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1.
Exp Parasitol ; 217: 107948, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32698076

RESUMO

Immunomodulation is an emerging concept to combat infection in recent years. Immunomodulators like arabinosylated-lipoarabinomannan (Ara-LAM) and glycyrrhizic-acid (GA) possess anti-leishmanial property, whereas sodium-antimony-gluconate (SAG) is still considered as the first choice for chemotherapy against leishmaniasis. During infection, invasion of Leishmania donovani needs the potential requirement of Ca2+, which is further responsible for apoptosis in intracellular amastigotes. However, suppression of elevated intracellular calcium by the activation of plasma-membrane-calcium-ATPase (PMCA4) facilitates survival of L. donovani in the host. In the present study, SAG, Ara-LAM, and GA were found to evoke significant increase in intracellular Ca2+ in L. donovani infected macrophages by inhibiting PMCA4. Moreover, PMCA4 inhibition by TFP or PMCA4 siRNA elevated the level of PKCß, whereas calcium-independent upregulation of PKCζ remained unchanged in infected macrophages. Furthermore, application of immunomodulators in infected macrophages resulted in down-regulation of PKCζ, conversion of anti-inflammatory to pro-inflammatory cytokines and inhibition of PMCA4. Plasma membrane-associated ceramide which is known to be elevated during leishmaniasis, triggered upregulation of PMCA4 via PKCζ activation. Interestingly, immunomodulators attenuated ceramide generation, which resulted into reduced PKCζ activation leading to the decreased PMCA expression in infected macrophages. Therefore, our study elucidated the efficacy of SAG, Ara-LAM, and GA in the reduction of parasite burden in macrophages by suppressing PMCA activation through inhibition of ceramide mediated upregulation of PKCζ.


Assuntos
Antiprotozoários/uso terapêutico , ATPases Transportadoras de Cálcio/sangue , Membrana Celular/enzimologia , Fatores Imunológicos/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Animais , Gluconato de Antimônio e Sódio/farmacologia , Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/farmacologia , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Ceramidas/metabolismo , Meios de Cultura Livres de Soro , Densitometria , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Ácido Glicirrízico/farmacologia , Ácido Glicirrízico/uso terapêutico , Imipramina/farmacologia , Immunoblotting , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/uso terapêutico , Macrófagos/fisiologia , Camundongos , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Transfecção
2.
Parasit Vectors ; 12(1): 348, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300064

RESUMO

BACKGROUND: In the last decade, resistance to antimonials has become a serious problem due to the emergence of drug-resistant strains. Therefore, understanding the mechanisms used by Leishmania parasites to survive under drug pressure is essential, particularly for species of medical-veterinary importance such as L. amazonensis. METHODS: Here, we used RNA-seq technology to analyse transcriptome profiles and identify global changes in gene expression between antimony-resistant and -sensitive L. amazonensis promastigotes. RESULTS: A total of 723 differentially expressed genes were identified between resistant and sensitive lines. Comparative transcriptomic analysis revealed that genes encoding proteins involved in metabolism (fatty acids) and stress response, as well as those associated with antimony resistance in other Leishmania species, were upregulated in the antimony-resistant line. Most importantly, we observed upregulation of genes encoding autophagy proteins, suggesting that in the presence of trivalent stibogluconate (SbIII) L. amazonensis can activate these genes either as a survival strategy or to induce cell death, as has been observed in other parasites. CONCLUSIONS: This work identified global transcriptomic changes in an in vitro-adapted strain in response to SbIII. Our results provide relevant information to continue understanding the mechanism used by parasites of the subgenus Leishmania (L. amazonensis) to generate an antimony-resistant phenotype.


Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Resistência a Medicamentos/genética , Leishmania/efeitos dos fármacos , Leishmania/genética , Transcriptoma , DNA de Protozoário/genética , Perfilação da Expressão Gênica , Ontologia Genética , Fenótipo , Análise de Sequência de RNA , Regulação para Cima
3.
Artigo em Inglês | MEDLINE | ID: mdl-31160283

RESUMO

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative ß-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.


