RESUMEN
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.
Asunto(s)
Leishmania braziliensis/metabolismo , Neuropéptido Y/metabolismo , Sustancia P/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Quimiotaxis , Flagelos/ultraestructura , Leishmania braziliensis/fisiología , Leishmania braziliensis/ultraestructura , Macrófagos , RatonesRESUMEN
The protozoan parasite Leishmania braziliensis is a major causative agent of the neglected tropical diseases Cutaneous and Mucocutaneous Leishmaniases in the New World. There are no vaccines to prevent the infection and the treatment relies on few drugs that often display high toxicity and costs. Thus, chemotherapeutic alternatives are required. Histone Deacetylases (HDACs) are epigenetic enzymes involved in the control of chromatin structure. In this work, we tested an in-house library of 78 hydroxamic acid derivatives as putative inhibitors of L. braziliensis HDACs (HDACi). The compounds were evaluated in relation to the toxicity to the host cell macrophage and to the leishmanicidal effect against L. braziliensis during in vitro infection. Eight HDACi showed significant leishmanicidal effects and the top 5 compounds showed effective concentrations (EC50) in the range of 4.38 to 10.21 µM and selectivity indexes (SI) from of 6 to 21.7. Analyses by Transmission Electron Microscopy (TEM) indicated induction of apoptotic cell death of L. braziliensis amastigotes with a necrotic phenotype. An altered chromatin condensation pattern and cellular disorganization of intracellular amastigotes was also observed. A tight connection between the mitochondrion and nuclear protrusions, presumably of endoplasmic reticulum origin, was found in parasites but not in the host cell. In flow cytometry (FC) analyses, HDACi promoted parasite cell cycle arrest in the G2-M phase and no changes were found in macrophages. In addition, the direct effect of HDACi against the promastigotes showed apoptosis as the main mechanism of cell death. The FC results corroborate the TEM analyses indicating that the HDACi lead to changes in the cell cycle and induction of apoptosis of L. braziliensis. The production of nitric oxide by the infected macrophages was not altered after treatment with the top 5 compounds. Taken together, our results evidenced new HDACi as promising agents for the development of new treatments for American Tegumentary Leishmaniasis caused by L. braziliensis.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/enzimología , Leishmaniasis Cutánea/enzimología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Leishmania braziliensis/ultraestructura , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/patología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/ultraestructura , Ratones , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Células RAW 264.7RESUMEN
Toxicity and poor adherence to treatment that favors the generation of resistance in the Leishmania parasites highlight the need to develop better alternatives. Here, we evaluated the in vitro effectiveness of hydrazone derived from chromanes 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (TC1) and 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide (TC2) and the mixture of triterpene saponin hederagenin-3-O-(3,4-O-diacetyl-ß-D-xylopyranosyl-(1à3)-a-L- rhamnopyranosyl-(1à2)-a-L-arabinofuranoside, hederagenin-3-O-(3,4-O-diacetyl-a-L- arabinopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside and, hederagenin-3-O-(4-O-acetyl-ß-D-xylopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside from Sapindus saponaria (SS) on L. braziliensis and L. pifanoi. Mixtures of TC1 or TC2 with saponin were formulated for topical application and the therapeutic effectiveness was evaluated in the model for cutaneous leishmaniasis (CL) in golden hamster. The mode of action of these compounds was tested on various parasite processes and ultrastructural parasite modifications. TC1, TC2 and SS showed moderate cytotoxicity when tested independently but toxicity was improved when tested in combination. The compounds were more active against intracellular Leishmania amastigotes. In vivo studies showed that combinations of TC1 or TC2 with SS in 1:1 ratio (w/w) cured 100% of hamsters with no signs associated with toxicity. The compounds did cause changes in the mitochondrial activity of the parasite with a decrease in ATP levels and depolarization of membrane potential and overproduction of reactive oxygen species; nevertheless, these effects were not related to alterations in membrane permeability. The phagolysosome ultrastructure was also affected impacting the survival of Leishmania but the function of the lysosome nor the pH inside the phagolysosome did not change. Lastly, there was a protease inhibition which was directly related to the decrease in the ability of Leishmania to infect and multiply inside the macrophage. The results suggest that the combination of TC1 and TC2 with SS in a 1:1 ratio is capable of curing CL in hamsters. This effect may be due to the ability of these compounds to affect parasite survival and the ability to infect new cells.
