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1.
Biomed Pharmacother ; 133: 111049, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378956

RESUMO

Despite all efforts to provide new chemical entities to tackle leishmaniases, we are still dependent on a the limited drug arsenal, together with drawbacks like toxicity and drug-resistant parasites. Collaborative drug discovery emerged as an option to speed up the way to find alternative antileishmanial agents. This is the case of Medicines for Malaria Ventures - MMV, that promotes an open source drug discovery initiative to fight diseases worldwide. Here, we screened 400 compounds from 'Pathogen Box' (PBox) collection against Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis in Brazil. Twenty-three compounds were able to inhibit ≥ 80 % L. braziliensis growth at 5 µM. Six out of the PBox selected 23 compounds were found to be highly selective against L. braziliensis intracellular amastigotes with selectivity index varying from > 104 to > 746 and IC50s ranging from 47 to 480 nM. The compounds were also active against antimony-resistant L. braziliensis isolated from the field or laboratory selected mutants, revealing the potential on treating patients infected with drug resistant parasites. Most of the selected compounds were known to be active against kinetoplastids, however, two compounds (MMV688703 and MMV676477) were part of toxoplasmosis and tuberculosis 'PBox' disease set, reinforcing the potential of phenotyping screening to unveil drug repurposing. Here we applied a computational prediction of pharmacokinetic properties using the ADMET predictor pkCSM (http://biosig.unimelb.edu.au/pkcsm/). The tool offered clues on potential drug development needs and can support further in vivo studies. Molecular docking analysis identified CRK3 (LbrM.35.0660), CYP450 (LbrM.30.3580) and PKA (LbrM.18.1180) as L. braziliensis targets for MMV676604, MMV688372 and MMV688703, respectively. Compounds from 'Pathogen Box' thus represents a new hope for novel (or repurposed) small molecules source to tackle leishmaniases.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Descoberta de Drogas , Reposicionamento de Medicamentos , Resistência a Medicamentos , Leishmania braziliensis/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Animais , Antiprotozoários/química , Antiprotozoários/farmacocinética , Antiprotozoários/toxicidade , Simulação por Computador , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Leishmania braziliensis/crescimento & desenvolvimento , Modelos Biológicos , Modelos Químicos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Células THP-1
2.
PLoS One ; 15(5): e0232829, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379842

RESUMO

The diagnosis of American tegumentary leishmaniasis (ATL) still requires the design of more effective tools. Leishmania (Viannia) braziliensis is the causal agent of the 90% of Argentinean ATL cases. Considering the current knowledge, an ELISA based crude antigen (CA) for the diagnosis was designed. Ninety-nine subjects diagnosed as ATL, 27 as no-ATL, and 84 donors from non-ATL-endemic areas were included in this study. The current ATL diagnosis was based four techniques, dermal smear microscopic examination (parasitological test), PCR, Leishmanin skin test, and clinical records. We obtained CA extracts from promastigotes and amastigotes from macrophage cultures of different zymodemes of endemic Leishmania species circulating in the study area. Crude antigens from the 'local' main zymodeme of L. (V.) braziliensis showed the highest reactivity against anti-Leishmania antibodies compared to the other included species. The CA of amastigotes of this zymodeme was 3.4 fold more reactive than promastigotes one. Moreover, amastigote-membrane CA (MCA) were 3.6 fold more reactive than the soluble antigens. The MCA-ELISA reached a sensitivity and specificity of 98% (CI = 94.7%-100%) and 63.6% (53.9-73.1), respectively. When anti-Trypanosoma cruzi reactive sera were excluded, the specificity reached 98.4% (94.4-100), while the sensitivity was similar, with a positive predictive value (PV) of 98.6% (94.6-100) and negative PV of 96.3% (91.6-100). The performance of the MCA-ELISA results strongly contribute to the final diagnostic decision, since a non-reactive serological result almost discards the suspected ATL, because of its high negative PV. The developed MCA-ELISA showed a high diagnostic performance, which makes it a good candidate for ATL diagnosis, for seroprevalence studies, or for monitoring treatments efficacy.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Membrana Celular/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Afinidade de Anticorpos , Especificidade de Anticorpos , Argentina/epidemiologia , Doadores de Sangue , Doenças Endêmicas , Humanos , Leishmania braziliensis/crescimento & desenvolvimento , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Mucocutânea/sangue , Leishmaniose Mucocutânea/diagnóstico , Leishmaniose Mucocutânea/parasitologia , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Trypanosoma cruzi/imunologia
3.
Parasit Vectors ; 13(1): 9, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31915065

