RESUMEN

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Visceral/inmunología , Piel/inmunología , Adolescente , Adulto , Antígenos de Protozoos/farmacología , Linfocitos T CD4-Positivos/parasitología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/parasitología , Linfocitos T CD8-positivos/patología , Proliferación Celular , Citocinas/genética , Citocinas/inmunología , Citotoxicidad Inmunológica , Femenino , Citometría de Flujo , Expresión Génica , Granzimas/genética , Granzimas/inmunología , Humanos , Inmunofenotipificación , India , Células Asesinas Naturales/parasitología , Células Asesinas Naturales/patología , Leishmania donovani/inmunología , Leishmania donovani/patogenicidad , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/patología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/parasitología , Leucocitos Mononucleares/patología , Activación de Linfocitos , Masculino , Carga de Parásitos , Cultivo Primario de Células , Piel/parasitología , Piel/patología

RESUMEN

Cell-mediated and humoral immunity were explored in LiESAp-MDP vaccinated protected dogs versus susceptible placebo dogs 2 months and 8 months post-vaccination. As previously described, a strong and long-lasting cell-mediated immunity, critical for protection against Leishmania infantum was exclusively revealed in vaccinated dogs as confirmed by a positive response to the intradermal inoculation of leishmanin and by a significant higher anti-leishmanial activity of canine monocytes-derived macrophages. Moreover, our results support the view that cooperation of humoral antibody with cell-mediated immunity might be important in developing protective immunity in LiESAp-MDP vaccinated dogs. Anti-LiESAp serum samples were found functionally active in vitro, promoting (i) early killing of pretreated promastigotes and amastigotes, (ii) strong inhibitory effect on the in vitro growth of both parasitic developmental stages of L. infantum and (iii) most importantly, a significant inhibition of pretreated promastigotes in vitro infectivity in canine macrophages. However, anti-LiESAp antibody response was not implicated in the promastigotes-amastigotes differentiation process. In these experiments, we have added additional support to the concept that antibodies to Leishmania may be important in developing protective immunity.

Asunto(s)

Enfermedades de los Perros/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/veterinaria , Vacunas Antiprotozoos/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Proliferación Celular , Enfermedades de los Perros/prevención & control , Perros , Femenino , Hipersensibilidad Tardía/veterinaria , Inmunidad Celular , Inmunoglobulina G/sangre , Leishmania infantum/citología , Leishmania infantum/fisiología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/prevención & control , Macrófagos/inmunología , Masculino , Monocitos/fisiología , Vacunas Antiprotozoos/efectos adversos

RESUMEN

Extracts of seven medicinal plants used specifically against cutaneous leishmaniasis in the Madre de Dios region of Peru were evaluated in vitro against promastigote and axenic amastigote forms of Leishmania amazonensis. One of them showed interesting leishmanicidal activities (IC(50)=5 microg/ml in amastigotes). Bio-guided isolation of the stem bark's ethanol extract of Himatanthus sucuuba (Spruce ex Müll. Arg.) Woodson (Apocynaceae) afforded the spirolactone iridoids isoplumericin and plumericin. The latter showed a reduction of macrophage infection similar to that of the reference drug Amphotericin B (IC(50)=0.9 and 1 microM, respectively). These findings validate the traditional use of Himatanthus sucuuba in the treatment of cutaneous leishmaniasis (Uta) in Peru.

Asunto(s)

Antiprotozoarios , Apocynaceae/química , Indenos/farmacología , Iridoides/farmacología , Leishmania mexicana/efectos de los fármacos , Espironolactona/farmacología , Animales , Células 3T3 BALB , Bioensayo , Chlorocebus aethiops , Cristalografía por Rayos X , Etnobotánica , Humanos , Técnicas In Vitro , Indígenas Sudamericanos , Leishmania mexicana/crecimiento & desarrollo , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Espectroscopía de Resonancia Magnética , Masculino , Medicina Tradicional , Ratones , Ratones Endogámicos BALB C , Perú , Extractos Vegetales/química , Extractos Vegetales/farmacología , Células Vero