Asunto(s)
Hidrazonas/farmacología , Leishmania/efectos de los fármacos , Sapindus/química , Saponinas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/toxicidad , Hidrazonas/química , Hidrazonas/toxicidad , Leishmania/metabolismo , Leishmania/ultraestructura , Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestructura , Estadios del Ciclo de Vida/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Péptido Hidrolasas/efectos de los fármacos , Péptido Hidrolasas/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/toxicidad , Reinfección , Saponinas/química , Saponinas/toxicidadRESUMEN
BACKGROUND: Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES: Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS: In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION: This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.
Asunto(s)
Calpaína/genética , Genoma de Protozoos/genética , Leishmania braziliensis/química , Macrófagos Peritoneales/metabolismo , Animales , Western Blotting , Calpaína/efectos de los fármacos , Calpaína/metabolismo , Calpaína/ultraestructura , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/farmacología , Citometría de Flujo , Regulación de la Expresión Génica , Inmunohistoquímica , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestructura , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de VirulenciaRESUMEN
BACKGROUND Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.
Asunto(s)
Animales , Ratones , Leishmania braziliensis/química , Calpaína/genética , Macrófagos Peritoneales/metabolismo , Genoma de Protozoos/genética , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestructura , Inmunohistoquímica , Calpaína/efectos de los fármacos , Calpaína/metabolismo , Calpaína/ultraestructura , Inhibidores de Cisteína Proteinasa/farmacología , Regulación de la Expresión Génica , Western Blotting , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Virulencia , Microscopía Electrónica de Transmisión , Dipéptidos/farmacología , Citometría de Flujo , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE: To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS: The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS: Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS: PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.
Asunto(s)
Isoquinolinas/farmacología , Leishmania braziliensis/efectos de los fármacos , Leishmania major/efectos de los fármacos , Leishmania mexicana/efectos de los fármacos , Macrófagos/parasitología , Animales , Leishmania braziliensis/ultraestructura , Leishmania major/ultraestructura , Leishmania mexicana/ultraestructura , Dosificación Letal Mediana , Ratones , Ratones Endogámicos CBA , Microscopía Electrónica de Transmisión , Pruebas de Sensibilidad Parasitaria , Factores de TiempoRESUMEN
Calophyllum brasiliense (Clusiaceae) is a tree that grows mainly in the Atlantic Forest in Brazil. Its leaves possess antibacterial activity, cytotoxic activity against certain tumor cell lines, and antimicrobial activity in BALB/c mice infected with Leishmania (Leishmania) amazonensis.Aiming to identify ultrastructural changes and DNA fragmentation in Leishmania (Viannia) braziliensis, promastigotes were treated with a concentration of the dichloromethane extract and coumarin (-) mammea A/BB from C. brasiliense leaves that inhibited 50â% of the parasites (IC50), and were evaluated by transmission and scanning electron microscopy. Ultrastructural changes showed different levels of mitochondrial alterations, including mitochondrial swelling and a reduction in the density of the mitochondrial matrix. Induced DNA fragmentation, as observed by TUNEL, suggested that the extract and coumarin (-) mammea A/BB induced apoptosis-like cell death. These results suggest that the combination of C. brasiliense extract and coumarin (-) mammea A/BB can be considered a promising candidate for the development of new antiprotozoal agents, because of its significant leishmanicidal activity.