RESUMO

BACKGROUND: Prostaglandins (PG) are lipid mediators derived from arachidonic acid metabolism. They are involved in cellular processes such as inflammation and tissue homeostasis. PG production is not restricted to multicellular organisms. Trypanosomatids also synthesize several metabolites of arachidonic acid. Nevertheless, their biological role in these early-branching parasites and their role in host-parasite interaction are not well elucidated. Prostaglandin F2α synthase (PGF2S) has been observed in the Leishmania braziliensis secreted proteome and in L. donovani extracellular vesicles. Furthermore, we previously reported a positive correlation between L. braziliensis PGF2S (LbrPGF2S) expression and pathogenicity in mice. METHODS: LbrPGF2S gene expression and PGF2α synthesis in promastigotes were detected and quantified by western blotting and EIA assay kit, respectively. To investigate LbrPGF2S localization in amastigotes during bone marrow-derived macrophage infection, parasites expressing mCherry-LbrPGF2S were generated and followed by time-lapse imaging for 48 h post-infection. PGF2S homolog sequences from Leishmania and humans were analyzed in silico using ClustalW on Geneious v6 and EMBOSS Needle. RESULTS: Leishmania braziliensis promastigotes synthesize prostaglandin F2α in the presence of arachidonic acid, with peak production in the stationary growth phase under heat stress. LbrPGF2S is a cytoplasmic protein enriched in the secretory site of the parasite cell body, the flagellar pocket. It is an enzyme constitutively expressed throughout promastigote development, but overexpression of LbrPGF2S leads to an increase of infectivity in vitro. The data suggest that LbrPGF2S may be released from intracellular amastigotes into the cytoplasm of bone marrow-derived macrophages over a 48-hour infection period, using time-lapse microscopy and mCherry-PGF2S (mChPGF2S)-expressing parasites. CONCLUSIONS: LbrPGF2S, a parasite-derived protein, is targeted to the host cell cytoplasm. The putative transfer of this enzyme, involved in pro-inflammatory lipid mediator synthesis, to the host cell suggests a potential role in host-parasite interaction and may partially explain the increased pathogenicity associated with overexpression of LbrPGF2S in L. braziliensis. Our data provide valuable insights to help understand the importance of parasite-derived lipid mediators in pathogenesis.


Assuntos
Leishmania braziliensis/enzimologia , Leishmaniose Cutânea/parasitologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Interações Hospedeiro-Parasita , Humanos , Leishmania braziliensis/genética , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandinas/biossíntese , Proteínas de Protozoários/genética
4.
Sci Rep ; 9(1): 18951, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831818

RESUMO

Under stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently discovered in Leishmania. We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.


Assuntos
DNA de Protozoário , DNA Ribossômico , Loci Gênicos , Proteínas de Fluorescência Verde , Leishmania braziliensis , Leishmania mexicana , Microrganismos Geneticamente Modificados , Animais , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Leishmania braziliensis/citologia , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Leishmania mexicana/citologia , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Camundongos
5.
Artigo em Inglês | MEDLINE | ID: mdl-31555609

RESUMO

Leishmania (Viannia) braziliensis is responsible for the largest number of American tegumentary leishmaniasis (ATL) in Brazil. ATL can present several clinical forms including typical (TL) and atypical (AL) cutaneous and mucocutaneous (ML) lesions. To identify parasite and host factors potentially associated with these diverse clinical manifestations, we first surveyed the expression of two virulence-associated glycoconjugates, lipophosphoglycan (LPG) and the metalloprotease GP63 by a panel of promastigotes of Leishmania braziliensis (L. braziliensis) strains isolated from patients with different clinical manifestations of ATL and from the sand fly vector. We observed a diversity of expression patterns for both LPG and GP63, which may be related to strain-specific polymorphisms. Interestingly, we noted that GP63 activity varies from strain to strain, including the ability to cleave host cell molecules. We next evaluated the ability of promastigotes from these L. braziliensis strains to modulate phagolysosome biogenesis in bone marrow-derived macrophages (BMM), by assessing phagosomal recruitment of the lysosome-associated membrane protein 1 (LAMP-1) and intraphagosomal acidification. Whereas, three out of six L. braziliensis strains impaired the phagosomal recruitment of LAMP-1, only the ML strain inhibited phagosome acidification to the same extent as the L. donovani strain that was used as a positive control. While decreased phagosomal recruitment of LAMP-1 correlated with higher LPG levels, decreased phagosomal acidification correlated with higher GP63 levels. Finally, we observed that the ability to infect and replicate within host cells did not fully correlate with the inhibition of phagosome maturation. Collectively, our results revealed a diversity of strain-specific phenotypes among L. braziliensis isolates, consistent with the high genetic diversity within Leishmania populations.


