RESUMO
Detection and transduction of environmental signals, constitute a prerequisite for successful parasite invasion; i.e., Leishmania transmission, survival, pathogenesis and disease manifestation and dissemination, with diverse molecules functioning as inter-cellular signaling ligands. Receptors [i.e., G protein-coupled receptors (GPCRs)] and their associated transduction mechanisms, well conserved through evolution, specialize in this function. However, canonical GPCR-related signal transduction systems have not been described in Leishmania, although orthologs, with reduced domains and function, have been identified in Trypanosomatidae. These inter-cellular communication means seem to be essential for multicellular and unicellular organism's survival. GPCRs are flexible in their molecular architecture and may interact with the so-called receptor activity-modifying proteins (RAMPs), which modulate their function, changing GPCRs pharmacology, acting as chaperones and regulating signaling and/or trafficking in a receptor-dependent manner. In the skin, vasoactive- and neuro- peptides released in response to the noxious stimuli represented by the insect bite may trigger parasite physiological responses, for example, chemotaxis. For instance, in Leishmania (V.) braziliensis, sensory [Substance P, SP, chemoattractant] and autonomic [Vasoactive Intestinal Peptide, VIP, and Neuropeptide Y, NPY, chemorepellent] neuropeptides at physiological levels stimulate in vitro effects on parasite taxis. VIP and NPY chemotactic effects are impaired by their corresponding receptor antagonists, suggesting that the stimulated responses might be mediated by putative GPCRs (with essential conserved receptor domains); the effect of SP is blocked by [(D-Pro 2, D-Trp7,9]-Substance P (10-6 M)] suggesting that it might be mediated by neurokinin-1 transmembrane receptors. Additionally, vasoactive molecules like Calcitonin Gene-Related Peptide [CGRP] and Adrenomedullin [AM], exert a chemorepellent effect and increase the expression of a 24 kDa band recognized in western blot analysis by (human-)-RAMP-2 antibodies. In-silico search oriented towards GPCRs-like receptors and signaling cascades detected a RAMP-2-aligned sequence corresponding to Leishmania folylpolyglutamate synthase and a RAMP-3 aligned protein, a hypothetical Leishmania protein with yet unknown function, suggesting that in Leishmania, CGRP and AM activities may be modulated by RAMP- (-2) and (-3) homologs. The possible presence of proteins and molecules potentially involved in GPCRs cascades, i.e., RAMPs, signpost conservation of ancient signaling systems associated with responses, fundamental for cell survival, (i.e., taxis and migration) and may constitute an open field for description of pharmacophores against Leishmania parasites.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Leishmania , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Comunicação Celular , Humanos , Leishmania/metabolismo , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Substância P/farmacologiaRESUMO
Chemotactic responses play a significant role during Leishmania (V.) braziliensis differentiation through its life cycle and during infection. The aim of this description has been to portray the modified "two-chamber capillary chemotaxis assay" as a technique useful for quantitative in vitro evaluation of Leishmania chemotaxis after reviewing the methods described until now to assess chemotaxis in vitro in Leishmania sp. This valued simple and reproducible method convenient for parasite migration determination, was tested by the use of controlled changes in monosaccharide (D-glucose and D-fructose) concentrations as referent ligands. The validation of the method demonstrates that this technique is useful to evaluate the relationship existing between parasite migration towards the monosaccharides and sugar concentration. This means that within specific ranges, parasites attracted by the monosaccharide migrate towards more concentrated solutions and accumulate (higher number of parasites) at that spot. Interestingly, both the time course of the experiment and the osmolality of the solution influence parasite migration capacity. Our validation suggests that this improved methodology quantitatively evaluates taxis of Leishmania towards/against different substances. On the basis of our herein presented data, we conclude that this technique is a novel, rapid and reliable screening method to evaluate chemotaxis in Leishmania.â¢The two-chamber capillary chemotaxis assay was standardized for Leishmania.â¢The technique is useful to quantitatively evaluate in vitro chemotaxis in Leishmania.â¢Parasite migration was characterized by monosaccharide chemical gradients.â¢This assay is a novel, rapid and reliable screening method to evaluate chemotaxis. Contain between 1 and 3 bullet points highlighting the customization rather than the steps of the procedure.