RESUMEN

Antibodies (Ab) directed against a tryptophan-like epitope (WE) were previously detected in patients with human African trypanosomiasis (HAT). We investigated whether or not these Ab resulted from immunization against trypanosome antigen(s) expressing a WE. By Western blotting, we identified an antigen having an apparent molecular weight ranging from 60 to 65 kDa, recognized by purified rabbit anti-WE Ab. This antigen, present in trypomastigote forms, was absent in procyclic forms and Trypanosoma cruzi trypomastigotes. Using purified variable surface glycoproteins (VSG) from various trypanosomes, we showed that VSG was the parasite antigen recognized by these rabbit Ab. Anti-WE and anti-VSG Ab were purified from HAT sera by affinity chromatography. Immunoreactivity of purified antibodies eluted from affinity columns and of depleted fractions showed that WE was one of the epitopes borne by VSG. These data underline the existence of an invariant WE in the structure of VSG from several species of African trypanosomes.

Asunto(s)

Anticuerpos Antiprotozoarios/inmunología , Epítopos/aislamiento & purificación , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei gambiense/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunología , Animales , Anticuerpos Antiprotozoarios/aislamiento & purificación , Western Blotting , Cromatografía de Afinidad , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Femenino , Humanos , Ratones , Conejos , Trypanosoma cruzi/inmunología , Tripanosomiasis Africana/inmunología

RESUMEN

Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania, both in vitro and in vivo. We investigated the type of cell death process induced by NO for the intracellular amastigote stage of the protozoa Leishmania. Specific detection methods revealed a rapid and extensive cell death with morphological features of apoptosis in axenic amastigotes exposed to NO donors, in intracellular amastigotes inside in vitro - activated mouse macrophages and also in activated macrophages of regressive lesions in a leishmaniasis-resistant mouse model. We extended our investigations to the dog, a natural host-reservoir of Leishmania parasites, by demonstrating that co-incubation of infected macrophages with autologous lymphocytes derived from dogs immunised with purified excreted-secreted antigens of Leishmania resulted in a significant NO-mediated apoptotic cell death of intracellular amastigotes. From the biochemical point of view, NO-mediated Leishmania amastigotes apoptosis did not seem to be controlled by caspase activity as indicated by the lack of effect of cell permeable inhibitors of caspases and cysteine proteases, in contrast to specific proteasome inhibitors, such as lactacystin or calpain inhibitor I. Moreover, addition of the products of two NO molecular targets, cis-aconitase and glyceraldehyde-3-phosphate dehydrogenase, also had an inhibitory effect on the cell death induced by NO. Interestingly, activities of these two enzymes plus 6-phosphogluconate dehydrogenase, parasitic enzymes involved in both glycolysis and respiration processes, are overexpressed in amastigotes selected for their NO resistance. This review focuses on cell death of the intracellular stage of the pathogen Leishmania induced by nitrogen oxides and gives particular attention to the biochemical pathways and the molecular targets potentially involved. Questions about the role of Leishmania amastigotes NO-mediated apoptosis in the overall infection process are raised and discussed.

Asunto(s)

Apoptosis/fisiología , Leishmania/fisiología , Leishmaniasis/parasitología , Estadios del Ciclo de Vida/fisiología , Óxido Nítrico/fisiología , Animales , Perros , Humanos , Leishmania/citología , Leishmania/crecimiento & desarrollo , Ratones , Fenotipo

RESUMEN

We previously documented the induction of Leishmania amastigote apoptosis by trivalent antimony (SbIII) and nitric oxide (NO). We demonstrate here that SbIII-resistant amastigotes were resistant to NO toxicity when delivered extracellularly by NO donors or intracellularly via macrophage activation. Shared biochemical targets for SbIII and NO resistance in Leishmania are discussed.

Asunto(s)

Tartrato de Antimonio y Potasio/farmacología , Antiprotozoarios/farmacología , Leishmania infantum/efectos de los fármacos , Leishmania infantum/crecimiento & desarrollo , Estadios del Ciclo de Vida , Óxido Nítrico/toxicidad , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Resistencia a Medicamentos , Concentración 50 Inhibidora , Interferón gamma/farmacología , Leishmania infantum/citología , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/parasitología

RESUMEN

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.

