RESUMO
The present study aimed to evaluate the antileishmanial effect, the mechanisms of action and the association with miltefosine of Vernonia brasiliana essential oil against Leishmania infantum promastigotes. This essential oil was obtained by hydrodistillation and its chemical composition was determined by gas chromatography-mass spectrometry (GC-MS). The antileishmanial activity against L. infantum promastigotes and cytotoxicity on DH82 cells were evaluated by MTT colorimetric assay. Ultrastructural alterations were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential, in the production of reactive oxygen species, and analysis of apoptotic events were determined by flow cytometry. The association between the essential oil and miltefosine was evaluated using the modified isobologram method. The most abundant component of the essential oil was ß-caryophyllene (21.47 %). Anti-Leishmania assays indicated an IC50 of 39.01⯱â¯1.080⯵g/mL for promastigote forms after 72â¯h of treatment. The cytotoxic concentration for DH82 cells was 63.13⯱â¯1.211⯵g/mL after 24â¯h of treatment. The effect against L. infantum was proven through the ultrastructural changes caused by the oil, such as kinetoplast and mitochondrial swelling, vesicles in the flagellar pocket, discontinuity of the nuclear membrane, nuclear fragmentation and condensation, and loss of organelles. It was observed that the oil leads to a decrease in the mitochondrial membrane potential (35.10 %, pâ¯=â¯0.0031), increased reactive oxygen species production, and cell death by late apoptosis (17.60 %, pâ¯=â¯0.020). The combination of the essential oil and miltefosine exhibited an antagonistic effect. This study evidences the antileishmanial action of V. brasiliana essential oil against L. infantum promastigotes.
Assuntos
Antiprotozoários/farmacologia , Apoptose/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Óleos Voláteis/farmacologia , Óleos de Plantas/farmacologia , Sesquiterpenos Policíclicos/farmacologia , Vernonia , Animais , Antiprotozoários/isolamento & purificação , Antiprotozoários/toxicidade , Linhagem Celular , Cães , Interações Medicamentosas , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/metabolismo , Leishmania infantum/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Óleos Voláteis/isolamento & purificação , Óleos Voláteis/toxicidade , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Óleos de Plantas/isolamento & purificação , Óleos de Plantas/toxicidade , Sesquiterpenos Policíclicos/isolamento & purificação , Sesquiterpenos Policíclicos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Vernonia/químicaRESUMO
Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.
Assuntos
Leishmania infantum/química , Leishmania mexicana/química , Proteínas de Membrana/análise , Proteômica/métodos , Proteínas de Protozoários/análise , Animais , Membrana Celular/química , Cromatografia Líquida , Biologia Computacional , Cricetinae , Transportador de Glucose Tipo 2/análise , Interações Hospedeiro-Parasita , Leishmania infantum/metabolismo , Leishmania infantum/patogenicidade , Leishmania infantum/ultraestrutura , Leishmania mexicana/ultraestrutura , Macrófagos Peritoneais/parasitologia , Espectrometria de Massas , Mesocricetus , Metaloendopeptidases/análise , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais , Espectrometria de Massas em Tandem , VirulênciaRESUMO
Background: The leishmaniases are a group of parasitic diseases caused by trypanosomatids belonging to the genus Leishmania, members of the class Kinetoplastida, order Trypanosomatidae, family Trypanosomatidae. Despite innumerous wildspecies that are infected, the domestic dog is a potential reservoir of Leishmania infantum in urban areas, which expandsthe transmission pathway to humans. When infected, the dog becomes Visceral Canine Leishmaniasis (CVL), which ischaracterized by a diverse clinical picture that ranges from asymptomatic to non-specific signs, such as skin lesions, lymphadenomegaly, weight loss, splenomegaly, and/or ocular lesions, thus impairing accurate and rapid diagnosis. In Brazil, itis considered a public health problem since it is endemic in certain regions. Therefore, parasitological, serological, andmolecular methods can be used for the detection of the disease. However, the possibility of serological cross-reaction andthe occurrence of co-infection with other trypanosomatids decreases the specificity rate to below 100%, which suggeststhe use of more accurate diagnostic tools. Several molecular targets and starting samples for leishmaniasis diagnosis arealready standardized, but there is lack of data allowing the evaluation of the target, as well as which biological material ismore efficient for the molecular diagnosis of CVL. The sensitivity of PCR may vary with DNA quality, primer type, parasitemia level, and number of target copies per cell. The spleen, blood, liver, aspirate of bone marrow and lymph nodes arethe most frequently used for molecular diagnosis of CVL. The present study aimed to evaluate and compare three protocolsof the polymerase chain reaction (PCR) for the molecular diagnosis of CVL in different biological samples removed fromanimals...(AU)
Assuntos
Animais , Cães , Leishmania/isolamento & purificação , Leishmania/ultraestrutura , Leishmania infantum/ultraestrutura , Reação em Cadeia da Polimerase/veterináriaRESUMO
Background: The leishmaniases are a group of parasitic diseases caused by trypanosomatids belonging to the genus Leishmania, members of the class Kinetoplastida, order Trypanosomatidae, family Trypanosomatidae. Despite innumerous wildspecies that are infected, the domestic dog is a potential reservoir of Leishmania infantum in urban areas, which expandsthe transmission pathway to humans. When infected, the dog becomes Visceral Canine Leishmaniasis (CVL), which ischaracterized by a diverse clinical picture that ranges from asymptomatic to non-specific signs, such as skin lesions, lymphadenomegaly, weight loss, splenomegaly, and/or ocular lesions, thus impairing accurate and rapid diagnosis. In Brazil, itis considered a public health problem since it is endemic in certain regions. Therefore, parasitological, serological, andmolecular methods can be used for the detection of the disease. However, the possibility of serological cross-reaction andthe occurrence of co-infection with other trypanosomatids decreases the specificity rate to below 100%, which suggeststhe use of more accurate diagnostic tools. Several molecular targets and starting samples for leishmaniasis diagnosis arealready standardized, but there is lack of data allowing the evaluation of the target, as well as which biological material ismore efficient for the molecular diagnosis of CVL. The sensitivity of PCR may vary with DNA quality, primer type, parasitemia level, and number of target copies per cell. The spleen, blood, liver, aspirate of bone marrow and lymph nodes arethe most frequently used for molecular diagnosis of CVL. The present study aimed to evaluate and compare three protocolsof the polymerase chain reaction (PCR) for the molecular diagnosis of CVL in different biological samples removed fromanimals...