RESUMO
Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. METHODS: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. RESULTS: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. CONCLUSION: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis.
Assuntos
Humanos , Masculino , Feminino , Estratégias de Saúde , Incerteza , COVID-19 , Pesquisadores , Emoções , Pandemias , ConstrangimentoRESUMO
Cell-cell interaction and active migration (and invasion) of parasites into skin host-cell(s) are key steps for successful infection by Leishmania. Chemotaxis constitutes a primordial chapter of Leishmania-host cell interaction, potentially modulated by neuropeptides released into the skin due, for example, to the noxious stimuli represented by the insect bite. Herein we have evaluated in vitro the effect of sensory (Substance P, SP) and autonomic (Vasoactive Intestinal Peptide, VIP, and Neuropeptide Y, NPY) neuropeptides on parasite taxis, and investigated the potential modulatory effect of SP on Leishmania (Viannia) braziliensis-macrophage interaction. We demonstrated that VIP (10-10 M) and NPY (10-9 M) are chemorepellent to the parasites, while SP (10-8 M) produces a chemoattractant response. SP did not affect macrophage viability but seems to impair parasite-macrophage interaction as it decreased promastigote adherence to macrophages. As this effect is blocked by ([D-Pro 2, D-Trp7,9]-Substance P (10-6 M), the observed action may be mediated by neurokinin-1 (NK1) transmembrane receptors. VIP and NPY repellent chemotactic effect is impaired by their corresponding receptor antagonists. Additionally, they suggest that SP may be a key molecule to guide promastigote migration towards, and interaction, with dendritic cells and macrophage host cells.
Assuntos
Leishmania braziliensis/metabolismo , Neuropeptídeo Y/metabolismo , Substância P/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Quimiotaxia , Flagelos/ultraestrutura , Leishmania braziliensis/fisiologia , Leishmania braziliensis/ultraestrutura , Macrófagos , CamundongosRESUMO
Markers to diagnose chemoresistance in infecting Leishmania parasites are urgently required. This is fundamental for patients who do not heal during or after treatment, as they are unresponsive, or patients who relapse at the end of the therapy, suffering from therapeutic failure. Glucose utilization is an indicator of cell viability that closely associates with metabolic activity. In Leishmania, glucose is a source of carbon atoms and is imported into the cell through specific transporters. In experimentally developed chemoresistant Leishmania parasites a significant decrease of the expression of glucose transporters as well as in the cellular accumulation glucose has been described. Alternatively, the electrical membrane potential is an essential parameter for the formation of the electromotive force needed for the acquisition of important nutrients and solutes (e.g., glucose) by cells, and changes in glucose concentration are suggested to constitute a physiological adaptation associated with a chemoresistant phenotype of Leishmania parasites. Here we describe easy methods to measure glucose uptake and the membrane potential in isolates from patient suffering leishmaniasis. Correlation between both parameters might be helpful to identify chemoresistant parasites. Results suggest that the measured kinetics of glucose utilization rate can be correlated with the plasma membrane potential and together used to differentiate between the performance of wild-type and reference parasites on the one hand and parasites isolated from patients with therapeutic failure on the other.