Asunto(s)

Antígenos de Protozoos/administración & dosificación , Perros/inmunología , Perros/parasitología , Interferón gamma/biosíntesis , Leishmania infantum/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , Óxido Nítrico/metabolismo , Animales , Antígenos de Protozoos/aislamiento & purificación , Apoptosis , Técnicas de Cocultivo , Femenino , Inmunización , Interleucina-4/biosíntesis , Leishmania infantum/citología , Leishmania infantum/patogenicidad , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/veterinaria , Masculino

RESUMEN

Antibodies directed against nitrosylated epitopes have been found in sera from patients suffering from human African trypanosomiasis (HAT) but not in sera from control subjects living in the same endemic area or African control subjects living in France. We conjugated amino acids to albumin by glutaraldehyde (conjugates) and then nitrosylated the conjugates. Both conjugates and nitrosylated conjugates were analysed by enzyme-linked immunosorbent assay (ELISA). We detected antibodies directed against nitrosylated L-cysteine and L-tyrosine conjugates; antibody levels were higher in stage II patients than in stage I. Patients with severe clinical signs had higher antibody levels, and antibody levels were highest in patients with major neurological signs. Antibody response was only associated with the IgM isotype. We evaluated antibody specificity and avidity by competition experiments using conjugates and nitrosylated conjugates. Avidity was around 2 x10(-6) m for the S-nitroso-cysteine epitope and 2 x 10(-8) m for the S-nitroso-tyrosine epitope. Detection of circulating antibodies to S-nitroso-cysteine and S-nitroso-tyrosine epitopes provides indirect evidence for nitric oxide (NO) involvement in HAT and their levels are correlated with disease severity.

Asunto(s)

Autoanticuerpos/sangre , Compuestos Nitrosos/inmunología , Tripanosomiasis Africana/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Autoantígenos/inmunología , Proteínas Portadoras/inmunología , Cisteína/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Glutaral/inmunología , Humanos , Óxido Nítrico/inmunología , Índice de Severidad de la Enfermedad , Tirosina/inmunología

RESUMEN

Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modulation of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50,90/10,10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12,28,35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote-promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasite's life cycle.

Asunto(s)

Antimonio/farmacología , Antiprotozoarios/farmacología , Resistencia a Medicamentos , Leishmania infantum/efectos de los fármacos , Animales , Línea Celular , Enfermedades Endémicas , Humanos , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Leishmaniasis Visceral/parasitología , Estadios del Ciclo de Vida , Pruebas de Sensibilidad Parasitaria , Fenotipo

RESUMEN

The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes of Leishmania infantum at low concentrations (10 microg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 microg/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.

Asunto(s)

Antimonio/farmacología , Antiparasitarios/farmacología , Fragmentación del ADN/efectos de los fármacos , ADN Protozoario/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Animales , Apoptosis , ADN Protozoario/metabolismo , Leishmania infantum/genética , Pruebas de Sensibilidad Parasitaria

RESUMEN

The in vitro growth of promastigote cells of Leishmania amazonensis was found to strongly depend on interactions among strains that differed in their pentamidine resistance. In particular, the growth of resistant strains was reduced when they shared the same environment with a less-resistant strain.

Asunto(s)

Antiprotozoarios/farmacología , Leishmania/efectos de los fármacos , Pentamidina/farmacología , Animales , Farmacorresistencia Microbiana , Leishmania/crecimiento & desarrollo , Especificidad de la Especie

RESUMEN

Protocols for DNA electroporation in Leishmania promastigote cells are well established. More recently, in vitro culture of axenic Leishmania amastigotes became possible. We have established conditions for DNA transformation of axenically grown Leishmania infantum amastigotes. Parameters for DNA electroporation of Leishmania axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 microM (0.12 microg/ml), 55 microg/ml, 95 microg/ml, and 0.12 microg/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferase-expressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs.