Asunto(s)
Antiprotozoarios/farmacología , Calophyllum/química , Cumarinas/farmacología , Leishmania braziliensis/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Extractos Vegetales/farmacología , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Antiprotozoarios/química , Antiprotozoarios/aislamiento & purificación , Brasil , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cumarinas/química , Cumarinas/aislamiento & purificación , Fragmentación del ADN/efectos de los fármacos , Leishmania braziliensis/ultraestructura , Leishmaniasis Cutánea/parasitología , Cloruro de Metileno , Ratones , Ratones Endogámicos BALB C , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Hojas de la Planta/químicaRESUMEN
In order to evaluate the in vitro leishmanicidal activity of N,N'-Squaramides derivatives, compounds that feature both hydrogen bond donor and acceptor groups and are capable of multiple interactions with complementary sites, against Leishmania infantum, Leishmania braziliensis and Leishmania donovani a series of 18compounds was prepared and assayed on extracellular and intracellular parasite forms. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. Changes in metabolite excretion by 1H-NMR and the ultrastructural alterations occurring in the parasites treated using transmission electron microscopy (TEM), was analyzed. Compounds 1, 7, 11, 14 and 17 were the more active and less toxic. Infection rates showed that the order of effectiveness was 17 > 11 > 14 > 7 for both L. infantum and L. braziliensis and in the same way, the compound 1 for L. donovani. All these compounds have altered the typical structure of the promastigotes, glycosomes and mitochondria. These severe modifications by the compounds are the ultimate reasons for the alterations observed in the excretion products. The Squaramide 17 (3-(butylamino)-4-((3-(dimetilamino)propyl)(methyl)amino)cyclobut-3-en-1,2-dione) was clearly the most efficient of all compounds. The data appear to confirm that the severe modifications generated in organelles such as glycosomes or mitochondria by the compounds are the ultimate reasons for the alterations observed in the excretion products of all species. The activity, stability, low cost of starting materials, and straightforward synthesis make amino squaramides appropriate molecules for the development of an affordable anti-leishmanial agent.
Asunto(s)
Leishmania braziliensis/efectos de los fármacos , Leishmania donovani/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Macrófagos/parasitología , Quinina/análogos & derivados , Animales , Línea Celular , Citometría de Flujo , Concentración 50 Inhibidora , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestructura , Leishmania donovani/metabolismo , Leishmania donovani/ultraestructura , Leishmania infantum/metabolismo , Leishmania infantum/ultraestructura , Macrófagos/efectos de los fármacos , Ratones , Microscopía Electrónica de Transmisión , Quinina/química , Quinina/farmacología , Quinina/toxicidadRESUMEN
Leishmaniasis is a group of diseases that presents various clinical manifestations. Many studies have shown that the parasite plays an important role in the clinical manifestations and prognosis of this disease. The cutaneous and mucosal forms of American tegumentary leishmaniasis (ATL) are associated with Leishmania (Viannia) braziliensis, which exhibits intraspecific genetic polymorphisms and various clinical manifestations. The present study focused on four different L. braziliensis strains that were isolated from patients with distinct Glucantime(®) treatment responses. The isolates were described based on their molecular, biological, and infective characteristics. Growth patterns in culture medium and different grow phases were analyzed, MID-Logarithimic (Mid-LOG), Logarithimic (LOG) and Stationary (STAT) phases. Complement resistance was evaluated using guinea pig serum. Infection to murine peritoneal macrophages, cytokine and nitric oxide were analyzed. Ultrastructural features were determined by transmission electron microscopy, and molecular characteristics were determined based on random amplified polymorphic DNA (RAPD). All of the L. braziliensis isolates showed typical growth and similar complement sensitivity patterns. Markedly lower infectivity indexes were observed for all strains in the LOG phase, with different cytokine profiles. The ultrastructure analysis revealed distinct differences between the MID-LOG, LOG, and STAT phases. The RAPD results showed a divergence between the isolates of the L. braziliensis. The in vitro characterization of L. braziliensis isolates from humans with different treatment responses using various parameters enabled us to observe differences among the isolates. Molecular and in vivo characterizations are currently under study to improve understanding of the parasite-host interaction that can imply in the clinical manifestation differences.