Assuntos
Antiprotozoários/farmacologia , Glucose/análise , Leishmania/metabolismo , Leishmaniose/diagnóstico , Testes de Sensibilidade Parasitária/métodos , Adaptação Fisiológica , Antiprotozoários/uso terapêutico , Biomarcadores/análise , Biomarcadores/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Resistência a Medicamentos/fisiologia , Glucose/metabolismo , Humanos , Leishmania/citologia , Leishmania/efeitos dos fármacos , Leishmania/isolamento & purificação , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Falha de TratamentoRESUMO
The Calcitonin-Like Receptor (CLR) belongs to the classical seven-transmembrane segment molecules coupled to heterotrimeric G proteins. Its pharmacology depends on the simultaneous expression of the so-called Receptor Activity Modifier Proteins (RAMP-) -1, -2 and -3. RAMP-associated proteins modulate glycosylation and cellular traffic of CLR, therefore determining its pharmacodynamics. In higher eukaryotes, the complex formed by CLR and RAMP-1 is more akin to bind Calcitonin Gene-Related Peptide (CGRP), whereas those formed by CLR and RAMP-2 or RAMP-3, bind preferentially Adrenomedullin (AM). In lower eukaryotes, RAMPs, or any homologous protein, have not been identified until now. Herein we demonstrated a negative chemotactic response elicited by CGRP (10-9 and 10-8â¯M) and AM (10-9 to 10-5â¯M). Whether or not this response is receptor mediated should be verified, as well as the expression of a 24â¯kDa band in Leishmania, recognized by western blot analysis by the use of (human-)-RAMP-2 antibodies as detection probes. Queries with human RAMP-2 and RAMP-3 protein sequences in blastp against Leishmania (Viannia) braziliensis predicted proteome, allowed us to detect two sequence alignments in the parasite: A RAMP-2-aligned sequence corresponding to Leishmania folylpolyglutamate synthase (FPGS), and a RAMP-3 aligned protein, a hypothetical Leishmania protein with yet unknown function. The presence of homologous of these proteins was described in-silico in other members of the Trypanosomatidae. These preliminary and not yet complete data suggest the feasibility that both CGRP and Adrenomedullin activities may be regulated by homologs of RAMP- (-2) and (-3) in these parasites.
Assuntos
Adrenomedulina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Leishmania , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Sequência de Aminoácidos , Quimiotaxia/fisiologia , Simulação por Computador , Humanos , Leishmania/química , Leishmania/metabolismo , Leishmania/fisiologia , Estágios do Ciclo de Vida/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/química , Proteína 3 Modificadora da Atividade de Receptores/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Este trabajo utilizó un modelo macrófago humano-amastigote como herramienta para recrear in vitro la infección causada por aislados de pacientes con fracaso terapéutico y valorar su utilidad en la identificación de aislados de Leishmania con fenotipo quimio-resistente. Objetivos: (1) Evaluar un modelo in vitro de macrófago humano-amastigote y (2) Determinar su utilidad en la identificación de aislados de Leishmania con fenotipo quimio-resistente. Métodos: Se evaluó un protocolo de purificación basado en la capacidad de los monocitos de adherirse al plástico. Monocitos purificados de sangre humana fueron infectados con promastigotes metacíclicos de especies de referencia y aislados de Leishmania de tres pacientes con falla terapéutica a antimoniales. Se determinó el porcentaje de infección inicial y el efecto leishmanicida de glucantime, anfotericinaB y pentamidina; se correlacionó la capacidad leishmanicida con los niveles de producción de óxido nítrico en cada condición estudiada. Resultados: Los resultados sugieren que el modelo macrófago humano-amastigote empleado recrea in vitro la infección causada por especies de referencia, o con aislados de pacientes con fracaso terapéutico. Adicionalmente sugieren que en monocitos infectados (1) con el aislado VE98MR no puede definirse una IC50 para glucantime ni para pentamidina y (2) con el aislado VE96ZC no puede definirse una IC50 para glucantime mas si para pentamidina. De igual forma, se evidencia una disminución efectiva del porcentaje de infección susceptible a anfotericina-B, para todos los aislados y cepas de referencia. El efecto leishmanicida no se correlaciona con aumentos significativos de la producción de óxido nítrico. Conclusiones: El modelo macrófago humano-amastigote empleado constituye una prueba de concepto que permitió identificar como aislados potencialmente quimio-resistentes a L. (L.) amazonensis (VE98MR) y L. (L.) mexicana (VE96ZC), mas no al aislado L. (L.) amazonensis (VE2000MM)(AU)