Asunto(s)

Antiprotozoarios/farmacología , Vida Libre de Gérmenes/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria/métodos , Transformación Genética , Animales , Línea Celular , ADN Recombinante/genética , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Electroporación , Genes Reporteros , Humanos , Leishmania infantum/genética , Leishmania infantum/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Transfección

RESUMEN

Trypanosoma brucei gambiense, a causative agent of sleeping sickness, induced a dose-dependent production of tumor necrosis factor (TNF)-alpha by human macrophages in vitro. TNF-alpha was also induced in the Mono Mac 6 cell line, which indicates a direct effect of parasite components on macrophages. Parasite-soluble factors were also potent inducers of TNF-alpha. The addition of anti-TNF-alpha to cocultures of macrophages and parasites increased the number of trypanosomes and their life span, whereas irrelevant antibodies had no effect. TNF-alpha may have a direct role (i.e., direct trypanolytic activity) and/or an indirect one, such as TNF-alpha-mediated induction of cytotoxic molecules. A direct dose-dependent lytic effect of TNF-alpha on purified parasites was observed. This lytic effect was inhibited by anti-TNF-alpha. These data suggest that, as in experimental trypanosomiasis, TNF-alpha is involved in parasite growth control in human African trypanosomiasis.

Asunto(s)

Macrófagos/inmunología , Trypanosoma brucei gambiense/inmunología , Tripanosomiasis Africana/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Antígenos de Protozoos/inmunología , Línea Celular , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Humanos , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei gambiense/crecimiento & desarrollo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

In a previous paper we have demonstrated that the induction, by direct drug pressure, of a resistance to Sb(III) antimony at physiological concentration in the amastigote stage of the parasite, led to a high cross-resistance to Sb(V) species in the form of Glucantime. In this paper, further chemoresistant clones were characterized. Axenic amastigotes of Leishmania infantum were adapted to survive in culture medium containing 4, 20, 30 and 120 microg/ml of potassium antimonyl tartrate Sb(II). These mutants were 12, 28, 35 and 44-fold more resistant to Sb(III) than the parental wild-type clone. They were able to resist at concentrations of Glucantime Sb(V) as high as 160 microg/ml when growing in THP-1 cells. We have investigated the efficacy of second line drugs in clinical use (pentamidine and amphotericin B) on the antimony-resistant mutants. Amphotericin B was toxic for both wild-type and chemoresistant mutants at concentrations ranging from 0.05 to 0.15 microM. Pentamidine which is extensively used when the first course of antimonial pentavalent compounds is unsuccessful, was more toxic for all the chemoresistant organisms than for the wild-type clone. In the same way, chemoresistant amastigotes growing within THP-1 cells were more susceptible to pentamidine than the wild-type clone. Our results showed that the resistance of the mutants was restricted to the antimony containing drugs and did not led to a cross-resistance against the other clinically relevant drugs. These results confirmed that these two drugs (pentamidine and amphotericin B) are good candidates to treat pentavalent antimonial unresponsiveness.

Asunto(s)

Antiprotozoarios/farmacología , Leishmania infantum/efectos de los fármacos , Meglumina/farmacología , Compuestos Organometálicos/farmacología , Anfotericina B/farmacología , Animales , Tartrato de Antimonio y Potasio/farmacología , Línea Celular , Resistencia a Medicamentos/genética , Humanos , Leishmania infantum/genética , Antimoniato de Meglumina , Mutación , Pentamidina/farmacología

RESUMEN

Following purification by affinity chromatography, a Leishmania major S-hexylglutathione- binding protein of molecular mass 66kDa was isolated. The immune serum against the parasite 66kDa polypeptide when used to screen a L. major cDNA library could identify clones encoding for the human v-fos transformation effector homologue, namely ribosomal protein S3a, and thus was named LmS3a-related protein (LmS3arp). A 1027bp cDNA fragment was found to contain the entire parasite gene encoding for a highly basic protein of 30kDa calculated molecular mass sharing homology to various ribosomal S3a proteins from different species. Using computer methods for a multiple alignment and sequence motif search, we found that LmS3arp shares a sequence homology to class theta glutathione S-transferase mainly in a segment containing critical residues involved in glutathione binding. These new findings are discussed in the light of recent published data showing multiple function(s) of the ribosomal proteins S3a.