Assuntos
Antiprotozoários/farmacologia , Trypanosoma brucei brucei/metabolismo , Adenosina Trifosfatases/metabolismo , Anfotericina B/farmacologia , Gluconato de Antimônio e Sódio/farmacologia , Leishmania/parasitologia , Paromomicina/farmacologia , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Proteínas R-SNARE/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
4.
BMC Genomics ; 19(1): 843, 2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30486770

RESUMO

BACKGROUND: Leishmaniasis is a neglected tropical disease with diverse clinical phenotypes, determined by parasite, host and vector interactions. Despite the advances in molecular biology and the availability of more Leishmania genome references in recent years, the association between parasite species and distinct clinical phenotypes remains poorly understood. We present a genomic comparison of an atypical variant of Leishmania donovani from a South Asian focus, where it mostly causes cutaneous form of leishmaniasis. RESULTS: Clinical isolates from six cutaneous leishmaniasis patients (CL-SL); 2 of whom were poor responders to antimony (CL-PR), and two visceral leishmaniasis patients (VL-SL) were sequenced on an Illumina MiSeq platform. Chromosome aneuploidy was observed in both groups but was more frequent in CL-SL. 248 genes differed by 2 fold or more in copy number among the two groups. Genes involved in amino acid use (LdBPK_271940) and energy metabolism (LdBPK_271950), predominated the VL-SL group with the same distribution pattern reflected in gene tandem arrays. Genes encoding amastins were present in higher copy numbers in VL-SL and CL-PR as well as being among predicted pseudogenes in CL-SL. Both chromosome and SNP profiles showed CL-SL and VL-SL to form two distinct groups. While expected heterozygosity was much higher in VL-SL, SNP allele frequency patterns did not suggest potential recent recombination breakpoints. The SNP/indel profile obtained using the more recently generated PacBio sequence did not vary markedly from that based on the standard LdBPK282A1 reference. Several genes previously associated with resistance to antimonials were observed in higher copy numbers in the analysis of CL-PR. H-locus amplification was seen in one cutaneous isolate which however did not belong to the CL-PR group. CONCLUSIONS: The data presented suggests that intra species variations at chromosome and gene level are more likely to influence differences in tropism as well as response to treatment, and contributes to greater understanding of parasite molecular mechanisms underpinning these differences. These findings should be substantiated with a larger sample number and expression/functional studies.


Assuntos
Genoma , Leishmania donovani/genética , Leishmania donovani/patogenicidade , Aneuploidia , Gluconato de Antimônio e Sódio/farmacologia , Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Sequência de Bases , Cromossomos/genética , Dosagem de Genes , Ontologia Genética , Heterozigoto , Homozigoto , Humanos , Mutação INDEL/genética , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/isolamento & purificação , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Fases de Leitura Aberta/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Virulência/efeitos dos fármacos , Virulência/genética
5.
Infect Dis Poverty ; 7(1): 108, 2018 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-30340519

RESUMO

BACKGROUND: Ethiopia has the highest number of visceral leishmaniasis (VL) cases after Sudan in Sub-Saharan Africa. However, there was lack of comprehensive data on VL treatment outcome despite the huge burden of the diseases in the country. Hence, we aimed to perform a systematic review and meta-analysis on this topic to obtain stronger evidence on treatment outcomes of VL from the existing literature in Ethiopia. METHODS: The Cochrane guidelines to conduct meta-analysis following the Preferred Reporting Items for Systematic review and Meta-Analysis statement was used to conduct a computerized systematic search of the PubMed, Google Scholar, and ScienceDirect databases. Random effects model was used to combine studies showing heterogeneity of Cochrane Q P < 0.10 and I2 > 50. Treatment outcomes were assessed at end of treatment and at 6 months follow-up. Subgroup analyses were performed on treatment outcomes based on the different antileishmanial treatment options and patients' HIV status. RESULTS: Fifteen studies were included in the final analyses. At end of treatment, an overall treatment success rate of 82.6% was noticed. At 6 months follow-up, the overall treatment success rate was 72.2%. For patients treated with sodium stibogluconate (SSG), the treatment success rates at the end of treatment and at six-month follow-up were 81.5% and 80.7%, respectively. Multiple doses of liposomal-amphotericin B (L-AMB) had treatment success rates of 96.7 and 71-100% at the end of treatment and at 6 months follow-up, respectively. The combination of SSG with paromomycin (PM) gave treatment success rates of up to 90.1% at the end of treatment. HIV-infected individuals were found to have a higher mortality (odds ratio = 4.77, 95% CI: 1.30-17.43, P = 0.009) rate at 6 months follow-up. CONCLUSIONS: SSG alone has shown lower treatment efficacy in the management of VL when compared to combination of SSG with PM and multiple doses of L-AMB. The combination of SSG with PM gave good treatment success rates with shorter duration of treatment. Hence, the combination of SSG with PM should be used preferentially over SSG monotherapy. Multiple doses of L-AMB showed great efficacy especially among patients with complications, severe disease, HIV co-infection, and intolerance to the adverse effects of antimonials. HIV-infected individuals had a worse prognosis than their HIV-negative counterparts.