Assuntos
Glicoesfingolipídeos/metabolismo , Interações Hospedeiro-Patógeno , Leishmania braziliensis/imunologia , Leishmaniose Mucocutânea/imunologia , Leishmaniose Mucocutânea/parasitologia , Metaloendopeptidases/metabolismo , Fagossomos/metabolismo , Animais , Células Cultivadas , Evasão da Resposta Imune , Leishmania braziliensis/crescimento & desenvolvimento , Proteína 1 de Membrana Associada ao Lisossomo/antagonistas & inibidores , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos Endogâmicos C57BL , Biogênese de Organelas
6.
Colloids Surf B Biointerfaces ; 183: 110421, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31401463

RESUMO

Using the electron paramagnetic resonance (EPR) of spin-labeled stearic acid and a spin label chemically attached to the membrane proteins, the interaction of miltefosine (MIL) and the ionic surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) with the plasma membrane of Leishmania (L.) amazonensis promastigotes was studied. The spin-label EPR data indicated that the four compounds studied have the ability to increase the molecular dynamics of membrane proteins to a large extent. Compared to the other compounds, SDS produced the smallest increases in dynamics and demonstrated the lowest antileishmanial activity and cytotoxicity to J774.A1 macrophages. The activities of the other three compounds were not different from each other, but CTAC had a stronger activity against L. amazonensis promastigotes at higher cellular concentrations (> 1 × 109 cells/mL) and was the most effective against L. amazonensis-infected macrophages. However, CTAC was also the most cytotoxic to macrophages. By measuring the IC50/CC50 values for assays of different cell concentrations, we estimated the membrane-water partition coefficient (KM/W) as well as the concentrations in the membrane (cm50) and aqueous phase (cw50) of the compounds at their IC50/CC50. Compared to the other compounds, SDS showed the lowest value of KM/W and the highest value of cm50. In all experiments in this study, the data for the zwitterionic molecules HPS and MIL were not significantly different.


Assuntos
Antiprotozoários/farmacologia , Cetrimônio/farmacologia , Citotoxinas/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Antiprotozoários/química , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cetrimônio/química , Citotoxinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Concentração Inibidora 50 , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Simulação de Dinâmica Molecular , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Compostos de Amônio Quaternário/química , Dodecilsulfato de Sódio/química , Marcadores de Spin , Ácidos Esteáricos/química , Tensoativos/química
7.
Arch Pharm (Weinheim) ; 352(6): e1800299, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31012160

RESUMO

To identify new agents for the treatment of American cutaneous leishmaniasis, a series of eight 1,4-bis(substituted benzalhydrazino)phthalazines was evaluated against Leishmania braziliensis and Leishmania mexicana parasites. These compounds represent a disubstituted version of the 1-chloro-4-(monoaryl/heteroarylhydranizyl)phthalazine that exhibited a significant response against L. braziliensis according to our previous findings. Two disubstituted phthalazines 3b and 3f were identified as potential antileishmanial agents against L. braziliensis parasites, exhibiting a submicromolar IC50 response of 2.37 and 7.90 µM on the promastigote form, and of 1.82 and 4.56 µM against intracellular amastigotes, respectively. In particular, compound 3b showed interesting responses against amastigote isolates from reference, glucantime-resistant and clinical human strains, which were by far superior to the biological response found for the glucantime drug. With regard to the toxicity results, both 3b and 3f exhibited moderate LD50 values against murine macrophages (BMDM), with good selectivity indexes on promastigotes and intracellular amastigotes of L. braziliensis. A comparison of biological response was established between the monosubstituted and disubstituted versions of these benzalhydrazino-phthalazines. Easy synthetic procedure and significant response against amastigote strains including against resistant lines made compound 3b a potential candidate for further pharmacokinetic and in vivo experiments as antileishmanial agent, and as a platform for further structural optimization. Mechanism-of-action studies and molecular docking simulations discarded to inhibition of superoxide dismutase as possible mode of action.


