Leishmanicidal activity of the alkaloid-rich fraction from Guatteria latifolia.

Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania and includes cutaneous, mucocutaneous and visceral clinical forms. The drugs currently available for leishmaniasis treatment are pentavalent antimonials, amphotericin B and miltefosine, which present high toxicity, elevated cost and development of parasite resistance. The natural products constitute an important source of substances with leishmanicidal potential. Here we evaluated in vitro the anti-Leishmania amazonensis activity of crude extracts of branches, leaves and fruits of Guatteria latifolia. The branch extract (GCE) exhibited promising leishmanicidal activity against promastigotes (IC50 51.7 µg/ml), and was submitted to fractionation guided by in vitro assays. Among the seven subfractions obtained, GF1 and GF2 were the most actives against promastigotes with IC50 25.6 and 16 µg/ml, respectively. Since GCE, GF1 and GF2 were not toxic for macrophages, next, we tested their effect on intracellular amastigotes, and the IC50 values obtained were, respectively 30.5, 10.4 and 7.4 µg/ml, after 24 h treatment. The selectivity index for GCE, GF1 and GF2 were >6.5, >19.2 and > 27, respectively. Additionally, GCE, GF1 and GF2 affected the division pattern of the promastigotes by increasing 6.7, 9.4 and 7-fold the cells in Sub-G0/G1 phase, and decreasing 1.6, 2.5 and 1.8-fold the cells in G0/G1 phase, respectively. To assess the GCE and GFs capacity to modulate microbicidal mechanisms of macrophages, nitric oxide (NO) and TNF-α production were tested. Our results indicated that at the IC50s GCE, GF1 and GF2 decreased NO production of infected macrophages stimulated with IFN-γ and LPS, besides, only GF1 decreased the production of TNF-α. Our data warrant further studies of GCE, GF1 and GF2 to identify active compounds against Leishmania parasites.

DNA extracellular traps are part of the immune repertoire of Periplaneta americana.

Extracellular traps (ETs), web-like structures composed of DNA and histones, are released by innate immune cells in a wide range of organisms. ETs capture microorganisms, thereby avoiding their spread, and also concentrate antimicrobial molecules, which helps to kill microbes. Although vertebrate innate immune systems share homology with the insect immune system, ETosis have yet to be characterized in insects. Here, we report that the hemocytes of the hemimetabolous insect Periplaneta americana release ETs upon in vitro stimulation. We further discuss the relationship between ETs and nodulation and in controlling bacterial spread in vivo.

Cell surface carbohydrate of Leishmania mexicana amazonensis: differences between infective and non-infective forms.

The cell surface carbohydrates of Leishmania mexicana amazonensis (amastigotes and promastigotes, both infective and non-infective forms) were comparatively analyzed by agglutination assay employing 28 highly purified lectins, and by binding assay using 125I-labeled lectins. Among the D-GalNAc binding lectins, Bandeiraea simplicifolia-I, Dolichos biflorus, Phaseolus vulgaris and Glycine max were highly specific for the amastigotes, while that from Maclura aurantiaca selectively agglutinated promastigotes. The lectins from Wistaria floribunda, Phaseolus lunatus (D-GalNAc), Arachis hypogaea (D-Gal) and Triticum vulgaris (D-GlcNAc) were selective for the infective forms (both amastigotes and promastigotes), not reacting with the non-infective ones. Conversely, no parasite agglutination occurred with the L-fucose binding lectins Lotus tetragonolobus and Ulex europaeus-I. Binding studies with 125I-labeled lectins from Wistaria floribunda, Triticum vulgaris and Arachis hypogaea were performed to find whether unagglutinated non-infective promastigotes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the infective promastigotes.

Fibronectin shedding by Leishmania may influence the parasite-macrophage interaction.