Asunto(s)
Antiprotozoarios/uso terapéutico , Leishmania braziliensis/fisiología , Leishmaniasis Cutánea/parasitología , Meglumina/uso terapéutico , Compuestos Organometálicos/uso terapéutico , Adulto , Anciano , Animales , Brasil , Proteínas del Sistema Complemento/farmacología , Citocinas/metabolismo , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Femenino , Cobayas , Humanos , Leishmania braziliensis/genética , Leishmania braziliensis/inmunología , Leishmania braziliensis/ultraestructura , Leishmaniasis Cutánea/tratamiento farmacológico , Macrófagos Peritoneales/parasitología , Masculino , Antimoniato de Meglumina , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
Cutaneous leishmaniasis has an estimated incidence of 1.5 million new cases per year and the treatment options available are old, expensive, toxic, and difficult to administer. Chalcones have shown good activity against several species of Leishmania. However few studies have discussed the mechanisms of action and drug target of this group of compounds in Leishmania. The synthetic chalcones that were evaluated in the present study were previously shown to exhibit activity against Leishmania (Viannia) braziliensis. The objective of the present study was to identify ultrastructural and morphological changes in L. (V.) braziliensis after treatment with three synthetic chalcones (1-3). Promastigotes were treated with chalcones 1-3 and evaluated by transmission and scanning electron microscopy. Cellular and nuclear morphology of the parasites, changes in membrane permeability, and DNA fragmentation in agarose electrophoresis gel were also investigated after exposure to synthetic chalcones. All three synthetic chalcones (1-3) induced ultrastructural alterations in mitochondria, intense vacuolization, two nuclei with rounding of parasites, and cellular and nuclear shrinkage. Chalcones 1-3 also induced no changes in membrane permeability, and presence of nucleosome-sized DNA fragments. Synthetic chalcones 1-3 induced ultrastructural and morphological changes, suggesting that chalcones 1-3 induce apoptosis-like cell death. Further studies should be conducted to elucidate other aspects of the action of these chalcones against Leishmania spp. and their use for the treatment of cutaneous leishmaniasis.
Asunto(s)
Chalconas/farmacología , Leishmania braziliensis/efectos de los fármacos , Animales , Apoptosis , Permeabilidad de la Membrana Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Chalconas/síntesis química , Cricetinae , Fragmentación del ADN/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Leishmania braziliensis/ultraestructura , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Vacuolas/efectos de los fármacosRESUMEN
As leishmanioses constituem um grupo de doenças crônicas causadas por protozoários pertencentes ao gênero Leishmania. Tendo em vista a complexidade e ineficácia dos tratamentos atuais, o desenvolvimento de novas drogas menos tóxicas ainda é uma necessidade. Na prospecção de possíveis agentes quimioterápicos contra as leishmanioses, as lectinas apresentam-se como candidatos promissores por apresentarem um amplo espectro de atividades biológicas. No presente trabalho nós investigamos o potencial leishmanicida e imunomodulador da lectina Onil. Nossos resultados demonstraram que a Onil apresentou baixa toxidade sobre células do exsudato peritoneal (CEP) de camundongos (CC50= 317,5 ± 0,6 µg/mL), foi efetiva ao inibir o crescimento de formas promastigotas de L. braziliensis (IC50=150,58± 0,78 µg/mL), mostrou-se mais seletiva para o parasito do que para célula do hospedeiro (ISe=2,1). No entanto, não foi capaz de inibir a sobrevivência das amastigotas no interior das CEPs. A lectina Onil causou alterações ultraestruturais no flagelo, bem como mostrou um efeito sobre a divisão celular de formas promastigotas. A marcação das células tratadas com Anexina V (AV) e Iodeto de Propídio (IP) mostrou uma pequena subpopulação de células apresentava marcação para AV/IP