This work used a human-amastigote macrophage model as a tool to recreate in vitro infection caused by isolates from patient's with therapeutic failure and assess its usefulness in the identification of chemo-resistant Leishmania isolates. Objectives: (1) Evaluate in vitro a human-amastigote macrophage model and (2) determine its usefulness in the identification of Leishmania isolates with chemo-resistant phenotype. Methods: A purification protocol based on the ability of monocytes to adhere to plastic was evaluated. Monocytes purified from human blood were infected with metacyclic promastigotes of reference species and Leishmania isolates from three patients with antimonial therapeutic failure. The percentage of initial infection and the leishmanicidal effect of glucantime, amphotericin-B and pentamidine were determined; the leishmanicidal capacity was correlated with the levels of nitric oxide production in each condition studied. Results: Results suggest that the human-amastigote macrophage model recreates in vitro the infection caused by reference species, or isolates from patients with therapeutic failure. In addition, they suggest that (1) an effective IC50 for glucantime and pentamidine could not be defined in monocytes infected with the isolate VE98MR and (2) an effective IC50 for pentamidine but nor for glucantime could be defined in monocytes infected with the isolate VE96ZC. On the contrary, an effective decrease in the percentage of infection susceptible to amphotericin-B was observed for all isolates and reference strains. The leishmanicidal effect did not correlate with significant increases in nitric oxide production. Conclusion: The human-amastigote macrophage model used constitutes a proof of concept to identify as potentially chemo-resistant isolates L. (L.) amazonensis (VE98MR) and L. (L.) mexicana (VE96ZC), but not L (L.) amazonensis (VE2000MM)(AU)
Assuntos
Humanos , Masculino , Feminino , Técnicas In Vitro/métodos , Leishmaniose Cutânea/fisiopatologia , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/virologia , Medicina Tropical , Saúde Pública , Tratamento Farmacológico , Ativação de MacrófagosRESUMO
Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of "resistance" related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined.
Assuntos
Resistência a Medicamentos , Leishmania/efeitos dos fármacos , Leishmania/patogenicidade , Leishmaniose/tratamento farmacológico , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Quimioterapia Combinada , Humanos , Leishmania/genética , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/patogenicidade , Leishmaniose/imunologia , Leishmaniose/parasitologia , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Epidemiologia Molecular , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Falha de TratamentoRESUMO
Trypanosoma brucei rhodesiense and T. brucei gambiense, the causative agents of Human African Trypanosomiasis, are transmitted by tsetse flies. Within the vector, the parasite undergoes through transformations that prepares it to infect the human host. Sequentially these developmental stages are the replicative procyclic (in which the parasite surface is covered by procyclins) and trypo-epimastigote forms, as well as the non-replicative, infective, metacyclic form that develops in the vector salivary glands. As a pre-adaptation to their life in humans, metacyclic parasites begin to express and be densely covered by the Variant Surface Glycoprotein (VSG). Once the metacyclic form invades the human host the parasite develops into the bloodstream form. Herein the VSG triggers a humoral immune response. To avoid this humoral response, and essential for survival while in the bloodstream, the parasite changes its cover periodically and sheds into the surroundings the expressed VSG, thus evading the consequences of the immune system activation. Additionally, tools comparable to quorum sensing are used by the parasite for the successful parasite transmission from human to insect. On the other hand, the human host promotes clearance of the parasite triggering innate and adaptive immune responses and stimulating cytokine and chemokine secretion. All in all, the host-parasite interaction is extremely active and leads to responses that need multiple control sites to develop appropriately.
RESUMO
Leishmaniasis is a public health problem in tropical and subtropical areas of the world, including Venezuela. The incidence of treatment failure and the number of cases with Leishmania-HIV co-infection underscore the importance of developing alternative, economical and effective therapies against this disease. The work presented here analyzed whether terpenoids derived from betulin are active against New World Leishmania parasites. Initially we determined the concentration that inhibits the growth of these parasites by 50% or IC50, and subsequently evaluated the chemotactic effect of four compounds with leishmanicidal activity in the sub-micromolar and micromolar range. That is, we measured the migratory capacity of Leishmania (V.) braziliensis in the presence of increasing concentrations of compounds. Finally, we evaluated their cytotoxicity against the host cell and their effect on the infectivity of L. (V.) braziliensis. The results suggest that (1) compounds 14, 17, 18, 25 and 27 are active at concentrations lower than 10 µM; (2) compound 26 inhibits parasite growth with an IC50 lower than 1 µM; (3) compounds 18, 26 and 27 inhibit parasite migration at pico- to nanomolar concentrations, suggesting that they impair host-parasite interaction. None of the tested compounds was cytotoxic against J774.A1 macrophages thus indicating their potential as starting points to develop compounds that might affect parasite-host cell interaction, as well as being leishmanicidal.