Fibronectin (FN) is a large extracellular matrix protein involved in the endocytosis of several types of particles by different phagocytes. Here we investigated the role of FN in the entry and destruction of Leishmania amazonensis promastigotes (flagellated form) by murine resident peritoneal macrophages. We also studied the lateral mobility of this protein on the surface of the parasite cells using a immunogold technique. We compared the effects of addition and depletion of FN on infective and non-infective populations of Leishmania promastigotes. The invasion by the latter but not by the former, was increased by FN, and the uptake of these cells was more sensitive to FN depletion from the culture medium. We also observed enhanced killing of intracellular infective promastigotes upon FN addition to the macrophage cultures. Immunocytochemical localization of FN on the surface of the flagellates revealed that the parasite cells released bound FN by membrane shedding in a constitutive fashion. Therefore we conclude that FN removal by shedding may be part of a physiological mechanism by which the parasites evade intracellular destruction by host cells.

Trichomonas vaginalis and Tritrichomonas foetus secrete neuraminidase into the culture medium.

Supernatants taken from axenic cultures of Trichomonas vaginalis and Tritrichomonas foetus contain a neuraminidase activity, the detection of which is augmented when the trichomonad culture media are supplemented with 30% supernatant of confluent epithelial cultures. The enzyme was active against human erythrocytes, which became highly reactive to peanut agglutinin lectin. The specificity of the enzyme was checked by using a substrate specific to neuraminidase: 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuramic acid.

Nuclear and cytoplasmic lectin binding sites in promastigotes of Leishmania.

We demonstrated the presence of intracellular lectin binding sites in promastigotes of Leishmania mexicana amazonensis. Direct and indirect lectin-gold techniques were used on Lowicryl K4M-embedded cells. The nuclear compartment was labeled by most lectins. Nucleoli were mainly labeled by WFH (Wistaria floribunda hemagglutinin) and LFA (Limax flavus agglutinin) specific for D-galactose/N-acetyl-D-galactosamine (D-Gal/D-GalNAc) and sialic acid, respectively. Sections treated with the fetuin-gold complex without previous lectin incubation also exhibited labeled nucleoli, although less intensely, suggesting the presence not only of sialic acid but also of a sialic acid-specific endogenous carbohydrate binding molecule in Leishmania nuclei. Surprisingly, the Golgi complex was never labeled, whereas the endoplasmic reticulum was frequently labeled, especially by RCA (Ricinus communis agglutinin; D-GalNAc/D-Gal) and WGA (wheat germ agglutinin; D-GlcNAc). The kinetoplast, a DNA-containing structure located within the mitochondrion, was generally labeled towards its extremities, where previous studies have shown the presence of ribonucleoproteins. Some possible biological roles for these intracellular glycoconjugates are discussed.

Increased leishmanicidal activity of alveolar macrophages from mature horses with mild equine asthma / Aumento da atividade leishmanicida de macrófagos alveolares de equinos adultos com asma equina leve

Alveolar macrophages (AMs) are an essential part of defense mechanisms within the lungs and their phagocytic activity is important for organ homeostasis. The phagocytic ability of AMs obtained from bronchoalveolar lavage from 17 mature mixed-breed pleasure horses (8 healthy and 9 diagnosed with mild equine asthma) was studied through assays with Leishmania (Viannia) braziliensis promastigotes, which enabled the calculation of a phagocytic index (PI) and a survival index (SI). Results indicate that phagocytic activity of AMs in asthma affected horses is similar to healthy horses, while leishmanicidal activity is significantly increased in horses with asthma.(AU)

Os macrófagos alveolares (MAs) são uma parte essencial dos mecanismos de defesa dentro dos pulmões e sua atividade fagocítica é importante para a homeostase desse órgão. A capacidade fagocitária dos MAs obtidos do lavado broncoalveolar de 17 equinos adultos, sem raça definida (oito saudáveis e nove com diagnóstico de asma equina leve), foi estudada por meio de ensaios com promastigotas de Leishmania (Viannia) braziliensis. Foi calculado o índice fagocítico e o índice de sobrevivência. Os resultados indicam que a atividade fagocítica de MAs em cavalos com asma é semelhante a cavalos saudáveis, enquanto a atividade leishmanicida está significativamente aumentada em cavalos com essa enfermidade.(AU)

The presence of a symbiotic bacterium in Strigomonas culicis is related to differential ecto-phosphatase activity and influences the mosquito-protozoa interaction.

