CD4+ T Cell-Dependent Macrophage Activation Modulates Sustained PS Exposure on Intracellular Amastigotes of Leishmania amazonensis

Leishmania amazonensis amastigotes can make use of surface-exposed phosphatidylserine (PS) molecules to promote infection and non-classical activation of macrophages (MΦ), leading to uncontrolled intracellular proliferation of the parasites. This mechanism was quoted as apoptotic mimicry. Moreover, the amount of PS molecules exposed on the surface of amastigotes correlates with the susceptibility of the host. In this study, we tested whether host cellular responses influence PS expression on intracellular amastigotes. We found that the level of PS exposure on intracellular amastigotes was modulated by CD4+ T cell and MΦ activation status in vitro and in vivo. L. amazonensis infection generated a Th1/Th2-mixed cytokine profile, providing the optimal MΦ stimulation that favored PS exposure on intracellular amastigotes. Maintenance of PS exposed on the parasite was dependent on low, but sustained, levels of nitric oxide and polyamine production. Amastigotes obtained from lymphopenic nude mice did not expose PS on their surface, and adoptive transfer of CD4+ T cells reversed this phenotype. In addition, histopathological analysis of mice treated with anti-PS antibodies showed increased inflammation and similarities to nude mouse lesions. Collectively, our data confirm the role of pathogenic CD4+ T cells for disease progression and point to PS as a critical parasite strategy to subvert host immune responses

Exposure of Phosphatidylserine on Leishmania amazonensis Isolates Is Associated with Diffuse Cutaneous Leishmaniasis and Parasite Infectivity

Diffuse cutaneous leishmaniasis (DCL) is a rare clinical manifestation of leishmaniasis, characterized by an inefficient parasite-specific cellular response and heavily parasitized macrophages. In Brazil, Leishmania (Leishmania) amazonensis is the main species involved in DCL cases. In the experimental model, recognition of phosphatidylserine (PS) molecules exposed on the surface of amastigotes forms of L. amazonensis inhibits the inflammatory response of infected macrophages as a strategy to evade the host immune surveillance. In this study, we examined whether PS exposure on L. amazonensis isolates from DCL patients operated as a parasite pathogenic factor and as a putative suppression mechanism of immune response during the infection. Peritoneal macrophages from F1 mice (BALB/c×C57BL/6) were infected with different L. amazonensis isolates from patients with localized cutaneous leishmaniasis (LCL) or DCL. DCL isolates showed higher PS exposure than their counterparts from LCL patients. In addition, PS exposure was positively correlated with clinical parameters of the human infection (number of lesions and time of disease) and with characteristics of the experimental infection (macrophage infection and anti-inflammatory cytokine induction). Furthermore, parasites isolated from DCL patients displayed an increased area in parasitophorous vacuoles (PV) when compared to those isolated from LCL patients. Thus, this study shows for the first time that a parasite factor (exposed PS) might be associated with parasite survival/persistence in macrophages and lesion exacerbation during the course of DCL, providing new insights regarding pathogenic mechanism in this rare chronic disease

Cooperation between Apoptotic and Viable Metacyclics Enhances the Pathogenesis of Leishmaniasis

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PSPOS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PSPOS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNELPOS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PSPOS and PS-negative (PSNEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PSNEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PSPOS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen

Ação da própolis sobre a desaminação de aminoácidos e a fermentação ruminal Effect of the propolis on amino acids deamination and ruminal fermentation