Assuntos
Leishmaniose Visceral/epidemiologia , Gluconato de Antimônio e Sódio/farmacologia , Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Coinfecção , Feminino , Infecções por HIV , Humanos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/mortalidade , Leishmaniose Visceral/parasitologia , Masculino , Razão de Chances , Resultado do Tratamento
6.
Turkiye Parazitol Derg ; 42(1): 11-19, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29780014

RESUMO

OBJECTIVE: Two pentavalent antimonials, meglumine antimoniate (Glucantime®, France) and sodium stibogluconate (Pentostam®, England), are used to treat cutaneous leishmaniasis (CL) in Turkey. The present study, serving as a guidebook for young researchers, aims to provide basis for conducting drug resistance tests and active ingredient scanning in in vitro and in vivo models. METHODS: A CL isolate kept in liquid nitrogen was initially thawed and genotyped by real-time polymerase chain reaction (PCR) using ITS1 prob. In vitro and in vivo tests were conducted to determine drug resistance against meglumine antimoniate and sodium stibogluconate. Hemocytometry and XTT (sodium 3,39-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate) methods were used to investigate in vitro drug resistance. CL mouse models were used to analyze in vivo drug resistance. RESULTS: The isolate was determined as Leishmania tropica by genotyping by PCR on the internal transcribed spacer 1 (ITS1) gene region. In in vitro drug resistance tests, sodium stibogluconate was observed to be more effective than meglumine antimoniate, but there was no statistically significant difference between the two (p > 0.05). It was observed that the footpad lesions of the animals started to shrink afterward the 5th week of infection following treatment with these agents, and parasitologic recovery was observed at the end of 3 months. CONCLUSIONS: With an aim to be used as a guidebook for young researchers, active ingredient scanning and drug resistance tests in both in vitro and in vivo models were presented in the current study.


Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Leishmania tropica/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Animais , Resistência a Medicamentos , Humanos , Antimoniato de Meglumina , Camundongos , Testes de Sensibilidade Microbiana/normas , Projetos Piloto , Guias de Prática Clínica como Assunto , Turquia
7.
PLoS Negl Trop Dis ; 12(3): e0006310, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29561842

RESUMO

BACKGROUND: In Algeria, the treatment of visceral and cutaneous leishmanioses (VL and CL) has been and continues to be based on antimony-containing drugs. It is suspected that high drug selective pressure might favor the emergence of chemoresistant parasites. Although treatment failure is frequently reported during antimonial therapy of both CL and VL, antimonial resistance has never been thoroughly investigated in Algeria. Determining the level of antimonial susceptibility, amongst Leishmania transmitted in Algeria, is of great importance for the development of public health policies. METHODOLOGY/PRINCIPAL FINDINGS: Within the framework of the knowledge about the epidemiology of VL and CL amassed during the last 30 years, we sampled Leishmania isolates to determine their susceptibility to antimony. We analyzed a total of 106 isolates including 88 isolates collected between 1976 and 2013 in Algeria from humans, dogs, rodents, and phlebotomines and 18 collected from dogs in France. All the Algerian isolates were collected in 14 localities where leishmaniasis is endemic. The 50% inhibitory concentrations (IC50) of potassium antimony tartrate (the trivalent form of antimony, Sb(III)) and sodium stibogluconate (the pentavalent form of antimony, Sb(V)) were determined in promastigotes and intramacrophage amastigotes, respectively. The epidemiological cutoff (ECOFF) that allowed us to differentiate between Leishmania species causing cutaneous or visceral leishmaniases that were susceptible (S+) or insusceptible (S-) to the trivalent form of antimony was determined. The computed IC50 cutoff values were 23.83 µg/mL and 15.91 µg/mL for VL and CL, respectively. We report a trend of increasing antimony susceptibility in VL isolates during the 30-year period. In contrast, an increase in the frequency of S- phenotypes in isolates causing CL was observed during the same period. In our study, the emergence of S- phenotypes correlates with the inclusion of L. killicki (syn: L. tropica) isolates that cause cutaneous leishmaniasis and that have emerged in Algeria during the last decade. CONCLUSION/SIGNIFICANCE: Our results provide insight into the spatiotemporal dynamics of Leishmania antimony susceptibility in Algeria. We highlight the need for the future implementation of an effective methodology to determine the antimony susceptibility status of Leishmania isolates to detect the emergence of and prevent the dissemination of drug-resistant strains.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Leishmania/isolamento & purificação , Argélia/epidemiologia , Animais , Gluconato de Antimônio e Sódio/farmacologia , Cães , Resistência a Medicamentos , Humanos , Concentração Inibidora 50 , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Psychodidae/parasitologia , Roedores/parasitologia
8.
World J Microbiol Biotechnol ; 34(3): 38, 2018 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-29460068