Strigomonas culicis is a monoxenous trypanosomatid that co-evolves with a symbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges between both partners. S. culicis infects and colonizes the Aedes aegypti mosquito midgut, reaches its hemocoel and then invades the salivary glands. An artificial aposymbiotic strain is unable to colonize insects, reinforcing the idea that the bacterium influences the protozoan surface composition and cell interaction. Here, we report the characterization of the hydrolytic activity of ecto-phosphatases evaluated in symbiont-bearing and aposymbiotic strains of S. culicis by incubating the protozoa with p-nitrophenyl phosphate (pNPP) at different pH levels, in the presence of phosphatase inhibitors, and with several divalent metals. The symbiont-bearing and aposymbiotic cells differ in their ecto-phosphatase enzymes, based on their activities and specificities. Furthermore, the ability of the protozoan to bind to the mosquito midgut and salivary glands was impaired by ecto-phosphatase inhibition. Taken together, our data suggest that the symbiont influences the host protozoan ecto-phosphatase activity and indicate a possible role of this enzyme during mosquito tissue colonization by S. culicis.

Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis.

Leishmaniasis is a tropical disease caused by protozoan parasites of the genus Leishmania which affects 12 million people worldwide. The discovery of drugs for the treatment of leishmaniasis is a pressing concern in global health programs. The aim of this study aim was to evaluate the leishmanicidal effect of piperine and its derivatives/analogues on Leishmania amazonensis. Our results showed that piperine and phenylamide are active against promastigotes and amastigotes in infected macrophages. Both drugs induced mitochondrial swelling, loose kinetoplast DNA, and led to loss of mitochondrial membrane potential. The promastigote cell cycle was also affected with an increase in the G1 phase cells and a decrease in the S-phase cells, respectively, after piperine and phenylamide treatment. Lipid analysis of promastigotes showed that piperine reduced triglyceride, diacylglycerol, and monoacylglycerol contents, whereas phenylamide only reduced diacylglycerol levels. Both drugs were deemed non toxic to macrophages at 50 µM as assessed by XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt), Trypan blue exclusion, and phagocytosis assays, whereas low toxicity was noted at concentrations higher than 150 µM. None of the drugs induced nitric oxide (NO) production. By contrast, piperine reduced NO production in activated macrophages. The isobologram analysis showed that piperine and phenylamide acted synergistically on the parasites suggesting that they affect different target mechanisms. These results indicate that piperine and its phenylamide analogue are candidates for development of drugs for cutaneous leishmaniasis treatment.

Leishmanicidal effect of LLD-3 (1), a nor-triterpene isolated from Lophanthera lactescens.

Leishmanicidal activity of 6alpha, 7alpha, 15beta, 16beta, 24-pentacetoxy-22alpha-carbometoxy-21beta,22beta-epoxy-18beta-hydroxy-27,30-bisnor-3,4-secofriedela-1,20 (29)-dien-3,4 R-olide (LLD-3 (1)) isolated from Lophanthera lactescens Ducke, a member of the Malpighiaceae, was demonstrated against intramacrophage amastigote forms (IC(50) of 0.41mug/mL). The in vitro leishmanicidal effect of Glucantime, the first choice drug for leishmaniasis treatment, was increased by LLD-3 (1) association. The leishmanicidal effect of LLD-3 (1) was not due to stimulation of nitric oxide production by macrophages. LLD-3 (1) was also not cytotoxic for mouse peritoneal macrophages or B cells as assessed by the XTT and Trypan blue exclusion assays. LLD-3 (1) was unable to affect proliferation of naïve or activated B and T cells, as well as the B cells immunoglobulin synthesis. Cellularity of different tissues, liver and kidney functions were not altered in mice treated with LLD-3 (1), as well as the histology pattern of different organs. Our results add LLD-3 (1) as a potential drug candidate for treatment of leishmaniasis.

Leishmania amazonensis: early proteinase activities during promastigote-amastigote differentiation in vitro.