Asunto(s)

Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Genes Protozoarios/genética , Leishmania major/genética , Proteínas Protozoarias , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/inmunología , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Biblioteca de Genes , Glutatión/metabolismo , Leishmania major/química , Leishmania major/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Radioisótopos de Azufre

RESUMEN

To study the antigens secreted by promastigote and amastigote forms of Leishmania infantum which are able to induce a humoral response in human patients and dogs, we have carried out immunoprecipitation assays with different supernatants of in vitro cultured parasites, metabolically labelled with [35S]methionine, using serum samples from human patients and dogs. In addition, some metabolic labelling experiments were performed daily during the in vitro culture parasite's life cycle to follow the time course excretion-secretion of parasitic antigens. The results demonstrated that the two different hosts developed an antibody response against secreted antigens of both stages of Leishmania infantum. Nevertheless, the humoral response directed against the excreted-secreted antigens of the promastigote forms was qualitatively and quantitatively different when we compare the human and the dog immune responses. On the other hand, when the excreted-secreted antigens of the amastigote forms are immunoprecipitated with either human or canine immune serum, the humoral response is similar. In addition, the time course study showed that excretion-secretion of antigens was qualitatively and quantitatively modulated during the parasitic in vitro life cycle.

Asunto(s)

Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Enfermedades de los Perros/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/veterinaria , Animales , Formación de Anticuerpos , Antígenos de Protozoos/biosíntesis , Enfermedades de los Perros/sangre , Enfermedades de los Perros/parasitología , Perros , Humanos , Leishmaniasis Visceral/sangre , Pruebas de Precipitina

RESUMEN

The elimination of serum or of serum-derived macromolecules that supplant the fetal calf serum requirement from Leishmania culture media could decrease costs and improve the feasibility of large-scale production of well-defined parasite material. We report a completely defined medium, without serum-derived protein and/or macromolecules as a serum substitute, of common, available, and inexpensive constituents that can be used in place of serum-supplemented media for the continuous in vitro cultivation of promastigote forms of various Leishmania species. Typical promastigote morphology was observed in Giemsa-stained smears, regardless of the strain analyzed. Electrophoretic analysis showed that the proteinase patterns of aserically grown promastigote forms were similar to those obtained in serum-supplemented RPMI 1640 medium for all Leishmania studied. Similar antigenic profiles were recognized in immunoblots by sera from hosts with visceral or cutaneous leishmaniasis after growing promastigotes in the two different culture media. For parasites causing both cutaneous and visceral leishmaniasis, the absence of serum and macromolecules in the culture medium did not markedly change their in vitro infectivity for resident mouse macrophages and their virulence in animals compared with parasites cultivated in nondefined medium. Serum-free technology will be increasingly important in providing stability and reproducibility as research using promastigote moves closer to therapeutic applications.

Asunto(s)

Medio de Cultivo Libre de Suero , Leishmania/crecimiento & desarrollo , Animales , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/metabolismo , Femenino , Immunoblotting , Cinética , Leishmania/enzimología , Leishmania/patogenicidad , Leishmaniasis Cutánea/parasitología , Leishmaniasis Visceral/parasitología , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos BALB C , Pase Seriado , Virulencia

RESUMEN

The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leishmania species, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of Leishmania infantum cultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC50s], about 1.8 mg/ml and 22 microg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes of L. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plus m-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantum amastigotes (IC50s, of 0.54 and 1.32 microg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to the Leishmania amastigote stage.

Asunto(s)

Gluconato de Sodio Antimonio/farmacología , Antimonio/química , Antiprotozoarios/farmacología , Leishmania infantum/efectos de los fármacos , Leishmania infantum/crecimiento & desarrollo , Animales , Gluconato de Sodio Antimonio/química , Línea Celular , Cresoles/farmacología , Resistencia a Medicamentos , Humanos , Meglumina/farmacología , Pruebas de Sensibilidad Microbiana

RESUMEN

In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (e.g. logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [35S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunofluorescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.

Asunto(s)

Antígenos de Protozoos/análisis , Proteínas de Unión al ADN/análisis , Proteínas Fúngicas/análisis , Histona Desacetilasas , Leishmania major/inmunología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Transactivadores/análisis , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Peso Molecular , Pruebas de Precipitina , Homología de Secuencia de Aminoácido , Sirtuina 2 , Sirtuinas

RESUMEN

Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 microM pentamidine, while clone LmPENT20 was resistant to 20 microM pentamidine. Resistance indexes (50% inhibitory concentration [IC50] after drug presure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wild-type clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 microM pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC50s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drug-resistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

Asunto(s)

Antiprotozoarios/farmacología , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/crecimiento & desarrollo , Pentamidina/farmacología , Animales , Resistencia a Medicamentos , Leishmania mexicana/ultraestructura , Fenotipo