O papel modulatório do ferro sobre a resposta imune na Leishmaniose Visceral

Iron is an essential element for many cellular functions, including the immune response against intracellular pathogens. In this study, we aimed evaluate the effect of iron on IRP2, IFN-γ, TNF-α, IL-6, IL-10, MIG and IP10 expression in PBMC and assess the effect of the spleen parasite load on the expression of these genes in the spleen of L. infantum naturally infected dogs. Blood sample from 7 DTH+ donor was collected and PBMC was obtained. The cells were cultivated in absence (iron chelator desferroximane, DFO 10 μM supplemented media) or in presence of iron (hemin 6 mM) for 1 h, followed by stimulation with Leishmania infatum antigen for 4 h. 44 dog spleen samples were obtained and parasite load in this organ was determinate by qPCR. Gene expression was analyzed by qPCR and cytokine production quantified by flow cytometry. In antigen stimulated cells, genes involved in immune response are significantly more expressed in presence of iron. T CD4+ and TCD8+ lymphocytes produces IFN-γ, TNF-α and IL-10 possibly in iron dependent pathway. Monocytes antigen stimulated reduced TNF-α, IL-6 and IL-10 production in presence of iron. We found spleen of infected dogs IRP2 expression increases according to parasite load in that organ, while an inverse profile was found for IFN-γ, TNF-α e IL-10 expression. These results suggest that T lymphocytes depends on iron to produce IFN-γ, TNF-α and IL-10, while iron seems to inhibit cytokine production in monocytes. So, we propose an immunoregulatory mechanism carried out by iron during L. infantum infection in humans and dogs

Lectina da esponja marinha Tedania ignis: purificação, caracterização e interação com leishmanias

Lectin obtained from the marine sponge Tedania ignis was purified and characterized by extraction of soluble proteins (crude extract) in 50mM Borax, pH 7.5. The purification procedure was carried out by crude extract precipitation with ammonium sulfate 30% (FI). The precipitated was resuspended in the same buffer and fractionated with acetone 1.0 volume (F1.0). A lectin was purified from this specific fraction by using an affinity chromatography Sepharose 6B. This lectin preferentially agglutinated human erythrocytes from B type previously treated with papain enzyme. The hemagglutinating activity lectin was dependent of divalent Mn2+ cation and was inhibited by the carbohydrates galactose, xylose and fructose. SDS-PAGE analysis indicated a molecular mass of the lectin around 45 kDa. This protein showed stability until 40°C for 1 h. Further, it showed activity between pH 2.5 and 11.5, with an enhanced activity at pH 7.5. Leishmania chagasi promastigotes stained with Coomassie brilliant blue R-250 were agglutinated by F1,0 and in the presence of galactose this interaction was abolished. These results show that this lectin could be implicated in defense procedures and it will can be used as biological tools in studies with this protozoon

Co-infecção pelo vírus da hepatite G em pacientes infectados pelo Leihsmania chagasi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmania and Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis , in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

Leishmaniamamazonensis DNA in wild females of Lutzomyia cruzi (Diptera: Psychodidae) in the state of Mato Grosso do Sul, Brazil

Studies on natural infection by Leishmania spp of sandflies collected in endemic and nonendemic areas can provide important information on the distribution and intensity of the transmission of these parasites. This study sought to investigate the natural infection by Leishmania in wild female sandflies. The specimens were caught in the city of Corumbá, state of Mato Grosso do Sul (Brazil) between October 2012-March 2014, and dissected to investigate flagellates and/or submitted to molecular analysis to detect Leishmania DNA. A total of 1,164 females (77.56% of which were Lutzomyia cruzi ) representing 11 species were investigated using molecular analysis; 126 specimens of Lu. cruzi were dissected and also submitted to molecular analysis. The infection rate based on the presence of Leishmania DNA considering all the sandfly species analysed was 0.69%; only Leishmania (Leishmania) amazonensis was identified in Lu. cruzi by the molecular analysis. The dissections were negative for flagellates. This is the first record of the presence of L. (L.) amazonensis DNA in Lu. cruzi, and the first record of this parasite in this area. These findings point to the need for further investigation into the possible role of this sandfly as vector of this parasite.

Ocorrência de tripanossomatídeos em morcegos (Mammalia : Chiroptera) no Distrito Federal, Brasil

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Zoologia, 2016.