Leishmania proteinase activity is known as parasite differentiation marker, and has been considered relevant for leishmanial survival and virulence. These properties suggest that Leishmania proteinases can be promising targets for development of anti-leishmania drugs. Here, we analyze the activities of four proteinases during the early phase of the Leishmania amazonensis promastigotes differentiation into amastigotes induced by heat shock. We have examined activities of cysteine-, metallo-, serine-, and aspartic-proteinase by hydrolysis of specific chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme. Our results show that metallo-, serine-, and aspartic-proteinases activities were down-regulated during the shock-induced transformation of promastigotes into amastigotes. In contrast, cysteine-proteinase activity increased concomitantly with the promastigote differentiation. Immunocytochemical localization using two anti-cysteine-proteinase monospecific rabbit antibodies detected the enzyme in several cell compartments of both parasite stages. Our results show different proteinase activity modulation and expression during the early phases of the shock-induced parasite transformation.

Lectin-binding properties of different Leishmania species.

Carbohydrate cell-surface residues on stationary promastigotes of 19 isolates of Leishmania were studied with a panel of 27 highly purified lectins, which were specific for N-acetyl-D-glucosamine, D-mannose, L-fucose, D-galactose, N-acetyl-D-galactosamine, and sialic acid. The specificity of the cell-surface carbohydrates was analyzed by agglutination and radioiodinated lectin-binding assays. L. (L.) amazonensis and L. (L.) donovani were agglutinated by 12 and 10 of the 27 lectins used, respectively. Artocarpus integrifolia lectin (Jacalin) was incapable of agglutinating the tested species of the donovani complex, and this result was confirmed by radioiodinated Jacalin-binding assays. Jacalin had an average of 3.8 x 10(6) receptors/L. (L) amazonensis promastigote and bound with an association constant of 5 x 10(6) M(-1).

Involvement of the macrophage mannose-6-phosphate receptor in the recognition of Leishmania mexicana amazonensis.

Significant differences were found in the ability of resident mouse peritoneal macrophages to ingest amastigote and promastigote forms of Leishmania mexicana amazonensis. Differences in the association index of the parasites to the macrophages were also found between infective and non-infective promastigotes. Evidence was obtained suggesting that the macrophage receptor, which recognizes mannose-6-phosphate-containing units found in lysosomal enzymes, is involved in the association with the macrophage of promastigotes, but not of amastigotes. Addition of mannose-6-phosphate, its structural analogue fructose-1-phosphate, Hansenula holstii phosphomannan or the mannose-6-phosphate-containing lysosomal enzyme alpha-D-mannosidase to the interaction medium, markedly inhibits the association of the parasites with macrophages.

The comparative fine structure and surface glycoconjugate expression of three life stages of Leishmania major.

The cellular ultrastructure and surface glycoconjugate expression of three life stages of Leishmania major were compared. Noninfective logarithmic phase promastigotes (LP) are immature cells bearing a thin cell coat, short flagellum, small and empty flagellar pocket, and a loose cytoplasm filled with profiles of ER and large Golgi complex. LP also contain subpopulations of maturing cells containing less ER and Golgi and synthesizing cytoplasmic granules of different size, number, and electron-density. Infective or metacyclic promastigotes (MP) are fully differentiated nondividing forms with a thickened, prominent cell coat, long flagellum, distended flagellar pocket filled with secretory material, and few cytoplasmic organelles other than abundant electron-dense granules. Tissue amastigotes also contain electron-dense cytoplasmic granules, their flagellar pockets are also enlarged and contain secretory material, but they lack a discernable cell coat. Immunogold labeling of GP63 on the cell surface was extensive only on amastigotes. Promastigote GP63 appeared to be masked by the presence of a densely packed lipophosphoglycan (LPG) coat which was extensively labeled on the entire surface of MP and LP. An elongated, developmentally modified form of LPG was abundantly labeled only on MP. LPG was poorly labeled on amastigotes, arguing that the promastigote cell coat is a stage-specific structure which is lost during intracellular transformation.

Effect of a phorbol ester on basic surface properties of trichomonads.

The effect of nanomolar concentrations of 12-O-tetradecanoilphorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus was evaluated by means of measurements of the parasites' surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites' net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.

Anionic site behavior in Leishmania and its role in the parasite-macrophage interaction.