Assuntos
Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Ftalazinas/farmacologia , Animais , Antiprotozoários/química , Antiprotozoários/toxicidade , Células Cultivadas , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/metabolismo , Dose Letal Mediana , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ftalazinas/química , Ftalazinas/toxicidade , Relação Estrutura-Atividade , Superóxido Dismutase/metabolismo
8.
RNA Biol ; 16(5): 639-660, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30689499

RESUMO

Leishmaniasis is a worldwide public health problem caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the most important species responsible for tegumentary leishmaniases in Brazil. An understanding of the molecular mechanisms underlying the success of this parasite is urgently needed. An in-depth study on the modulation of gene expression across the life cycle stages of L. braziliensis covering coding and noncoding RNAs (ncRNAs) was missing and is presented herein. Analyses of differentially expressed (DE) genes revealed that most prominent differences were observed between the transcriptomes of insect and mammalian proliferative forms (6,576 genes). Gene ontology (GO) analysis indicated stage-specific enriched biological processes. A computational pipeline and 5 ncRNA predictors allowed the identification of 11,372 putative ncRNAs. Most of the DE ncRNAs were found between the transcriptomes of insect and mammalian proliferative stages (38%). Of the DE ncRNAs, 295 were DE in all three stages and displayed a wide range of lengths, chromosomal distributions and locations; many of them had a distinct expression profile compared to that of their protein-coding neighbors. Thirty-five putative ncRNAs were submitted to northern blotting analysis, and one or more hybridization-positive signals were observed in 22 of these ncRNAs. This work presents an overview of the L. braziliensis transcriptome and its adjustments throughout development. In addition to determining the general features of the transcriptome at each life stage and the profile of protein-coding transcripts, we identified and characterized a variety of noncoding transcripts. The novel putative ncRNAs uncovered in L. braziliensis might be regulatory elements to be further investigated.


Assuntos
Perfilação da Expressão Gênica/métodos , Leishmania braziliensis/crescimento & desenvolvimento , RNA de Protozoário/genética , Análise de Sequência de RNA/métodos , Animais , Brasil , Biologia Computacional/métodos , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Humanos , Insetos/parasitologia , Leishmania braziliensis/genética , Mamíferos/parasitologia , RNA não Traduzido/genética
9.
Am J Trop Med Hyg ; 100(2): 306-310, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30628567

RESUMO

Local therapies have been proposed as safe and effective alternatives to systemic drugs in cutaneous leishmaniasis (CL), especially among less severe cases. However, they are not widely available and used in endemic places, including Colombia, which has a high burden of disease. Further complicating the uptake of local therapies is that different treatment guidelines have been established by the World Health Organization (WHO) and Pan American Health Organization (PAHO). Using data from a large referral center in Colombia, we determined the proportion of patients who would be eligible for and potentially benefit from local therapies according to both international guidelines. The sample included 1,891 confirmed cases of CL aged ≥ 12 years, mostly infected with Leishmania Viannia panamensis (91%, n = 601/660), between 2004 and 2014. Overall, 57% of the sample had one lesion, whereas another 31% had two to three lesions. For 74% of patients, all lesions were in an area other than head or neck. The maximum lesion size was ≤ 3 cm for 58% and < 5 cm for 88% of the sample. Based on our data, up to 56% of patients could have been eligible for local therapies according to the WHO criteria. By contrast, only 23% were eligible according to the more restrictive PAHO criteria. Regardless, these data suggest that a substantial proportion of CL patients in Colombia may benefit from local therapies given their relatively mild presentation of disease and low risk of complications. Individualized risk-benefit assessment and guideline adjustments may increase local therapy eligibility and benefit a large number of patients.


Assuntos
Antimônio/uso terapêutico , Antiprotozoários/uso terapêutico , Leishmania braziliensis/efeitos dos fármacos , Leishmania guyanensis/efeitos dos fármacos , Leishmaniose Cutânea/terapia , Paromomicina/uso terapêutico , Pentamidina/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Colômbia/epidemiologia , Estudos Transversais , Crioterapia/métodos , Feminino , Humanos , Hipertermia Induzida/métodos , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/patogenicidade , Leishmania guyanensis/crescimento & desenvolvimento , Leishmania guyanensis/patogenicidade , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Guias de Prática Clínica como Assunto , Índice de Gravidade de Doença
10.
Exp Parasitol ; 198: 31-38, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30690024

RESUMO

Diverse spiro dihydroquinoline-oxindoles (JS series) were prepared using the BF3•OEt2-catalyzed imino Diels-Alder reaction between ketimine-isatin derivatives and trans-isoeugenol. Ten spiro-oxiindole derivatives were selected and evaluated at different stages of the life cycle of Leishmania braziliensis parasites, responsible for cutaneous leishmaniasis in South America. Among them, the 8'-ethyl-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one called JS87 was able to inhibit the growth of promastigotes without affecting the mammalian cells viability, and to decrease the number of intracellular amastigotes of L. braziliensis. This spiro compound was found to act through the alteration of parasite internal regulation by disrupting the regulatory volume decrease (RVD), and to affect the sterol biosynthetic pathway at level of squalene epoxidase (SE) enzyme. These results revealed that the spiro annulation between quinoline and oxindole scaffolds enhances the anti-leishmanial activity, and could assist in the development of potent quinoline-oxindole hybrids against Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis in South America.