compatíveis com o processo de morte celular por necrose/apoptose tardia. A marcação das células controles e tratadas com Onil com Rho 123 revelou que na grande maioria das células o potencial de membrana mitocondrial foi preservado. O tratamento com a lectina (75-300 µg/mL) não alterou significativamente a produção de NO e não induziu alterações na produção de citocinas em CEPs infectadas L. braziliensis. Por outro lado, uma intensa produção de citocinas associadas aos perfis Th1, Th2 e Th17 foi observada em CEPs não infectadas tratadas com 30 µg/mL da Onil. Nossos dados apontam para uma possível utilização da Onil como agente adjuvante ou como carreadora de drogas para o tratamento da leishmaniose cutânea
Leishmaniasis comprises a group of disease caused by protozoa belonging to the Leishmaniagenus. Taking in account the complexity and inefficiency of current treatments against leishmaniasis, the development of new, less toxic drugs is still needed. In a search of potential chemotherapeutic agents against leishmaniasis, lectins presented as promising candidates because for presenting a broad spectrum of biological activities. In this regard, in the present study we investigated the leishmanicidal and immunomodulatory activities of Onil in vitro. Our results demonstrated that Onil presented low toxicity to mice peritoneal exudate cells (PEC) (CC50= 317.5 ± 0.6 μg / mL), was effective to inhibit the growth of promastigotes of Leishmania braziliensis (IC50= 150.58 ± 0.78 μg / mL) and was shown to be more selective for the parasite than to the host cell, with SeI=2.1...
Asunto(s)
Ratones , Antiprotozoarios/farmacología , Leishmaniasis Cutánea , Lectinas/toxicidad , Lectinas/uso terapéutico , Leishmania braziliensis , Leishmania braziliensis/ultraestructura , Antiprotozoarios/toxicidad , Antiprotozoarios/uso terapéutico , Cíclidos/sangre , Exudados y TransudadosRESUMEN
The effects of the Securinega alkaloid (+)-phyllanthidine on Leishmania (L.) amazonensis and the first chemical investigation of Margaritaria nobilis L.f. (Phyllanthaceae) are described. Treating the parasites with this alkaloid caused a dose-dependent reduction in promastigote growth of 67.68% (IC50 82.37 µg/mL or 353 µM) and in amastigote growth of 83.96% (IC50 49.11 µg/mL or 210 µM), together with ultrastructural alterations in the promastigotes. No cytotoxic effect was detected in mammalian cells (CC50 1727.48 µg/mL or CC50 5268 µM). Classical chromatographic techniques and spectral methods led to the isolation and identification of betulinic acid, kaempferol, corilagin, gallic acid and its methyl ester, besides (+)-phyllanthidine from M. nobilis leaves and stems. Margaritaria nobilis is another source of the small group of Securinega alkaloids, together with other Phyllanthaceae (Euphorbiaceae s.l.) species. The low toxicity to macrophages and the effects against promastigotes and amastigotes are suggestive that (+)-phyllanthidine could be a promising antileishmanial agent for treating cutaneous leishmaniasis.
Asunto(s)
Antiprotozoarios/farmacología , Euphorbiaceae/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Leishmania braziliensis/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos , Fitoquímicos/farmacología , Alcaloides/aislamiento & purificación , Animales , Antiprotozoarios/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Ácido Gálico/aislamiento & purificación , Glucósidos/aislamiento & purificación , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Taninos Hidrolizables/aislamiento & purificación , Concentración 50 Inhibidora , Quempferoles/aislamiento & purificación , Leishmania braziliensis/crecimiento & desarrollo , Leishmania braziliensis/ultraestructura , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Triterpenos Pentacíclicos , Fitoquímicos/aislamiento & purificación , Extractos Vegetales/química , Cultivo Primario de Células , Triterpenos/aislamiento & purificación , Ácido BetulínicoRESUMEN
In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.
