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.
Assuntos
Animais , Feminino , Camundongos , Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Macrófagos/parasitologia , Terpenos/farmacologia , Antiprotozoários/química , Leishmania braziliensis/ultraestrutura , Leishmania infantum/ultraestrutura , Espectroscopia de Ressonância Magnética , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Testes de Sensibilidade Parasitária , Terpenos/químicaRESUMO
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.
Assuntos
Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Macrófagos/parasitologia , Terpenos/farmacologia , Animais , Antiprotozoários/química , Feminino , Concentração Inibidora 50 , Leishmania braziliensis/ultraestrutura , Leishmania infantum/ultraestrutura , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Testes de Sensibilidade Parasitária , Terpenos/químicaRESUMO
We have previously demonstrated that yangambin, a lignan obtained from Ocotea duckei Vattimo (Lauraceae), shows antileishmanial activity against promastigote forms of Leishmania chagasi and Leishmania amazonensis. The aim of this study was to determine the in vitro effects of yangambin against these parasites using electron and confocal microscopy. L. chagasi and L. amazonensis promastigotes were incubated respectively with 50 µg/mL and 65 µg/mL of pure yangambin and stained with acridine orange. Treated-parasites showed significant alterations in fluorescence emission pattern and cell morphology when compared with control cells, including the appearance of abnormal round-shaped cells, loss of cell motility, nuclear pyknosis, cytoplasm acidification and increased number of acidic vesicular organelles (AVOs), suggesting important physiological changes. Ultrastructural analysis of treated-promatigotes showed characteristics of cell death by apoptosis as well as by autophagy. The presence of parasites exhibiting multiples nuclei suggests that yangambin may also affect the microtubule dynamic in both Leishmania species. Taken together our results show that yangambin is a promising agent against Leishmania.
Assuntos
Furanos/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Lignanas/farmacologia , Ocotea/química , Laranja de Acridina , Animais , Cães , Corantes Fluorescentes , Furanos/química , Processamento de Imagem Assistida por Computador , Leishmania infantum/fisiologia , Leishmania infantum/ultraestrutura , Leishmania mexicana/fisiologia , Leishmania mexicana/ultraestrutura , Lignanas/química , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
In a search for novel antileishmanial drugs, we investigated the activity of the calcium channel blocker nimodipine against Leishmania spp. and explored the ultrastructural damages of parasites induced by nimodipine after a short period of incubation. Nimodipine was highly effective against promastigotes and intracellular amastigotes of Leishmania (L.) chagasi, with 50% inhibitory concentration values of 81.2 and 21.5 muM, respectively. Nimodipine was about fourfold more effective than the standard pentavalent antimony against amastigotes and showed a Selectivity Index of 4.4 considering its mammalian cells toxicity. Leishmania (L.) amazonensis and Leishmania (L.) major promastigotes were also susceptible to nimodipine in a range concentration between 31 and 128 muM. Ultrastructural studies of L. (L.) chagasi revealed intense mitochondria damage and plasma membrane blebbing, resulting in a leishmanicidal effect as demonstrated by the lack of mitochondrial oxidative metabolism. The amastigote-killing effect suggests other mechanism than macrophage activation, as no upregulation of nitric oxide was seen. This calcium channel blocker is an effective in vitro antileishmanial compound and if adequately studied could be used as a novel drug candidate or as a novel drug lead compound for drug design studies against leishmaniasis.
Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/ultraestrutura , Nimodipina/farmacologia , Animais , Antimônio/farmacologia , Antiprotozoários/toxicidade , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Células Cultivadas , Concentração Inibidora 50 , Macaca mulatta , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Nimodipina/toxicidade , Óxido Nítrico/metabolismoRESUMO
Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, L: -glutamine, and HEPES at 37 masculineC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host-parasite relationship as well as the process of pathogenesis.
Assuntos
Células de Kupffer/parasitologia , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/patogenicidade , Macrófagos Peritoneais/parasitologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultura/química , Leishmania infantum/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The current therapy for leishmaniasis, which affects annually about 2 million people, is far from satisfactory. All available drugs require parenteral administration and are potentially toxic. Plant essential oils have been traditionally used in folk medicine and appear as valuable alternative source for chemotherapeutic compounds. In this study, we demonstrated the effect of essential oils from Cymbopogon citratus, Lippia sidoides, and Ocimum gratissimum on growth and ultrastructure of Leishmania chagasi promastigote forms. Steam distillation was used to isolate the essential oils, and their constituents were characterized by gas chromatography coupled to mass spectrometry and nuclear magnetic resonance. All essential oils showed in vitro inhibitory action on L. chagasi promastigotes growth in a dose-dependent way, with IC(50)/72 h of 45, 89, and 75 microg/mL for C. citratus, L. sidoides, and O. gratissimum, respectively. Drastic morphological alterations were observed in all essential oil-treated parasites, including cell swelling, accumulation of lipid droplets in the cytoplasm, and increase of acidocalcisome volume. Furthermore, aberrant-shaped cells with multi-septate body were observed by scanning electron microscopy, suggesting an additional effect on cytokinesis. Taken together, our data show that these essential oils affect the parasite viability being the C. citratus essential oil the most effective against L. chagasi.
