Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay

Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS) exposure and its recognition by a specific receptor (PSR) were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane - an early hallmark of apoptosis - and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.

Anti-inflammatory and antinociceptive activities of an acid fraction of the seeds of Carpotroche brasiliensis (Raddi) (Flacourtiaceae)

Carpotroche brasiliensis is a native Brazilian tree belonging to the Oncobeae tribe of Flacourtiaceae. The oil extracted from its seeds contains as major constituents the same cyclopentenyl fatty acids hydnocarpic (40.5%), chaulmoogric (14.0%) and gorlic (16.1%) acids found in the better known chaulmoogra oil prepared from the seeds of various species of Hydnocarpus (Flacourtiaceae). These acids are known to be related to the pharmacological activities of these plants and to their use as anti-leprotic agents. Although C. brasiliensis oil has been used in the treatment of leprosy, a disease that elicits inflammatory responses, the anti-inflammatory and analgesic activities of the oil and its constituents have never been characterized. We describe the anti-inflammatory and antinociceptive activities of C. brasiliensis seed oil in acute and chronic models of inflammation and in peripheral and central nociception. The mixture of acids from C. brasiliensis administered orally by gavage showed dose-dependent (10-500 mg/kg) anti-inflammatory activity in carrageenan-induced rat paw edema, inhibiting both the edema by 30-40% and the associated hyperalgesia. The acid fraction (200 mg/kg) also showed significant antinociceptive activity in acetic acid-induced constrictions (57% inhibition) and formalin-induced pain (55% inhibition of the second phase) in Swiss mice. No effects were observed in the hot-plate (100 mg/kg; N = 10), rota-road (200 mg/kg; N = 9) or adjuvant-induced arthritis (50 mg/kg daily for 7 days; N = 5) tests, the latter a chronic model of inflammation. The acid fraction of the seeds of C. brasiliensis which contains cyclopentenyl fatty acids is now shown to have significant oral anti-inflammatory and peripheral antinociceptive effects.

Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity

Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10(7) parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/10(7) parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 µM). The IC50 values of SNAP determined to L. amazonensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a decreased capacity to survive (~40%) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species.

Role of Leishmania (Leishmania) amazonensis amastigote glycosphingolipids in macrophage infectivity

The role of glycosphingolipids (GSLs) present in amastigote forms of Leishmania (Leishmania) amazonensis during infection of macrophages was analyzed, with particular emphasis on GSLs presenting the terminal Galpß1-3Galpa disaccharide. Macrophage invasion by L. (L.) amazonensis amastigotes was reduced by 37% when the disaccharide Galpß1-3Galp (1 mM) was added to the culture medium. The putative macrophage receptor/lectin for ß-Gal-globotriaosylceramide (Galpß1-3Galpa1-4Galpß1-4Glc pß1-1Cer) and other structurally related GSLs from L. (L.) amazonensis amastigotes were analyzed by micelles and parasite binding assay to peritoneal macrophage proteins fractionated by SDS-PAGE under nonreducing conditions. Micelles containing purified amastigote GSLs or a suspention of L. (L.) amazonensis amastigotes fixed with 2% formaldehyde were incubated with nitrocellulose membrane containing the macrophage proteins transferred by Western blotting. Binding of micelles containing purified GSLs from amastigote forms or fixed L. (L.) amazonensis amastigotes to nitrocellulose membrane was probed using monoclonal antibody ST-3, which recognizes the glycoepitope Galpß1-3Galpa1-R present either in the micelle preparation or on the amastigote surface. Macrophage protein with molecular mass ~30 kDa bound the amastigote GSL and appeared to be a doublet on electrophoresis. The specificity of this interaction was confirmed using fixed L. (L.) chagasi amastigotes, which do not express GSLs such as ß-Galp-globotriaosylceramides, and which do not bind to 30-kDa protein.