Asunto(s)
Aminoácidos/farmacología , Quimiotaxis/efectos de los fármacos , Leishmania/fisiología , Aminoácidos/química , Aminoácidos Dicarboxílicos/farmacología , Aminoácidos Sulfúricos/farmacología , Flagelos/efectos de los fármacos , Flagelos/fisiología , Flagelos/ultraestructura , Compuestos Heterocíclicos/farmacología , Leishmania/efectos de los fármacos , Leishmania/ultraestructura , Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/fisiología , Leishmania braziliensis/ultraestructura , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/fisiología , Leishmania mexicana/ultraestructura , Concentración OsmolarRESUMEN
The antiprotozoal activity of some indazole-derived amines (2, 3, 5-8) as well as that of some simple structurally related 3-alkoxy-1-alkyl-5-nitroindazoles (1, 4) against promastigote and amastigote forms of Leishmania infantum and Leishmania braziliensis is reported. In some cases, these compounds showed in vitro activities against the different morphological forms of Leishmania similar to or higher than those of the reference drug glucantime; this fact, along with low unspecific cytotoxicities against macrophages shown by some of them, led to good selectivity indexes (SI). The high efficiency of some 5-nitroindazoles against the mentioned protozoa was confirmed by further in vitro studies on infection rates. Complementary analyses by (1)H NMR of the changes on the metabolites excreted by parasites after treatment with the more active indazole derivatives in many cases showed the decreased excretion of succinate and increased levels of acetate, lactate and alanine, as well as, in some cases, the appearance of glycine and pyruvate as new metabolites. Damage caused by indazoles at the glycosomal or mitochondrial level are consistent with these metabolic changes as well as with the huge ultrastructural alterations observed by transmission electron microscopy (TEM), especially affecting the mitochondria and other cytoplasmic organelles.
Asunto(s)
Antiprotozoarios/farmacología , Indazoles/farmacología , Leishmania braziliensis/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Acetatos/metabolismo , Alanina/metabolismo , Animales , Antiprotozoarios/química , Glicina/metabolismo , Técnicas In Vitro , Indazoles/química , Ácido Láctico/metabolismo , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestructura , Leishmania infantum/metabolismo , Leishmania infantum/ultraestructura , Leishmaniasis Visceral , Macrófagos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Ratones , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Orgánulos/efectos de los fármacos , Orgánulos/ultraestructura , Ácido Pirúvico/metabolismo , Ácido Succínico/metabolismoRESUMEN
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.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania braziliensis/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Pirazoles/farmacología , Superóxido Dismutasa/efectos de los fármacos , Animales , Antiprotozoarios/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Femenino , Humanos , Leishmania braziliensis/enzimología , Leishmania braziliensis/ultraestructura , Leishmania infantum/enzimología , Leishmania infantum/ultraestructura , Leishmaniasis/parasitología , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Macrófagos/efectos de los fármacos , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Modelos Moleculares , Poliaminas/química , Poliaminas/farmacología , Proteínas Protozoarias/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Pirazoles/química , Superóxido Dismutasa/metabolismoRESUMEN
BACKGROUND: Iron is an essential element for the survival of microorganisms in vitro and in vivo, acting as a cofactor of several enzymes and playing a critical role in host-parasite relationships. Leishmania (Viannia) braziliensis is a parasite that is widespread in the new world and considered the major etiological agent of American tegumentary leishmaniasis. Although iron depletion leads to promastigote and amastigote growth inhibition, little is known about the role of iron in the biology of Leishmania. Furthermore, there are no reports regarding the importance of iron for L. (V.) braziliensis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the effect of iron on the growth, ultrastructure and protein expression of L. (V.) braziliensis was analyzed by the use of the chelator 2,2-dipyridyl. Treatment with 2,2-dipyridyl affected parasites' growth in a dose- and time-dependent manner. Multiplication of the parasites was recovered after reinoculation in fresh culture medium. Ultrastructural analysis of treated promastigotes revealed marked mitochondrial swelling with loss of cristae and matrix and the presence of concentric membranar structures inside the organelle. Iron depletion also induced Golgi disruption and intense cytoplasmic vacuolization. Fluorescence-activated cell sorting analysis of tetramethylrhodamine ester-stained parasites showed that 2,2-dipyridyl collapsed the mitochondrial membrane potential. The incubation of parasites with propidium iodide demonstrated that disruption of mitochondrial membrane potential was not associated with plasma membrane permeabilization. TUNEL assays indicated no DNA fragmentation in chelator-treated promastigotes. In addition, two-dimensional electrophoresis showed that treatment with the iron chelator induced up- or down-regulation of proteins involved in metabolism of nucleic acids and coordination of post-translational modifications, without altering their mRNA levels. CONCLUSIONS: Iron chelation leads to a multifactorial response that results in cellular collapse, starting with the interruption of cell proliferation and culminating in marked mitochondrial impairment in some parasites and their subsequent cell death, whereas others may survive and resume proliferating.