The behavior of cationized ferritin (CF) binding sites on the surface of Leishmania mexicana amazonensis (amastigotes, infective and non-infective promastigotes) and their participation in the interaction with macrophages were evaluated. Glutaral-dehyde-fixed parasites treated with CF present a uniform labelling over the whole cell surface. However, living parasites displayed CF patches and caps. Capping was usually seen towards the anterior (flagellated) portion of the cells, where shedding phenomena took place. These processes were inhibited by sodium azide but not by low temperature (4 degrees C). CF treatment of non-infective promastigotes led to an increase in their uptake by macrophages, whereas the uptake of amastigotes or infective promastigotes was not significantly altered. The effect of CF on the parasite surface charge was analyzed by whole-cell microelectrophoresis. The mean electrophoretic mobility (EPM) of non-infective promastigotes was decreased by 26%, while once again the other parasite forms were not significantly affected. Transmission electron microscopy of mouse peritoneal macrophage cultures, fixed after interaction with CF-labelled parasites, revealed that both amastigotes and infective promastigotes quickly removed bound CF. Therefore CF was seen neither in parasite-macrophage attachment areas nor in parasitophorous vacuoles. On the contrary, non-infective promastigote-macrophage attachment areas were remarkably large and preferentially comprised CF-labelled membranes. These results strongly suggest an important participation of cell surface anionic sites in the L. mexicana amazonensis-macrophage interaction.

The role of the lipophosphoglycan of Leishmania in vector competence.

The surface lipophosphoglycans (LPG) of Leishmania promastigotes express stage- and species-specific polymorphisms that are defined by variations in the type and number of phosphorylated oligosaccharide repeats. We have studied how these polymorphic structures control the development of transmissible infections in the sandfly vector as well as the species-specificity of vectorial competence. Procyclic promastigotes displayed an inherent capacity to bind to midgut epithelial cells of a competent vector. This capacity was lost during their transformation of metacyclic promastigotes, permitting the selective release and anterior migration of infective-stage parasites for subsequent transmission by bite. Midgut attachment and release were found to be controlled by developmental modifications in terminally exposed saccharides on LPG, which, depending on the species of Leishmania, involved either substitution or capping of terminal side-chain sugars, loss of terminal side-chain sugars, substitution or loss of neutral capping sugars. The stage-specific terminal sugars involved in midgut adhesion are, in some cases, also species-specific, and the extent to which these differences affect midgut attachment, forcefully predicted vectorial competence.

The surface free energy of Leishmania mexicana amazonensis.

Surface charge of Leishmania mexicana amazonensis was investigated by direct zeta-potential determination and ultrastructural cytochemistry, and its surface tension was studied by measurements of the advancing contact angle formed by the parasite monolayers with drops of liquids of different polarities. Both virulent and avirulent promastigotes exhibited negatively charged surfaces with a zeta-potential of about -15 mV. Treatment of these cells with trypsin, alkaline phosphatase, or phospholipase C rendered their surfaces less negatively charged, whereas neuraminidase did not alter the parasite negativeness. Cytochemically, we could observe a reduction in the cationized ferritin binding after the parasite treatment with each of the former enzymes, but not with neuraminidase. The surface free energy of parasites was calculated by taken to account the London dispersion, the Keeson dipole-dipole, and the Debye dipole-induced forces, as well as the surface polarity of the parasites and their zeta-potentials, by considering their adhesion to polystyrene surfaces. The delta G values of -6.4 and -18.1 mJ.m-2 were obtained for avirulent and virulent promstigotes, respectively.

Cell surface charge and sugar residues of Crithidia fasciculata and Crithidia luciliae.

The surface charge of Crithidia fasciculata and Crithidia luciliae was analysed by measurement of the zeta-potential and labelling of the protozoan surface with cationized ferritin particles. Both trypanosomatids have a net negative surface charge, with a zeta-potential of -10.39 mV and -11.12 mV for C. luciliae and C. fasciculata, respectively. Enzyme treatment showed that phosphate groups, but not sialic acid, significantly contributed to the negative surface charge. Lectin-induced agglutination was used to analyse the presence of surface-exposed carbohydrates in C. fasciculata and C. luciliae. The cells did not agglutinate when incubated in the presence of lectins which recognized L-fucose, N-acetyl-D-glucosamine and sialic acid. However, lectins which bind to N-acetyl-D-galactosamine, D-galactose and D-mannose agglutinated both protozoa.