Leishmania (Leishmania) chagasi-infected mice as a model for the study of glomerular lesions in visceral leishmaniasis

Renal involvement in visceral leishmaniasis (VL) is very frequent but the pathogenesis of this nephropathy is poorly understood. In previous studies using dogs with VL we have detected new immunopathological elements in the glomeruli such as T cells and adhesion molecules. Although Leishmania (Leishmania) chagasi-infected dogs and hamsters are considered to be good models for VL, their use is limited for immunopathologic studies. The use of isogenic mouse strains susceptible to L. (L.) chagasi infection was an alternative but, on the other hand, the renal lesions of these animals have not yet been characterized. Thus, our purpose in the present study was to characterize mice infected with L. (L.) chagasi as a suitable model to study VL nephropathy. Kidney samples were obtained from control mice (N = 12) and from BALB/c mice (N = 24) injected intraperitoneally with 20 million L. (L.) chagasi amastigotes 7, 15, and 30 days after injection and processed for histopathological studies and detection of IgG deposits. Glomerular hypercellularity was clearly visible and, upon Mason's trichrome and periodic acid methenamine silver staining, a pattern suggestive of mesangial proliferative glomerulonephritis was observed in mice with VL. Time-dependent IgG deposits were also seen in infected mice. We consider L. (L.) chagasi-infected mice to be a suitable model for studies of the immunopathogenesis of glomerular lesions in VL.

Cardiac and pulmonary alterations in symptomatic and asymptomatic dogs infectednaturally with Leishmania (Leishmania) chagasi

Fifteen symptomatic and seven asymptomatic dogs infected naturally with Leishmania chagasi were examined in order to identify the presence of parasites and changes in heart and lung. Histopathological, cytological, and immunohistochemical analyses were performed on samples of heart and lung tissues. An inflammatory reaction characterized by inflammatory mononuclear, perivascular and intermuscular infiltrates was observed in both symptomatic and asymptomatic animals on histopathological analysis of the heart. In the lung, there was thickening of the alveolar septa due to congestion, edema, inflammatory infiltrate, and fibroblast proliferation. A focal reaction was observed although a diffuse reaction was present in both groups. On cytological examination, heart and lung imprints revealed amastigotes in two symptomatic animals and heart imprints were found in 1 asymptomatic dog. Immunoperoxidase staining showed amastigotes in the lung and heart of only 1 of 6 symptomatic animals examined. Within the ethical principles and limits of this research, it can be inferred that the study of heart and lung alterations in canine visceral leishmaniasis is increasingly important for understanding the problem related to humans. Dogs with visceral leishmaniasis were a good experimental model, since infection was caused by the same agent and the animals developed clinical, pathological and immunological alterations similar to those observed in humans.

Anti-Leishmania titers and positive skin tests in patients cured of kala-azar

Visceral leishmaniasis (VL), also known as kala-azar, is an important public health problem. If not treated, virtually all clinically symptomatic patients die within months. The diagnosis is based on the Montenegro skin test (MST) and anti-Leishmania titers. Nevertheless, the time required for cured individuals living in a leishmaniasis-endemic area to present a positive skin test and negative anti-Leishmania serology is known. To determine the cellular and humoral immune response profile in relation to different times post-VL cure, a cross-sectional study was conducted on subjects from a kala-azar endemic area in Paço do Lumiar, MA, Brazil, on the basis of 1995-2005 notifications reported by the National Health Foundation/Regional Coordination of Maranhão. We visited cured individuals with a history of VL within the last 10 years. Seventy-four subjects (30 females) ranging in age from 1 to 44 years were included, all of them symptom free at the time of the study. A cellular immune response was observed in 73 (98.6%) subjects, whereas no significant antibody titers were detected by indirect immunofluorescence (IIF) in the sera of 69 (93.2%) cases. Ten years post-cure, 39 (52%) subjects had a positive MST and negative IIF reaction, while in one subject the skin and anti-Leishmania serology tests were negative. Two other subjects were positive in both tests 1 year after cure. These data suggest that a cellular immune response may still be present in subjects cured of VL regardless of post-cure time, and that the parasite persists in the host after clinical cure of the disease. This would explain the persistence of significant Leishmania sp antibody titers in some subjects after treatment.