Assuntos
Antiprotozoários/farmacologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Macrófagos/parasitologia , Triterpenos/farmacologia , Animais , Antiprotozoários/química , Linhagem Celular , Humanos , Leishmania braziliensis/fisiologia , Macrófagos/efeitos dos fármacos , Camundongos , Triterpenos/químicaRESUMO
Besides infection with drug-resistant parasites, therapeutic failure in leishmaniasis may be caused by altered drug pharmacokinetics, re-infection, and host immunologic compromise. Our aim has been to evaluate if relapses that occur in patients suffering from diffuse cutaneous leishmaniasis (DCL) associate with changes in the fitness of infecting organisms. Therefore, in isolates from patients suffering DCL, we correlated glucose uptake and plasma membrane potential and compared the results with those obtained from reference strains. The data demonstrate that Leishmania parasites causing DCL incorporate glucose at an efficient rate, albeit without significant changes in the plasma membrane potential as their corresponding reference strains. The isolate that did not change its accumulation rate of glucose compared to its reference strain expressed a less polarized membrane potential that was insensitive to mitochondrial inhibitors, suggesting a metabolic dysfunction that may result in glycolysis being the main source of ATP. The results constitute a proof of concept that indicates that parasites causing DCL adapted well to drug pressure and expressed an increased fitness. That is, that in Leishmania mexicana and Leishmania amazonensis, parasites isolated from DCL patients, a strong modification of the parasite physiology might occur. As consequences, the parasites adapted well to drug pressure, increased their fitness, and they had an efficient glucose uptake rate albeit not significant changes in membrane potential as their corresponding reference strains. Further validation of the concepts herein established and whether or not the third isolate corresponds with a drug-resistant phenotype need to be demonstrated at the genetic level.
Assuntos
Antiprotozoários/uso terapêutico , Glucose/metabolismo , Leishmania/metabolismo , Leishmaniose Tegumentar Difusa/parasitologia , Potenciais da Membrana/fisiologia , Animais , Membrana Celular/fisiologia , Humanos , Leishmania/efeitos dos fármacos , Leishmania/isolamento & purificação , Leishmania mexicana/imunologia , Falha de TratamentoRESUMO
Chemotactic responses play a significant role during Leishmania differentiation, as well as in the course of parasite-host-cell interaction, a process that precedes a successful infection. The present study uses the modified "two-chamber capillary assay" to quantitatively evaluate the chemotactic properties and the toxic activities of methotrexate containing branched chain polymeric polypeptide conjugates in Leishmania. Our results demonstrate that this methodology quantitatively determines the taxis of Leishmania towards/against gradients of compounds. They also demonstrate that chemotaxis produced by the polypeptide-methotrexate conjugates depends on specific chemical characteristics. For example, the N-terminal amino acid (Ser or Glu) location at the branch significantly influences the elicited chemotaxis. Furthermore, the use of different attachment sites in the methotrexate conjugates (α- or γ-carboxylic groups) affect their chemotactic activity. Specific cytotoxic activities and cytostatic effects of the conjugates on parasites and on murine and human cells of the macrophage/monocyte system respectively, suggest that these ligands may be used as a group of anti-Leishmania substances acting selectively on Leishmania and different hosts.