Asunto(s)
2,2'-Dipiridil/farmacología , Quelantes/farmacología , Hierro/metabolismo , Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/crecimiento & desarrollo , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Muerte Celular , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/ultraestructura , Fragmentación del ADN , Expresión Génica/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Humanos , Etiquetado Corte-Fin in Situ , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestructura , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/fisiología , Proteínas Protozoarias/biosíntesisRESUMEN
We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log-phase promastigotes treated with 1 µM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 µM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 µM myriocin-treated parasites, respectively. High percentage of myriocin-treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 µM myriocin, respectively). Transmission electron microscopy following treatment with 1 µM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3-ketodihidrosphingosine to myriocin-treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid-free mutant, where addition of ethanolamine rescue wild-type parasite characteristics.
Asunto(s)
Citocinesis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ácidos Grasos Monoinsaturados/farmacología , Leishmania braziliensis/citología , Leishmania braziliensis/efectos de los fármacos , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Técnica del Anticuerpo Fluorescente Indirecta , Glicoesfingolípidos/metabolismo , Leishmania braziliensis/enzimología , Leishmania braziliensis/ultraestructura , Microscopía Electrónica de Transmisión , Esfingolípidos/metabolismoRESUMEN
OBJECTIVES: To evaluate the in vitro leishmanicidal activity of nine flavonoid derivatives from Delphinium staphisagria against L. infantum and L. braziliensis. DESIGN AND METHODS: The in vitro activity of compounds 1-9 was assayed on extracellular promastigote and axenic amastigote forms and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were carried on J774.2 macrophage cells using Glucantime as the reference drug. The mechanisms of action were analysed performing metabolite excretion and transmission electronic microscope ultrastructural alteration studies. RESULTS: Nine flavonoids showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania infantum and L. braziliensis. These compounds were nontoxic to mammalian cells and were effective at similar concentrations up to or lower than that of the reference drug (Glucantime). The results showed that 2(â³)-acetylpetiolaroside (compound 8) was clearly the most active. CONCLUSION: This study has demonstrated that flavonoid derivatives are active against L. infantum and L. braziliensis.
Asunto(s)
Antiprotozoarios/farmacología , Delphinium/química , Flavonoides/farmacología , Leishmania braziliensis/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Animales , Línea Celular , Flavonoides/aislamiento & purificación , Leishmania braziliensis/ultraestructura , Leishmania infantum/ultraestructura , Ratones , Microscopía Electrónica de TransmisiónRESUMEN
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.
Asunto(s)
Animales , Femenino , Ratones , Antiprotozoarios/farmacología , Leishmania braziliensis/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Macrófagos/parasitología , Terpenos/farmacología , Antiprotozoarios/química , Leishmania braziliensis/ultraestructura , Leishmania infantum/ultraestructura , Espectroscopía de Resonancia Magnética , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Pruebas de Sensibilidad Parasitaria , Terpenos/químicaRESUMEN
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.