Assuntos
Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Oxindois/farmacologia , Quinolinas/farmacologia , Compostos de Espiro/farmacologia , Animais , Antiprotozoários/química , Concentração Inibidora 50 , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Oxindois/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacologia , Quinolinas/química , Compostos de Espiro/química
11.
Free Radic Biol Med ; 129: 35-45, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30196081

RESUMO

Human leishmaniasis caused by Leishmania (Viannia) braziliensis can be presented as localized cutaneous leishmaniasis (LCL) or mucosal leishmaniasis (ML). Macrophages kill parasites using nitric oxide (NO) and reactive oxygen species (ROS). The aim of this study was to evaluate the ability of parasites obtained from patients with LCL or ML to produce and resist NO or ROS. Promastigotes and amastigotes from LCL or ML isolates produced similar amounts of NO in culture. Promastigotes from ML isolates were more resistant to NO and H2O2 than LCL parasites in a stationary phase, whereas amastigotes from LCL isolates were more resistant to NO. In addition, in the stationary phase, promastigote isolates from patients with ML expressed more thiol-specific antioxidant protein (TSA) than LCL isolates. Therefore it is suggested that infective promastigotes from ML isolates are more resistant to microbicidal mechanisms in the initial phase of infection. Subsequently, amastigotes lose this resistance. This behavior of ML parasites can decrease the number of parasites capable of stimulating the host immune response shortly after the infection establishment.


Assuntos
Antiprotozoários/farmacologia , Peróxido de Hidrogênio/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Estágios do Ciclo de Vida/efeitos dos fármacos , Óxido Nítrico/farmacologia , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Meios de Cultura/química , Feminino , Interações Hospedeiro-Parasita , Humanos , Imunidade Inata , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/isolamento & purificação , Leishmania braziliensis/metabolismo , Leishmaniose Tegumentar Difusa/imunologia , Leishmaniose Tegumentar Difusa/metabolismo , Leishmaniose Tegumentar Difusa/parasitologia , Leishmaniose Mucocutânea/imunologia , Leishmaniose Mucocutânea/metabolismo , Leishmaniose Mucocutânea/parasitologia , Estágios do Ciclo de Vida/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
12.
Photochem Photobiol Sci ; 17(4): 482-490, 2018 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-29582862

RESUMO

Leishmaniasis is a neglected disease present in more than 88 countries. The currently adopted chemotherapy faces challenges related to side effects and the development of resistance. Photodynamic therapy (PDT) is emerging as a therapeutic modality for cutaneous leishmaniasis. Zn(ii) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (ZnTE-2-PyP4+, ZnP) is a cationic, water-soluble, zinc porphyrin-based photosensitizer whose photodynamic effect on Leishmania braziliensis was analyzed by evaluating the number of visibly undamaged and motile cells, cell membrane integrity, mitochondrial membrane potential, and ultrastructural damage. Treatment of parasites with ZnP and light induced damage in up to 90% of L. braziliensis promastigote cells. Propidium iodide labeling suggested the loss of plasma membrane integrity. In samples treated with ZnP and light, a hyperpolarization of the mitochondrial membrane potential was also observed. Ultrastructural evaluation of promastigotes after photodynamic treatment indicated a loss of cytoplasmic material and the presence of vacuoles. Scanning electron microscopy showed wrinkling of the plasma membrane and a reduced cell volume. Additionally, the number of amastigotes per macrophage was reduced by about 40% after photodynamic application. The treatment showed no considerable toxicity against mammalian cells. Therefore, the results indicated that PDT associated with ZnTE-2-PyP4+ represents a promising alternative to cutaneous leishmaniasis treatment.


Assuntos
Antiparasitários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Metaloporfirinas/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Antiparasitários/química , Humanos , Leishmaniose Cutânea/tratamento farmacológico , Metaloporfirinas/química , Microscopia Eletrônica de Varredura , Testes de Sensibilidade Parasitária , Fármacos Fotossensibilizantes/química
13.
Protist ; 169(1): 107-121, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29482071