Foram objetivos deste trabalho determinar a ação in vitro da própolis sobre a atividade específica de produção de amônia (AEPA) ou atividade de desaminação de aminoácidos e sobre a fermentação ruminal em bovinos. A AEPA foi determinada utilizando-se líquido de rúmen e tampão de McDougall (1:4) contendo diferentes níveis de extrato de própolis e excesso de caseína hidrolisada. No estudo da ação da própolis in vivo sobre a fermentação ruminal e AEPA, foram utilizados quatro novilhos Holandeses, em dois períodos experimentais, sob dieta contendo 35% de concentrado, submetidos aos tratamentos controle e com extrato de própolis. O extrato de própolis obtido com etanol a 70% em água foi mais eficiente in vitro que a 99,5%, obtendo-se valores de até 78% de inibição da AEPA em relação ao controle. O extrato de própolis não afetou o consumo de matéria seca, o pH ruminal, as concentrações de amônia e de proteína microbiana e as proporções molares dos ácidos graxos voláteis (AGV), acético, propiônico e butírico no líquido de rúmen. Entretanto, o extrato de própolis aumentou a concentração de AGV totais e inibiu a AEPA pelos microrganismos ruminais, indicando que, apesar de não ter reduzido o nível ruminal de amônia, existe o potencial deste efeito ocorrer em outras situações, como em dietas contendo alta taxa de proteína degradável/carboidrato fermentescível, observado em pastagens novas de gramíneas ou pastagens de gramíneas consorciadas com leguminosas.<br>The objective of this work was to determine the in vitro effect of the propolis on the specific activity of ammonia production (SAAP) or activity deamination of amino acids and on ruminal fermentation in bovine. The SAAP was determined using ruminal fluid and McDougall buffer (1:4) with different levels of propolis extract and excess of hydrolyzed casein. In the study of the in vitro effect of the propolis on the ruminal fermentation and SAAP, four Holstein steers were used, in two experimental periods, fed a 35.0% concentrate diet and submitted to the control and propolis extract treatments. The propolis extract obtained with ethanol at 70% in water was in vitro more efficient than that obtained with ethanol at 99.5% in water. Values up to 78% of SAAP inhibition in relation to the control were obtained. The propolis extract did not affect dry matter intake, ruminal pH, ammonia and microbial protein concentrations. There were no differences for molar proportions of the volatile fat acid (VFA), acetic, propionic and butyric in the rumen fluid. However, the propolis extract increased the total VFA concentration as well as inhibited SAAP by the ruminal microorganisms, demonstrating that, in spite of not having reduced the ruminal ammonia level, it seems to exist potential of this effect to happen in other situations, as in diets with high rate of degradable protein/fermentable carbohydrate, observed in new grass pastures or grass pastures consociated with legumes

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 µg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death

Ação do extrato de própolis sobre a fermentação in vitro de diferentes alimentos pela técnica de produção de gases

Dois experimentos foram realizados procurando-se avaliar in vitro a eficiência do extrato de própolis em inibir a produção de gases oriundos da fermentação ruminal de diferentes alimentos. No primeiro experimento, incubaram-se 100 mg de matéria seca de feno de brachiária moído, em ausência (0,2 mL de solução alcoólica a 70% em água) ou presença de 0,2 mL de extrato de própolis (extração de 3 g de própolis em pedra triturada para cada 10 mL de álcool a 70%, durante dez dias, posteriormente diluída para 50% da mesma). O extrato de própolis, quando comparado ao tratamento controle, reduziu a produção final total e a produção final de gases para carboidratos fibrosos. A taxa de digestão específica para carboidratos fibrosos e carboidratos não-fibrosos foi superior, quando se utilizou o extrato de própolis. A redução da produção total de gases pode ser atribuída ao efeito da própolis em aumentar a concentração molar de propionato, com conseqüente diminuição da relação acetato:propionato. No experimento 2, procurou-se avaliar diferentes diluições de extrato de própolis (0; 13,7; 33,3; e 66,7%), em analogia à monensina sódica, adicionada para atingir 5,0 µM como concentração final nos tubos de incubação. Observou-se efeito significativo de tratamento, alimento e interação alimento:tratamento sobre o volume de gás proveniente dos carboidratos fibrosos e não-fibrosos. Não houve efeito do menor nível de própolis (13,7%) sobre nenhuma das dietas avaliadas, tanto para volume final de gases oriundos dos carboidratos fibrosos quanto não-fibrosos. Entretanto, o maior nível (66,7%) mostrou-se eficiente em todas as dietas, para ambos os carboidratos, inclusive suplantando a monensina, na maioria das vezes, quanto à menor produção final de gases

Clinical and immunologic data from patients with diffuse cutaneous leishmaniasis (DCL) and localized cutaneous leishmaniasis (LCL).

<p>LST, Leishmanin Skin Test. –, no nodules; +, few; ++, moderate; +++, intense; ++++, very intense.</p