Assuntos
Quimiotaxia/efeitos dos fármacos , Citostáticos/farmacologia , Citotoxinas/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Metotrexato/farmacologia , Polilisina/farmacologia , Animais , Antiprotozoários/farmacologia , Linhagem Celular , Humanos , Leishmania braziliensis/citologia , Macrófagos/efeitos dos fármacos , Metotrexato/química , Camundongos , Monócitos/efeitos dos fármacos , Polilisina/químicaRESUMO
CONTEXT: Euphorbiaceae plants exhibit anti-inflammatory and immunomodulatory properties. METHODS: We evaluated the activity of 14 extracts from seven Euphorbiaceae plants on primary immune cell cultures from healthy individuals. Peripheral blood mononuclear cells (PBMC) were exposed to the extracts w/o phytohaemagglutinin A or cycloheximide as agents that induce proliferation or apoptosis in PBMC, respectively. RESULTS: We found that five up to 14 Euphorbiaceae's extracts had the ability to modulate at least one of the immune parameters evaluated in this study. However, only the latex extracts of Euphorbia cotinifolia and Euphorbia tirucalli strongly induced both proliferation and apoptosis in PBMC. These extracts were further subfractioned by silica gel column chromatography. Two subfractions with enhanced activity in comparison to the crude extracts were obtained. Although these subfractions induced proliferation on both CD3(+) and CD3(-) cells, the most prominent effects were observed in the former subpopulation. Interestingly, the subfraction from E. tirucalli induced lymphocyte proliferation without the need of accessory cells; this ability was not inhibited by the carbohydrates d-galactose and α-Methyl-D-Mannopyranoside. CONCLUSIONS: Altogether, these results reveal the presence of novel candidates within the Euphorbia plants to induce proliferation and apoptosis in human lymphocytes, mainly in CD3(+) T cells.
Assuntos
Euphorbiaceae/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Extratos Vegetais/imunologia , Extratos Vegetais/farmacologia , Células Cultivadas , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Extratos Vegetais/isolamento & purificação , Folhas de Planta/imunologia , Cultura Primária de CélulasRESUMO
A source of chemotherapeutic failure in anti-infective therapies is the active movement of drugs across membranes, through ATP-binding cassette (ABC) transporters. In fact, simultaneous administration of therapeutic drugs with ABC transporter blockers has been invoked to be the way to actively prevent the emergence of drug resistance. Herein, we demonstrate that glucantime's efficacy in decreasing the infection rate of Leishmania-infected macrophages is strongly enhanced when used in combination with glibenclamide, a specific blocker of ABC transporters. Intracellular ABC transporters mediate glucantime sequestration in intracellular organelles. Their selective inhibition may effectively increase the cytoplasmic concentration of glucantime and its leishmanicidal activity. Our results reveal for the first time that glibenclamide targets in Leishmania major a compartment associated with a multivesicular system that is simultaneously labeled by the acidic marker LysoTracker-red and may represent the organelle where antimonials are sequestered. These results constitute a proof of concept that conclusively demonstrates the potential value that combination therapy with an ABC transporter blocker may have for leishmaniasis therapy.
Assuntos
Antiprotozoários/farmacologia , Glibureto/farmacologia , Leishmania major/efeitos dos fármacos , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Anfotericina B/farmacologia , Animais , Antiprotozoários/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Glibureto/metabolismo , Leishmania major/metabolismo , Leishmania major/ultraestrutura , Macrófagos Peritoneais/parasitologia , Meglumina/metabolismo , Antimoniato de Meglumina , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Compostos Organometálicos/metabolismoRESUMO
ATP-binding cassette (ABC) transporters encompass membrane transport proteins that couple the energy derived from ATP hydrolysis to the translocation of solutes across biological membranes. The functions of these proteins include ancient and conserved mechanisms related to nutrition and pathogenesis in bacteria, spore formation in fungi, and signal transduction, protein secretion and antigen presentation in eukaryotes. Furthermore, one of the major causes of drug resistance and chemotherapeutic failure in both cancer and anti-infective therapies is the active movement of compounds across membranes carried out by ABC transporters. Thus, the clinical relevance of ABC transporters is enormous, and the membrane transporters related to chemoresistance are among the best-studied members of the ABC transporter superfamily. As ABC transporter blockers can be used in combination with current drugs to increase their efficacy, the (possible) impact of efflux pump inhibitors is of great clinical interest. The present review summarizes the progress made in recent years in the identification, design, availability, and applicability of ABC transporter blockers in experimental scenarios oriented towards improving the treatment of infectious diseases caused by microorganisms including parasites.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/fisiologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias , Transportadores de Cassetes de Ligação de ATP/química , Bactérias/patogenicidade , Transporte Biológico , Desenho de Fármacos , Resistência a Múltiplos MedicamentosRESUMO
Leishmaniasis is a disease caused by at least 17 different species of protozoan Leishmania parasites and currently affects around 12 million people living mostly in tropical and subtropical areas. Failure to treat leishmaniasis successfully is often due to drug resistance. However, there are no cellular and molecular markers of chemoresistance against leishmanicidal drugs and the only reliable method for monitoring resistance of individual isolates is the in vitro amastigote/macrophage model. It is thus necessary to find cellular and molecular markers that can be used systematically to identify the drug-resistant phenotype of the infecting parasites. Until now, whether drug resistance in Leishmania compromises parasite proficiency, e.g. in terms of infectivity or metabolism, has not been systematically evaluated. Therefore, here we examine whether the physiological changes expressed by drug-resistant Leishmania reflect a modification of parasite vitality in drug-resistant compared with drug-sensitive parasites. Finally, the clinical implications of drug resistance in Leishmania are also discussed.