RESUMO

The present study demonstrates that the Leishmania (Viannia) braziliensis strain MCAN/BR/1998/R619 is composed of multiple subpopulations with measurable distinctions. Single parasites were separated from a culture of promastigotes in stationary phase by cell sorting and then cultivated as subpopulations. Subsequently, these subpopulations were evaluated for features of in vitro growth, infectivity to murine macrophages and proteinase gene expression. The first evidence of distinct characteristics was observed during the in vitro cultivation of isolated subpopulations, as distinct clusters of patterns were formed among the cultures, indicating the existence of quantifiable fluctuations in metrics. Further, when infecting murine macrophages, the subpopulations induced distinct patterns of production of immune response mediators. While some subpopulations mainly induced the production of IL-1ß, IL-6 and TNF-α, others induced the production of IL-12p70 and nitric oxide. Finally, amastigotes of these subpopulations had higher expression of proteinase genes than promastigotes. Additionally, cysteine proteinase, serine proteinase, metalloproteinase and aspartic proteinases were differentially expressed in promastigote and amastigote forms. These data suggest the existence of distinct profiles for the L. (V.) braziliensis MCAN/BR/1998/R619 strain and subpopulations that could drive the success of parasite adaptation to the environments that they inhabit.


Assuntos
Leishmania braziliensis/crescimento & desenvolvimento , Animais , Humanos , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Leishmania braziliensis/classificação , Leishmania braziliensis/genética , Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fator de Necrose Tumoral alfa/imunologia
14.
J Ethnopharmacol ; 216: 120-133, 2018 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-29391199

RESUMO

ETNOPHARMACOLOGICAL RELEVANCE: Thirty-eight Tacana medicinal plant species used to treat skin problems, including leishmania ulcers, skin infections, inflammation and wound healing, were collected in the community of Buena Vista, Bolivia, with the Tacana people. Twenty two species are documented for the first time as medicinal plants for this ethnic group living in the northern area of the Department of La Paz. AIM OF THE STUDY: To evaluate the leishmanicidal effect (IC50) and cytotoxicity (LD50) of the selected plants. To carry out bioguided studies on the active extracts. To assess the potential of Bolivian plant biodiversity associated with traditional knowledge in the discovery of alternative sources to fight leishmaniasis. MATERIALS AND METHODS: Seventy three ethanol extracts were prepared from 38 species by maceration and were evaluated in vitro against promastigotes of Leishmania amazonensis and L. braziliensis. Active extracts (IC50 ≤ 50 µg/mL) were fractionated by chromatography on Silica gel column and the fractions were assessed against the two Leishmania strains. The most active fractions and the crude extracts were evaluated against reference strains of L. amazonensis, L. braziliensis, L. aethiopica, two native strains (L. Lainsoni and L. braziliensis) and for cytotoxicity against HeLa cells. The chromatographic profile of the active fractions was obtained by reverse phase chromatography using HPLC. RESULTS: From the 73 extracts, 39 extracts (53.4%) were inactive and 34 showed activity. Thirteen species were sselected for bioguided studies. The crude extracts and their 36 fractions were evaluated against two Leishmania strains. The most active fraction were tested in a panel of five leishmania strains and for cytotoxicity. The Selective Index (SI = LD50/IC50) was calculated, and were generally low. Retention time and UV spectra were recorded for the active fractions by HPLC-DAD using a reverse phase column. Profiles were very different from each other, showing the presence of different compounds. CONCLUSION: Bolivian traditional knowledge from the Tacanba was useful to identify plants with effect on Leishmania promastigotes. Chromatographic bioguided studies showed stronger leishmanicidal and cytotoxic activity for the medium polar fraction. HPLC analysis showed different chromatographic profiles of the active fractions.


Assuntos
Leishmania braziliensis/efeitos dos fármacos , Medicina Tradicional , Extratos Vegetais/farmacologia , Plantas Medicinais , Tripanossomicidas/farmacologia , Bolívia , Sobrevivência Celular/efeitos dos fármacos , Etanol/química , Células HeLa , Humanos , Leishmania braziliensis/crescimento & desenvolvimento , Dose Letal Mediana , Testes de Sensibilidade Parasitária , Fitoterapia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/toxicidade , Plantas Medicinais/química , Plantas Medicinais/classificação , Plantas Medicinais/toxicidade , Solventes , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/toxicidade
15.
PLoS One ; 12(12): e0190294, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29281707