The dynamics of Brazilian protozoology over the past century

Brazilian scientists have been contributing to the protozoology field for more than 100 years with important discoveries of new species such as Trypanosoma cruzi and Leishmania spp. In this work, we used a Brazilian thesis database (Coordination for the Improvement of Higher Education Personnel) covering the period from 1987-2011 to identify researchers who contributed substantially to protozoology. We selected 248 advisors by filtering to obtain researchers who supervised at least 10 theses. Based on a computational analysis of the thesis databases, we found students who were supervised by these scientists. A computational procedure was developed to determine the advisors’ scientific ancestors using the Lattes Platform. These analyses provided a list of 1,997 researchers who were inspected through Lattes CV examination and allowed the identification of the pioneers of Brazilian protozoology. Moreover, we investigated the areas in which researchers who earned PhDs in protozoology are now working. We found that 68.4% of them are still in protozoology, while 16.7% have migrated to other fields. We observed that support for protozoology by national or international agencies is clearly correlated with the increase of scientists in the field. Finally, we described the academic genealogy of Brazilian protozoology by formalising the “forest” of Brazilian scientists involved in the study of protozoa and their vectors over the past century.

Spread of Phlebotominae in temperate climates: province of Córdoba, Argentina

We report the presence of the competent vector for Leishmania spp, Migonemyia migonei, and the Evandromyia cortelezzii-sallesi complex south of its known distribution in the central temperate region of Argentina, in the province of Córdoba. The persistence of this phlebotomine in the northern border of the province, its association with a case of cutaneous leishmaniasis, and the new record in the outskirts of the city of Córdoba, the second most populated in the country, strengthens the need for regular vector surveillance and a case detection-sensitive health system in vulnerable regions, even in temperate climates.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii (SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Anti-leishmanial effects of purified compounds from aerial parts of Baccharis uncinella C. DC. (Asteraceae)

Species of Baccharis exhibit antibiotic, antiseptic, wound-healing, and anti-protozoal properties, and have been used in the traditional medicine of South America for the treatment of several diseases. In the present work, the fractionation of EtOH extract from aerial parts of Baccharis uncinella indicated that the isolated compounds caffeic acid and pectolinaringenin showed inhibitory activity against Leishmania (L.) amazonensis and Leishmania (V.) braziliensis promastigotes, respectively. Moreover, amastigote forms of both species were highly sensible to the fraction composed by oleanolic + ursolic acids and pectolinaringenin. Caffeic acid also inhibited amastigote forms of L. (L.) amazonensis, but this effect was weak in L. (V.) braziliensis amastigotes. The treatment of infected macrophages with these compounds did not alter the levels of nitrates, indicating a direct effect of the compounds on amastigote stages. The results presented herein suggest that the active components from B. uncinella can be important to the design of new drugs against American tegumentar leishmaniases.

Leishmanicidal and cytotoxic activity of extracts and saponins from Ilex laurina (Aquifoliaceae)

Purpose: To evaluate the leishmanicidal and cytotoxic activity of alcohol and non-alcohol extracts and saponins from Ilex laurina . Methods: Extracts were obtained by percolation with solvents of different polarities: hexane, dichloromethane, ethyl acetate and ethanol. The ethyl acetate extract was subjected to silica gel column chromatography eluting with a step gradient of dichloromethane-methanol. All products were evaluated in vitro for leishmanicidal activity against amastigotes of leishmania panamensis and cytotoxicity on U- 937 cells. Results: Two saponins were isolated from the ethyl acetate extract. The ethyl acetate extract showed high leishmanicidal activity against intracellular amastigotes of L. panamensis (EC50, 7.5 ± 1.5 μg/mL) and low activity against axenic amastigotes (EC50, 52.8 ±1.6 μg/mL); this extract showed also high cytotoxicity (LC50, 57.7 ± 12.1 μg/mL). Saponin 2 exhibited high activity against intracellular amastigotes (EC50, 5.9 ± 0.5 μg/mL) but also showed high cytotoxicity on U-937 cells (EC50, 25.7 ± 6.1 μg/mL). This compound showed similar leishmanicidal activity and cytotoxicity to meglumine antimoniate and amphotericin B, respectively, drugs currently used for the treatment of leishmaniasis. Conclusions: Based on these results, Ilex laurina is a potential source of compounds that can lead to the development of new therapeutic alternatives against leishmaniasis.