Assuntos
Animais , Cães , Leishmania infantum/ultraestrutura , Leishmania/isolamento & purificação , Leishmania/ultraestrutura , Reação em Cadeia da Polimerase/veterináriaRESUMO
Plant products are an important source of bioactive agents against parasitic diseases, including leishmaniasis. Among these products, vegetable oils have gained ground in the pharmaceutical field. Here we report the development of nanoemulsions as a delivery system for copaiba and andiroba oils (nanocopa and nanoandi) in order to test their effects on Leishmania infantum and L. amazonensis. The nanocopa and nanoandi had an average particle size of 76.1 and 88.1, respectively with polydispersity index 0.14 to 0.16 and potential zeta -2.54 to -3.9. The data indicated toxic activity of nanocopa and nanoandi against promastigotes of both Leishmania species ultrastructural analyses by scanning electron microscopy revealed that exposition to nanoemulsions induced oval cell shape and retracted flagella. The treatment with nanocopa and nanoandi led to a reduction in L. infantum and L. amazonensis infection levels in macrophage cultures. The nanoemulsions treatment have significant beneficial effects on all the parameters evaluated in lesions induced by L. amazonensis (lesion size, parasite burden and histopathology) on BALB/c mice. The treatment of L. infantum-infected BALB/c mice with nanoemulsions also showed promising results reducing parasite burden in spleen and liver and improving histopathological features.
Assuntos
Fabaceae/química , Leishmania infantum/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Óleos Voláteis/administração & dosagem , Animais , Antiprotozoários/uso terapêutico , Emulsões , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Concentração Inibidora 50 , Intestinos/patologia , Rim/patologia , Leishmania infantum/ultraestrutura , Leishmania mexicana/ultraestrutura , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/patologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/patologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Nanopartículas/ultraestrutura , Óleos Voláteis/química , Fosforilcolina/análogos & derivados , Fosforilcolina/uso terapêutico , Estômago/patologiaRESUMO
BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.
Assuntos
Antimônio/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/ultraestrutura , Leishmaniose Visceral/parasitologia , Compostos de Sulfidrila/metabolismo , Butionina Sulfoximina/farmacologia , Resistência a Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Microscopia Eletrônica de Transmissão , Testes de Sensibilidade ParasitáriaRESUMO
Leishmania (Leishmania) infantum chagasi is one of the agents that cause visceral leishmaniasis. This disease occurs more frequently in third world countries, such as Brazil. The treatment is arduous, and is dependent on just a few drugs like the antimonial derivatives and amphotericin B. Moreover, these drugs are not only expensive, but they can also cause severe side effects and require long-term treatment. Therefore, it is very important to find new compounds that are effective against leishmaniasis. In the present work we evaluated a new group of synthetic amides against the promastigote and amastigote forms of L. infantum chagasi. The results showed that one of these amides in particular, presented very effective activity against the promastigotes and amastigotes of L. infantum chagasi at low concentrations and it also presented low toxicity for mammal cells, which makes this synthetic amide a promising drug for combating leishmaniasis.
Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Fenetilaminas/farmacologia , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Brasil , Linhagem Celular , Descoberta de Drogas , Leishmania/efeitos dos fármacos , Leishmania/ultraestrutura , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/fisiologia , Leishmania infantum/ultraestrutura , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Fenetilaminas/síntese química , Fenetilaminas/químicaRESUMO
The in vitro leishmanicidal activity and cytotoxicity of pyrazole-containing macrocyclic polyamines 1-4 was assayed on Leishmania infantum and Leishmania braziliensis species. Compounds 1-4 were more active and less toxic than glucantime and both infection rates and ultrastructural alterations confirmed that 1 and 2 were highly leishmanicidal and induced extensive parasite cell damage. Modifications in the excretion products of parasites treated with 1-3 were also consistent with substantial cytoplasm alterations. Compound 2 was highlighted as a potent inhibitor of Fe-SOD in both species, whereas its effect on human CuZn-SOD was poor. Molecular modelling suggested that 2 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the enzyme`s antioxidant features.
Assuntos
Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Pirazóis/farmacologia , Superóxido Dismutase/efeitos dos fármacos , Animais , Antiprotozoários/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Leishmania braziliensis/enzimologia , Leishmania braziliensis/ultraestrutura , Leishmania infantum/enzimologia , Leishmania infantum/ultraestrutura , Leishmaniose/parasitologia , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Poliaminas/química , Poliaminas/farmacologia , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Pirazóis/química , Superóxido Dismutase/metabolismoRESUMO
Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ's activity against L. infantum was confirmed, producing IC50 values of 42.71µg/mL against promastigotes and 6.78µg/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX(®) Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets.
Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/fisiologia , Estresse Oxidativo , Tiazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Concentração Inibidora 50 , Leishmania infantum/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Membranas Mitocondriais/ultraestrutura , Nitrocompostos , Testes de Sensibilidade Parasitária , Espécies Reativas de Oxigênio/análiseRESUMO
The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.