RESUMO

M. tuberculosis and parasites of the genus Leishmania present the type II fatty acid biosynthesis system (FASII). The pentacyano(isoniazid)ferrate(II) compound, named IQG-607, inhibits the enzyme 2-trans-enoyl-ACP(CoA) reductase from M. tuberculosis, a key component in the FASII system. Here, we aimed to evaluate the inhibitory activity of IQG-607 against promastigote and amastigote forms of Leishmania (Viannia) braziliensis isolated from patients with different clinical forms of L. braziliensis infection, including cutaneous, mucosal and disseminated leishmaniasis. Importantly, IQG-607 inhibited the proliferation of three different isolates of L. braziliensis promastigotes associated with cutaneous, mucosal and disseminated leishmaniasis. The IC50 values for IQG-607 ranged from 32 to 75 µM, for these forms. Additionally, IQG-607 treatment decreased the proliferation of intracellular amastigotes in infected macrophages, after an analysis of the percentage of infected cells and the number of intracellular parasites/100 cells. IQG-607 reduced from 58% to 98% the proliferation of L. braziliensis from cutaneous, mucosal and disseminated strains. Moreover, IQG-607 was also evaluated regarding its potential toxic profile, by using different cell lines. Cell viability of the lineages Vero, HaCat and HepG2 was significantly reduced after incubation with concentrations of IQG-607 higher than 2 mM. Importantly, IQG-607, in a concentration of 1 mM, did not induce DNA damage in HepG2 cells, when compared to the untreated control group. Future studies will confirm the mechanism of action of IQG-607 against L. braziliensis.


Assuntos
Compostos Ferrosos/farmacologia , Isoniazida/análogos & derivados , Leishmania braziliensis/efeitos dos fármacos , Animais , Isoniazida/farmacologia , Leishmania braziliensis/crescimento & desenvolvimento
16.
Pathog Dis ; 75(7)2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28830073

RESUMO

Platelet-activating factor (PAF) is produced by macrophages during inflammation and infections. We evaluated whether PAF is able to modulate the infection of human macrophages by Leishmania braziliensis, the main Leishmania sp. in Brazil. Monocyte-derived macrophages were incubated with promastigote forms in absence or presence of exogenous PAF. We observed that the treatment of macrophages with low concentrations of PAF prior to infection increased the phagocytosis of L. braziliensis. More importantly, exogenous PAF reduced the parasitism when it was added before, during or after infection. In addition, treatment with a PAF antagonist (PCA 4248) resulted in a significant increase of macrophage infection in a concentration-dependent manner, suggesting that endogenous PAF is important to control L. braziliensis infection. Mechanistically, while exogenous PAF increased production of reactive oxygen species (ROS) treatment with PCA 4248 reduced oxidative burst during L. braziliensis infection. The microbicidal effects of exogenous PAF were abolished when macrophages were treated with apocynin, an NADPH oxidase inhibitor. The data show that PAF promotes the production of ROS induced by L. braziliensis, suggesting that this lipid mediator may be relevant to control L. braziliensis infection in human macrophages.


Assuntos
Leishmania braziliensis/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Espécies Reativas de Oxigênio/agonistas , Acetofenonas/farmacologia , Di-Hidropiridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Humanos , Leishmania braziliensis/crescimento & desenvolvimento , Macrófagos/imunologia , Macrófagos/parasitologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fator de Ativação de Plaquetas/antagonistas & inibidores , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/efeitos dos fármacos
17.
PLoS One ; 12(7): e0180532, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28742826

RESUMO

It was recently hypothesized that Leishmania amastigotes could constitute a semi-quiescent stage characterized by low replication and reduced metabolic activity. This concept developed with Leishmania (Leishmania) mexicana and Leishmania (Leishmania) major models might explain numerous clinical and sub-clinical features of Leishmania (Viannia) braziliensis infections, like reactivation of the disease, non-response to chemotherapy or asymptomatic infections. We compared here in vitro the proliferative capability of L. (V.) braziliensis amastigotes and promastigotes, assessed the expression of key molecular parameters and performed metabolomic analysis. We found that contrary to the highly proliferative promastigotes, amastigotes (axenic and intracellular) do not show evidence of extensive proliferation. In parallel, amastigotes showed a significant decrease of (i) the kDNA mini-circle abundance, (ii) the intracellular ATP level, (iii) the ribosomal components: rRNA subunits 18S and 28S α and ribosomal proteins RPS15 and RPL19, (iv) total RNA and protein levels. An untargeted metabolomic study identified clear differences between the different life stages: in comparison to logarithmic promastigotes, axenic amastigotes showed (a) a strong decrease of 14 essential and non-essential amino acids and eight metabolites involved in polyamine synthesis, (b) extensive changes in the phospholipids composition and (c) increased levels of several endogenous and exogenous sterols. Altogether, our results show that L. (V.) braziliensis amastigotes can show a phenotype with negligible rate of proliferation, a lower capacity of biosynthesis, a reduced bio-energetic level and a strongly altered metabolism. Our results pave the way for further exploration of quiescence among amastigotes of this species.