Interleukin-10-dependent down-regulation of interferon-gamma response to Leishmania by Mycobacterium leprae antigens during the clinical course of a coinfection

We have described a case of a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, two opposite polar forms of these spectral diseases. In the present follow-up study, we investigated the effect of the addition of Mycobacterium leprae antigens on interferon-gamma (IFN-γ) production in Leishmania antigen-stimulated cultures of peripheral blood mononuclear cells (PBMC) from this patient. For this purpose, PBMC cultures were stimulated with crude L. braziliensis and/or M. leprae whole-cell antigen extracts or with concanavalin A. In some experiments, neutralizing anti-human interleukin (IL)-10 antibodies were added to the cultures. IFN-γ and IL-10 levels in culture supernatants were measured by ELISA. During active leprosy, M. leprae antigens induced 72.3% suppression of the IFN-γ response to L. braziliensis antigen, and this suppression was abolished by IL-10 neutralization. Interestingly, the suppressive effect of M. leprae antigen was lost after the cure of leprosy and the disappearance of this effect was accompanied by exacerbation of mucosal leishmaniasis. Considered together, these results provide evidence that the concomitant lepromatous leprosy induced an IL-10-mediated regulatory response that controlled the immunopathology of mucosal leishmaniasis, demonstrating that, in the context of this coinfection, the specific immune response to one pathogen can influence the immune response to the other pathogen and the clinical course of the disease caused by it. Our findings may contribute to a better understanding of the Leishmania/M. leprae coinfection and of the immunopathogenesis of mucosal leishmaniasis.

Otimização da determinação de colesterol por clae e teores de colesterol, lipídios totais e ácidos graxos em camarão rosa (Penaeus brasiliensis)

A produção de camarões no Brasil é expressiva com condições propícias para expansão. Apesar de ser bem apreciado em termos culinários e ser uma fonte rica de proteínas, o camarão é apontado como um alimento de alto conteúdo de colesterol. Considerando que o nível de colesterol sangüíneo humano é dependente não só do teor de colesterol, mas também da quantidade de gordura e do tipo de ácidos graxos na dieta, um estudo integrado destes três constituintes foi realizado em camarão rosa (Penaeus brasiliensis), tamanho médio proveniente de São Paulo. A extração e a determinação do teor de lipídios totais foram realizadas de acordo com método de Folch, Less & Stanley. O método para determinação de colesterol por cromatografia líquida de alta eficiência, com coluna C18 e detector por conjunto de diodos, foi estabelecido no nosso laboratório. Este método mostrou-se eficiente, rápido e simples. A composição de ácidos graxos foi obtida por cromatografia gasosa com coluna capilar de sílica fundida com DB-WAX. Os teores de colesterol e lipídios totais para camarão rosa médio foram 127 ± 9mg/100 g e 1,0 ± 0,1 g/100 g, respectivamente. Foram detectados oitenta e sete ácidos graxos, sendo 20:5w3 (EPA), 16:0, 22:6w3 (DHA), 18:0, 18:1w9, 16:1w7, 20:4w6 e 18:1w7 os principais. O teor de colesterol encontrado no camarão analisado é alto. Por outro lado, o teor de gordura é baixo e os níveis de ácidos graxos poliinsaturados, especialmente EPA e DHA, são altos.

Avaliação de condições de consumo da sardinha Sardinella brasiliensis

Não havendo informações suficientes quanto à qualidade da sardinha (Sardinella brasiliensis) comercializada em São Paulo-SP, amostras frescas, descongeladas (oferecidas durante o defeso) e processadas da referida espécie foram avaliadas quanto às condições de consumo, através das Substâncias Reativas ao Ácido Tiobarbitúrico (TBA) e do Nitrogênio de Bases Voláteis Totais (N-BVT). A sardinha fresca comercializada na CEAGESP (Companhia de Entrepostos e Armazéns Gerais do Estado de São Paulo) apresentou condição aceitável de consumo. Foram adequados os níveis de TBA encontrados (<0,43 mg de AM/kg), tendo como referência dados da literatura para pescado fresco, como também os de N-BVT, abaixo do limite legal vigente de 30 mg/100 g. O mesmo não ocorreu com as sardinhas fresca e descongelada comercializadas em feiras livres, quando avaliadas pelas TBA. As sardinhas salmourada e anchovada não apresentaram condição aceitável de consumo tendo em conta o N-BVT acima do permitido. Os resultados mostraram que a sardinha pode chegar à CEAGESP (comércio atacadista) em condições apropriadas de consumo, mas perder a qualidade na comercialização feita em feira livre. Foi enfatizada a necessidade de reavaliação do congelamento da sardinha e sua estocagem visando a comercialização durante o defeso da espécie.

