sandflies adaptation to the periurban setting.

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

BmajPLA2-II, a basic Lys49-phospholipase A2 homologue from Bothrops marajoensis snake venom with parasiticidal potential

Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100μg/mL. The venom and BmajPLA2-II presented IC50 of 0.14±0.08 and 6.41±0.64μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64±7.94 and >150μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.

Phlebotomine Sand Fly Composition (Diptera: Psychodidae) and Putative Vectors of American Cutaneous Leishmaniasis in Porto Velho Municipality, Western Amazon, Brazil

Abstract This study evaluated the species composition of sand flies and identified potential vectors of Leishmania Ross species in rural areas of Porto Velho, Rondônia State, Brazil. American cutaneous leishmaniasis (ACL) is one of the gravest threats to public health in this state. Sand flies were collected over the course of 2014 and 2015 using HP light traps. Polymerase chain reaction was performed by targeting the Leishmania mkDNA region. In total, 2,344 sand flies were collected, from which 45 species, nine subgenera, and five species group were identified. The most abundant species were Lutzomyia antunesi (Coutinho) (n = 597, 25.47%), Lutzomyia ubiquitalis (Mangabeira) (n = 496, 21.16%), and Lutzomyia octavioi (Vargas) (n = 199, 8.49%). The greatest diversity occurred in the forest environment where the most abundant species were L. antunesi (n = 588, 25.07%), L. ubiquitalis (n = 493, 21.02%), L. octavioi (n = 199, 8.49%), and Lutzomyia flaviscutellata (Mangabeira) (n = 132, 5.63%). Two pools of L. ubiquitalis were positive for Leishmania DNA, which suggests that L. ubiquitalis is a putative vector of leishmaniasis in the municipality of Porto Velho.

Evaluation of the antiplasmodial and leishmanicidal potential of Myrciaria dubia (Myrtaceae) extract

INTRODUCTION: Malaria and leishmaniasis are prevalent in tropical regions, which have environmental characteristics that are highly favorable to protozoa and vectors of these diseases; the transmission of these infections in sub-tropical regions, although recognized, represents only a small fraction of cases. Plants are constantly being used in the search for and acquisition of new drugs, and many compounds derived from them have been used to combat various diseases. In this study, we evaluated the action of the dichloromethanolic extract of Myrciaria dubia leaves against the protozoa Plasmodium falciparum, Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi through bioassays. METHODS The extract from M. dubia was tested for its anti-P. falciparum activity in an anti-histidine-rich protein II immunosorbent assay. The antileishmanial assays were performed using the resazurin method, while cytotoxicity against human hepatoma (HepG2) strain was determined using the colorimetric MTT [3-(4, 5-dimethyl-2- thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide] method. RESULTS The M. dubia extract presented a half-maximal inhibitory concentration equal to 2.35 (1.05)μg/mL for P. falciparum, 190.73 (6.41) μg/mL for L. amazonensis, and greater than equal to 200µg/mL for L. chagasi and L. braziliensis strains. The cytotoxic concentration for 50% of the cells was above 500μg/mL for HepG2, indicating no toxicity and greater selectivity against parasites. CONCLUSIONS The results obtained indicate the presence of antiplasmodial and leishmanicidal bioactive compounds in the dichloromethanolic extracts of M. dubia leaves, and point towards future studies to elucidate the mechanism of action for each physiological effect.

Semisynthesis, cytotoxicity, antimalarial evaluation and structure-activity relationship of two series of triterpene derivatives

In this report, we describe the semisynthesis of two series of ursolic and betulinic acid derivatives through designed by modifications at the C-3 and C-28 positions and demonstrate their antimalarial activity against chloroquine-resistant P. falciparum (W2 strain). Structural modifications at C-3 were more advantageous to antimalarial activity than simultaneous modifications at C-3 and C-28 positions. The ester derivative, 3β-butanoyl betulinic acid (7b), was the most active compound (IC50 = 3.4 µM) and it did not exhibit cytotoxicity against VERO nor HepG2 cells (CC50 > 400 µM), showing selectivity towards parasites (selectivity index > 117.47). In combination with artemisinin, compound 7b showed an additive effect (CI = 1.14). While docking analysis showed a possible interaction of 7b with the Plasmodium protease PfSUB1, with an optimum binding affinity of -7.02 kcal/mol, the rather low inhibition displayed on a Bacillus licheniformis subtilisin A protease activity assay (IC50 = 93 µM) and the observed accumulation of ring forms together with a delay of appearance of trophozoites in vitro suggests that the main target of 3β-butanoyl betulinic acid on Plasmodium may be related to other molecules and processes pertaining to the ring stage. Therefore, compound 7b is the most promising compound for further studies on antimalarial chemotherapy. The results obtained in this study provide suitable information about scaffolds to develop novel antimalarials from natural sources.