Leishmaniose visceral canina em cães atendidos em hospital veterinário particular do Distrito Federal, Brasil

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Medicina Tropical, 2016.

Flebotomíneos (Diptera: Psychodidae) em um sistema agroflorestal da Região Metropolitana de Natal, Rio Grande do Norte, Brasil

Sandflies stand out as important vectors of leishmaniasis. The females need to ingest blood meals, enabling them to transmit protozoa of the genus Leishmania, which may give rise to visceral leishmaniasis (VL) or American tegumentary leishmaniasis (ATL), in addition to transmitting other parasites. Leishmaniasis are important infirmities, distributed worldwide, whose infection results from the interaction of reservoir animals, the vector insect, parasitic protozoa and the healthy host. In the state of Rio Grande do Norte (RN), Brazil, these insects are important transmitters of VL, which usually presents in the most serious form. It occurs mainly in metropolitan areas, with the dog as its main reservoir and Lutzomyia longipalpis as the vector. ATL is most present in the highland areas of the state. In addition to hematophagia, engaged in by the females, both sexes need to ingest carbohydrates, which are essential to the sand flies energy requirements and may interfere in the development of Leishmania. The aim of this study was to determine the occurrence and abundance of sand flies in different environments on the farm belonging to the Empresa de Pesquisas Agropecuárias do RN (Institute of Agricultural Research of RN), in the municipality of Parnamirim, in order to relate this occurrence with climatological and biological references and eating habits. Three consecutive monthly collections were carried out with CDC traps in a fragment of the Atlantic Forest, in a residence, on a goat breeding farm and on cashew, dwarf and giant coconut, mango, banana, eucalyptus, acacia and bean plantations. A total of 1241 sandflies from eight species (Lutzomyia evandroi, Lutzomyia longipalpis, Lutzomyia shannoni, Lutzomyia sordellii Lutzomyia walkeri, Lutzomyia wellcomei, Lutzomyia whitmani, and Lutzomyia intermedia) were collected, most in the forest environment. L. longipalpis, the main VL transmitter, was confirmed as a species adapted to anthropic environments, whereas others such as L. wellcomei, the vector of ATL, occurred predominantly in forests. Carbohydrate characterization of the sand flies and plants of the region demonstrated that a number of exotic plants such as hay and eucalyptus may play some role in the adaptation of these species to modified environments. Breeding in laboratory showed a mean biological cycle of 53.5 days from egg to adulthood for L. shannoni and the possibility of diapause behavior in L. wellcomei. This study serves as a source of information that may contribute to the epidemiological vigilance of tegumentary and visceral leishmaniasis in the state, given that it analyzes the bioecology of transmitting species, as well as their potential to adapt to new environments

Assessment of the sensitivity and specificity of serological (IFAT) and molecular (direct PCR) techniques for diagnosis of leishmaniasis in lagomorphs using a Bayesian approach

Leishmaniasis, caused by Leishmania infantum, is a vector-borne zoonotic disease that is endemic to the Mediterranean basin. The potential of rabbits and hares to serve as competent reservoirs for the disease has recently been demonstrated, although assessment of the importance of their role on disease dynamics is hampered by the absence of quantitative knowledge on the accuracy of diagnostic techniques in these species. A Bayesian latent-class model was used here to estimate the sensitivity and specificity of the Immuno-fluorescence antibody test (IFAT) in serum and a Leishmania-nested PCR (Ln-PCR) in skin for samples collected from 217 rabbits and 70 hares from two different populations in the region of Madrid, Spain. A two-population model, assuming conditional independence between test results and incorporating prior information on the performance of the tests in other animal species obtained from the literature, was used. Two alternative cut-off values were assumed for the interpretation of the IFAT results: 1/50 for conservative and 1/25 for sensitive interpretation. Results suggest that sensitivity and specificity of the IFAT were around 70–80%, whereas the Ln-PCR was highly specific (96%) but had a limited sensitivity (28.9% applying the conservative interpretation and 21.3% with the sensitive one). Prevalence was higher in the rabbit population (50.5% and 72.6%, for the conservative and sensitive interpretation, respectively) than in hares (6.7% and 13.2%). Our results demonstrate that the IFAT may be a useful screening tool for diagnosis of leishmaniasis in rabbits and hares. These results will help to design and implement surveillance programmes in wild species, with the ultimate objective of early detecting and preventing incursions of the disease into domestic and human populations.