Assuntos
Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Leishmaniose Cutânea/parasitologia , Metaboloma , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Vias Biossintéticas , Células Cultivadas , Feminino , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , RNA de Protozoário/análise , RNA de Protozoário/metabolismo , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/metabolismo
18.
Am J Trop Med Hyg ; 96(5): 1155-1159, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28500815

RESUMO

AbstractCutaneous leishmaniasis (CL) by Leishmania braziliensis is associated with decreasing cure rates in Brazil. Standard treatment with pentavalent antimony (Sbv) cures only 50-60% of the cases. The immunopathogenesis of CL ulcer is associated with high interferon-γ and tumor necrosis factor (TNF) production. Pentoxifylline, a TNF inhibitor, has been successfully used in association with Sbv in mucosal and cutaneous leishmaniasis. This randomized, double-blind, and placebo-controlled trial aimed to evaluate the efficacy and safety of oral pentoxifylline plus Sbv versus placebo plus Sbv in patients with CL in Bahia, Brazil. A total of 164 patients were randomized in two groups to receive the combination or the monotherapy. Cure rate 6 months after treatment was 45% in the pentoxifylline group and 43% in the control group. There was also no difference between the groups regarding the healing time (99.7 ± 66.2 days and 98.1 ± 72.7 days, respectively). Adverse events were more common in the pentoxifylline group (37.8%), versus 23% in the placebo group. This trial shows that Sbv combined therapy with pentoxifylline is not more effective than Sbv monotherapy in the treatment of CL caused by L. braziliensis.


Assuntos
Antimônio/uso terapêutico , Antiprotozoários/uso terapêutico , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Pentoxifilina/uso terapêutico , Adolescente , Adulto , Brasil , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Humanos , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Interferon gama/imunologia , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/patogenicidade , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia
19.
Molecules ; 22(5)2017 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-28481276

RESUMO

A series of fifty arylideneketones and thiazolidenehydrazines was evaluated against Leishmania infantum and Leishmania braziliensis. Furthermore, new simplified thiazolidenehydrazine derivatives were evaluated against Trypanosoma cruzi. The cytotoxicity of the active compounds on non-infected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their anti-parasitic effects. Seven thiazolidenehydrazine derivatives and ten arylideneketones had good activity against the three parasites. The IC50 values for T. cruzi and Leishmania spp. ranged from 90 nM-25 µM. Eight compounds had multi-trypanocidal activity against T. cruzi and Leishmania spp. (the etiological agents of cutaneous and visceral forms). The selectivity of these active compounds was better than the three reference drugs: benznidazole, glucantime and miltefosine. They also had low toxicity when tested in vivo on zebrafish. Trying to understand the mechanism of action of these compounds, two possible molecular targets were investigated: triosephosphate isomerase and cruzipain. We also used a molecular stripping approach to elucidate the minimal structural requirements for their anti-T. cruzi activity.


Assuntos
Doença de Chagas/tratamento farmacológico , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Visceral/dietoterapia , Tripanossomicidas , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Linhagem Celular , Doença de Chagas/metabolismo , Doença de Chagas/patologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrazinas , Cetonas , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/patologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/patologia , Camundongos , Tiazolidinas , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Peixe-Zebra
20.
Photodiagnosis Photodyn Ther ; 18: 325-330, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28457848

RESUMO

The cutaneous leishmaniasis is caused by the protozoan of the genus Leishmania. It is considered by WHO as a public health issue and a neglected disease, which affects rural workers and it is also a risk to travelers in endemic areas. The conventional treatment is toxic and leads to severe side effects. The photodynamic therapy has been studied as an alternative treatment to cutaneous leishmaniasis. This study aimed to evaluate the methylene blue internalization and the impact of the PDT in the viability and morphology of Leishmania major and Leishmania braziliensis promastigote in culture medium. The fluorescence microscopy was used to determine the MB localization. To evaluate the mitochondrial activity (MTT), viability (Trypan blue test) and the morphological alterations both species were incubated with the MB in concentrations starting in 500µg/ml, in serial dilution, until 7,8µg/ml. The fluorescence microscopy demonstrated that the MB is internalized by both species after one hour of incubation. The MB presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 70% in the higher concentrations tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. The results presented in this study are an indicative that the MB is a photosensitizer with promising potential to clinical application, besides its low cost.


Assuntos
Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania major/efeitos dos fármacos , Leishmania major/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Azul de Metileno/administração & dosagem , Fotoquimioterapia/métodos , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Leishmania braziliensis/efeitos da radiação , Leishmania major/efeitos da radiação , Estágios do Ciclo de Vida/efeitos da radiação , Fármacos Fotossensibilizantes/administração & dosagem , Resultado do Tratamento
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