Assuntos
Antiprotozoários/farmacologia , Resistência a Medicamentos , Leishmania/efeitos dos fármacos , Leishmania/metabolismo , Leishmaniose/parasitologia , Animais , Previsões , Glucose/metabolismo , Humanos , Cinética , Leishmania/crescimento & desenvolvimento , Testes de Sensibilidade ParasitáriaRESUMO
We compared growth rate, cell glucose turnover and expression of ATP-binding-cassette (ABC) transporters in Leishmania amazonensis (LTB0016; LTB) versus LTB(160) selected for resistance against the ABC transporter blocker glibenclamide. Additionally, we evaluated the influence of drug-resistance on Leishmania sensitivity against 2-mercaptoacetate and 2-deoxyglucose. Our data demonstrate that (1) LTB(160) and LTB constitutively express ABC transporters for neutral substrates, (2) glibenclamide resistance induces the expression of organic anion ABC transporters, members of the drug resistance associated transporters subfamily, (3) LTB(160) parasites use less glucose as energy substrate and exhibit a slower glucose uptake than LTB cells, and (4) LTB(160) parasites are less sensitive to 2-mercaptoacetate and 2-deoxyglucose than the glibenclamide-sensitive Leishmania LTB. Together these and previous results indicate that the metabolic adaptations expressed in drug-resistant LTB(160) differ from those described for mammalian drug resistant cells and constitute general mechanisms that underlie drug resistance in Leishmania and may be helpful for identifying alternative strategies to circumvent drug resistance in leishmaniasis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Resistência a Medicamentos/fisiologia , Glibureto/farmacologia , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Adaptação Fisiológica , Animais , Desoxiglucose/farmacologia , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Glucose/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Tioglicolatos/farmacologiaRESUMO
El papel de las células dendríticas es fundamental en la señalización de la infección por Leishmania desde la piel hasta los ganglios linfáticos; sin embargo, la proliferación de Leishmania en estas células está restringida. En el macrófago, célula hospedera de Leishmania por excelencia, los parásitos proliferan irrestrictamente y el control de la infección depende directamente de los niveles de óxido nítrico. Debido a que la disponibilidad de hierro celular modula la actividad de la enzima óxido nítrico sintetasa del macrófago y para analizar las causas de la restricción de la proliferación de Leishmania en células dendríticas, en este trabajo evaluemos la influencia del hierro libre intracelular sobre la funcionalidad de células de dendríticas y la infección por Leishmania major. Los resultados demuestran que la en células de Langerhans de la piel expuestas a quelantes de hierro la infección por Leishmania major y la expresión del receptor de transferina disminuyen, sin alterar la producción de óxido nítrico y las otras propiedades funcionales de la célula. La incubación de células dendríticas de médula ósea con quelantes de hierro no afecta la infección por Leishmania major ni la producción de óxido nítrico de la célula. Estos resultados demuestran que la restricción de la proliferación de Leishmania en células dendríticas es independiente de los niveles de hierro celular y de los niveles de óxido nítrico.