Avaliação da utilização de embalagem em atmosfera modificada sobre a conservação de sardinhas (Sardinella brasiliensis)

A vida útil dos alimentos perecíveis conservados em atmosfera normal é limitada principalmente pelo efeito do oxigênio atmosférico e o crescimento de microorganismos aeróbios produtores de alterações, que promovem mudanças de odor, sabor, cor e textura, conduzindo à perda da qualidade. A modificação da atmosfera prolonga significativamente a vida útil dos alimentos, quando comparados à refrigeração, podendo chegar a um aumento de três a quatro vezes. O trabalho teve como objetivo avaliar o efeito da embalagem de sardinhas (Sardinella brasiliensis) em atmosfera modificada. As amostras foram divididas em 8 lotes, embaladas com aproximadamente 1,5 L de ar (100%), CO2 (100%), 50/50 CO2/O2 e vácuo e termo-seladas, sendo armazenadas por um período de 22 dias. A cada 2 dias as amostras foram avaliadas quanto ao pH, bases voláteis totais (BVT) e contagens totais de microrganismos heterotróficos aeróbios mesófilos. Os valores de pH aumentaram durante o tempo de estocagem, com exceção das amostras armazenadas em 100 e 50% CO2. A evolução de BVT foi compatível com outros parâmetros, como pH e contagem de bactérias totais, sendo sua evolução mais lenta nas amostras em que ocorreu o uso de atmosfera modificada. Os microrganismos mesófilos, nas atmosferas enriquecidas com CO2, apresentaram fases de latência e tempos de duplicação maiores, quando comparados com os das amostras em que não foi aplicado o método. Sob o ponto de vista microbiológico, as embalagens enriquecidas com CO2 demonstraram ser o melhor método de conservação, como também os resultados de BVT se encontraram dentro dos limites de 30 mg.100 g -1 de acordo com o estabelecido pela RIISPOA8. Recomenda-se o uso da atmosfera de 100% de CO2 como forma de conservação da sardinha por apresentar melhores parâmetros de vida útil.

The effect of acid hydrolysis on the technological functional properties of pinhão (Araucaria brasiliensis) starch

Technological functional properties of native and acid-thinned pinhão (seeds of Araucária angustifolia, Brazilian pine) starches were evaluated and compared to those of native and acid-thinned corn starches. The starches were hydrolyzed (3.2 mol.L-1 HCl, 44 ºC, 6 hours) and evaluated before and after the hydrolysis reaction in terms of formation, melting point and thermo-reversibility of gel starches, retrogradation (in a 30-day period and measurements every three days), paste freezing and thawing stability (after six freezing and thawing cycles), swelling power, and solubility. The results of light transmittance (%) of pastes of native and acid-thinned pinhão starches was higher (lower tendency to retrogradation) than that obtained for corn starches after similar storage period. Native pinhão starch (NPS) presented lower syneresis than native corn starch (NCS) when submitted to freeze-thaw cycles. The acid hydrolysis increased the syneresis of the two native varieties under storage at 5 ºC and after freezing and thawing cycles. The solubility of NPS was lower than that of native corn starch at 25, 50, and 70 ºC. However, for the acid-thinned pinhão starch (APS), this property was significantly higher (p < 0.05) when compared to that of acid-thinned corn starch (ACS). From the results obtained, it can be said that the acid treatment was efficient in producing a potential fat substitute from pinhão starch variety, but this ability must be further investigated.