Assuntos
Cymbopogon/química , Leishmania infantum/efeitos dos fármacos , Lippia/química , Ocimum/química , Óleos Voláteis/isolamento & purificação , Animais , Tamanho Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Citoplasma/ultraestrutura , Cromatografia Gasosa-Espectrometria de Massas , Concentração Inibidora 50 , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/fisiologia , Leishmania infantum/ultraestrutura , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Óleos Voláteis/químicaRESUMO
Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30 min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi.
Assuntos
Leishmania infantum/microbiologia , Serratia marcescens/fisiologia , Animais , Aderência Bacteriana/fisiologia , Biofilmes , Concanavalina A/química , Relação Dose-Resposta a Droga , Fímbrias Bacterianas/fisiologia , Flagelos/microbiologia , Flagelos/ultraestrutura , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Interações Hospedeiro-Patógeno , Cinética , Leishmania infantum/metabolismo , Leishmania infantum/ultraestrutura , Manose/metabolismo , Manose/farmacologia , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Microscopia de Interferência , Serratia marcescens/ultraestruturaRESUMO
Investigation of the bioactive crude extract from the sponge Plakortis angulospiculatus from Brazil led to the isolation of plakortenone ( 1) as a new polyketide, along with five known polyketides ( 2- 6) previously isolated from other Plakortis sponges. The known polyketides were tested in antileishmanial, antitrypanosomal, antineuroinflammatory, and cytotoxicity assays. The results show that plakortide P ( 3) is a potent antiparasitic compound, against both Leishmania chagasi and Trypanosona cruzi, and exhibited antineuroinflammatory activity. The known polyketides 2- 6 were tested for cytotoxicity against four human cancer cell lines, but displayed only moderate cytotoxic activity.
Assuntos
Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antiprotozoários/isolamento & purificação , Antiprotozoários/farmacologia , Macrolídeos/isolamento & purificação , Macrolídeos/farmacologia , Plakortis/química , Animais , Anti-Inflamatórios não Esteroides/química , Antineoplásicos/química , Antiprotozoários/química , Brasil , Cricetinae , Ensaios de Seleção de Medicamentos Antitumorais , Eritrócitos/efeitos dos fármacos , Humanos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/ultraestrutura , Macrolídeos/química , Macrófagos/efeitos dos fármacos , Biologia Marinha , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Inflamação Neurogênica/tratamento farmacológico , Óxido Nítrico/biossíntese , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.
Assuntos
Leishmania infantum/crescimento & desenvolvimento , Psychodidae/parasitologia , Animais , Linhagem Celular/parasitologia , Humanos , Leishmania infantum/ultraestrutura , Microscopia Eletrônica de Transmissão , Psychodidae/citologiaRESUMO
The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6 percent) and on day 4 in the J774 cells (51 percent). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.
Assuntos
Humanos , Animais , Leishmania infantum/crescimento & desenvolvimento , Psychodidae/citologia , Linhagem Celular/parasitologia , Leishmania infantum/ultraestrutura , Microscopia Eletrônica , Psychodidae/parasitologiaRESUMO
Tecidual reaction at the inoculation site of L. (L.)chagasi promastigotes in hamsters depleted and non-depleted of complement was studied within 2, 6, 12, 24, 48 and 72 hours of infection. The inflammatory reaction was characterized by early predominance of polymorphonuclear cells (PMN) at 2, 6 and 12 hours of infection, mixed infiltrate of PMN and mononuclear cells (MN) at 24 hours, followed by predominance of MN at 48 and 72 hours after infection. The group depleted of complement showed a higher number of PMN at 2 hours and lower numbers of MN at 72 hours after infection (P < 0.0001). In the depleted group the phagocytosis by PMN was lower at 2 and 24 hours and by MN was lower at 24, 48 and 72 hours after infection. Electron microscopy showed extracellular intact and degenerated parasites, and lysed intracellular parasites, in PMN; and, rarely, preserved intracellular parasites in MN at 2, 6 and 12 hours after infection. The groups examined at 24, 48 and 72 hours of infection showed only cellular and parasite debris in mononuclear inflammatory cells. C3b deposits were detected by immunofluorescence in the interstitium and in the cytoplasm of inflammatory cells in non-depleted group at 2, 6 and 12 hours of infection. No immunoglobulin was detected in either group. Visceralization was detected 240 days after infection. The complement system has an important role in the inflammatory reaction and phagocytosis. The ultrastructural findings showed that the escape of the parasite probably occurs soon after inoculation.