Anti-quorum sensing activity of phenolic extract from Eugenia brasiliensis (Brazilian cherry)

Abstract The aim of this study was to assess the anti-quorum sensing activity of phenolic extracts from grumixama (Eugenia brasiliensis), also known as Brazilian cherry, in concentrations that did not interfere with bacterial growth. The pulp phenolic compounds were extracted by using solid phage extraction in a mini-collumn C18 and quantified by spectrophotometry. The anti-quorum sensing activity was evaluated by testing the inhibition of violacein production in Chromobacterium violaceum and by evaluating the swarming motility in Aeromonas hydrophila and Serratia marcescens, both phenotypes regulated by quorum sensing. The phenolic extract strongly inhibited the production of violacein in C. violaceum, reducing its production in comparison with a control with no extract. No inhibition of growth was observed at the concentrations tested for quorum sensing inhibition. Confirming the quorum sensing inhibition phenotype, the extract was also able to inhibit swarming motility in S. marcescens and in A. hydrophila, although in the later the effect was marginal. Overall, these results indicate that phenolic extract from E. brasiliensis presents quorum sensing inhibitory activity most likely due to the presence of fruit phenolics which have been implicated as quorum sensing inhibitors in Gram negative bacteria.

Efeito do substrato e da temperatura na germinação de sementes de cataia (Drimys brasiliensis miers. winteraceae)

O objetivo deste trabalho foi avaliar a germinação de sementes de cataia (Drimys brasiliensis) sob diferentes temperaturas e tempos de estratificação. As sementes foram submetidas ao método de estratificação em casa de vegetação, sem controle de temperatura, durante 30, 60 e 90 dias. Foram testados os substratos papel de filtro, areia, vermiculita e ágar a 1% e as temperaturas constantes de 17ºC, 25ºC e 30ºC, na presença de luz contínua e temperatura alternada de 20-30ºC, sendo 20ºC por 16 horas no escuro e 30ºC por 8 horas em presença de luz. O vigor foi avaliado por meio do índice de velocidade de germinação (I.V.G.). As sementes apresentaram grau de umidade inicial de 7% (base úmida). Após 30, 60, 90 dias de estratificação o grau de umidade passou para 11,3%, 27,7% e 46,2%, respectivamente. Os valores da porcentagem de germinação foram de 67% e 52% nos tratamentos com 30 e 60 dias de estratificação, respectivamente, obtidos na temperatura de 17ºC. Os valores de germinação obtidos com 30 dias de estratificação foram 68% para os substratos ágar, areia, e papel de filtro e 61% para o substrato vermiculita. No tratamento com 60 dias de estratificação, os valores de porcentagem de germinação foram 56%, 58% e 54% nos substratos ágar, areia e papel de filtro, respectivamente, não diferindo estatisticamente entre si. As sementes mais vigorosas foram as estratificadas durante 60 dias e colocadas para germinar nos substratos ágar, areia e papel de filtro, na temperatura de 17ºC, onde foram obtidos os valores 0,55, 0,61 e 0,65 para I.V.G., respectivamente. As sementes apresentam dormência por imaturidade embrionária e a estratificação em areia durante 60 dias é um método eficiente para superar a dormência das sementes de cataia. Os maiores valores de velocidade e percentagem de germinação em sementes de cataia são obtidos na temperatura constante de 17ºC com o uso dos substratos ágar, areia e papel de filtro.

Caracterização morfolóica de frutos e sementes de cataia (Drimys brasiliensis Miers. - Winteraceae)

Este trabalho teve como objetivo caracterizar morfologicamente as estruturas externas e internas dos frutos e das sementes de cataia (Drimys brasiliensis Miers.). Para a descrição dos frutos foram observados detalhes externos e internos do pericarpo, referentes à textura, consistência, pilosidade, brilho, forma, número de sementes por fruto e deiscência. Para as sementes foram analisadas as seguintes variáveis externas: dimensões, cor, textura, consistência, forma, posição do hilo e da micrópila. Para as características internas, verificou-se a presença ou ausência de endosperma, o tipo, a forma, a cor, o desenvolvimento embrionário e posição dos cotilédones, do eixo-hipocótilo-radícula e da plúmula. Durante o desenvolvimento embrionário, foram encontrados embriões que variaram de acordo com a diferenciação celular, evidenciando seus aspectos morfológicos, nos estágios de coração, torpedo e maduro. Os frutos de D. brasiliensis são múltiplos, livres, constituídos por cinco frutíolos, bagas, indeiscentes e polispérmicos. As sementes são reniformes e têm grande quantidade de endosperma, e apresentam dormência por imaturidade embrionária.