RESUMO

We evaluated, for the first time, the leishmanicidal potential of decanethiol functionalized silver nanoparticles (AgNps-SCH) on promastigotes and amastigotes of different strains and species of Leishmania: L. mexicana and L. major isolated from different patients suffering from localized cutaneous leishmaniasis (CL) and L. mexicana isolated from a patient suffering from diffuse cutaneous leishmaniasis (DCL). We recorded the kinetics of promastigote growth by daily parasite counting for 5 days, promastigote mobility, parasite reproduction by CFSE staining's protocol and promastigote killing using the propidium iodide assay. We also recorded IC50's of promastigotes and amastigotes, therapeutic index, and cytotoxicity by co-culturing macrophages with AgNps-SCH or sodium stibogluconate (Sb) used as reference drug. We used Sb as a reference drug since it is used as the first line treatment for all different types of leishmaniasis. At concentrations 10,000 times lower than those used with Sb, AgNps-SCH had a remarkable leishmanicidal effect in all tested strains of parasites and there was no toxicity to J774A.1 macrophages since > 85% were viable at the concentrations used. Therapeutic index was about 20,000 fold greater than the corresponding one for Sb treated cells. AgNps-SCH inhibited > 80% promastigote proliferation in all tested parasites. These results demonstrate there is a high leishmanicidal potential of AgNps-SCH at concentrations of 0.04 µM. Although more studies are needed, including in vivo testing of AgNps-SCH against different types of leishmaniasis, they can be considered a potential new treatment alternative.


Assuntos
Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Nanopartículas Metálicas/química , Prata/farmacologia , Animais , Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Cinética , Leishmania/crescimento & desenvolvimento , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Prata/administração & dosagem
9.
Exp Parasitol ; 176: 30-45, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28263760

RESUMO

Resistance of human pathogens like Leishmania to drugs is a growing concern where the multidrug-resistant phenotype renders chemotherapy ineffective. The acquired resistance of Leishmania to antimony has promoted intense research on the mechanisms involved but the question has not been resolved yet. In this study we have explored host-pathogen- drug interactions leading to identification of pharmacological determinants of host macrophages that resist the sodium antimony gluconate (SAG) mediated intracellular parasite killing. mRNA profiling of mammalian host stage amastigotes of sodium antimony gluconate (SAG) 'sensitive' and 'resistant' parasite lines was carried out using Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. Patient sera was used to identify immunogenic proteins by two-dimensional gel analysis (2DE) and mass spectrometric analysis (LC-MS/MS). Immunofluorescence microscopy confirmed the identities on 'sensitive' and 'resistant' parasite lines. A total of nine immunogenic proteins whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins are altered in expression levels in the 'resistant' parasites. Global expression profiling using microarrays revealed this regulation was not reflected by changes in the levels of the cognate mRNAs. Following identification of proteins by mass spectrometry, one such regulated protein, enolase, was chosen for more detailed analysis. Immunofluorescence microscopy employing antisera against this enzyme confirmed that its level was differentially regulated in the 'resistant' isolate. We show that high serum level of immunoreactive protein is associated with 'resistant' phenotype. Differentially expressed proteins with immunomodulatory activities were found to be associated with the 'resistant phenotype'.


Assuntos
Antígenos de Protozoários/análise , Resistência a Medicamentos/imunologia , Genômica , Epitopos Imunodominantes/análise , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Proteômica , Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Western Blotting , Eletroforese em Gel Bidimensional , Humanos , Soros Imunes/imunologia , Imunoglobulina G/imunologia , Índia , Leishmania donovani/imunologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Espectrometria de Massas , Microscopia de Fluorescência , Proteínas de Protozoários/imunologia
10.
Antimicrob Agents Chemother ; 60(9): 5262-75, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27324767

RESUMO

The mechanisms underlying the drug resistance of Leishmania spp. are manifold and not completely identified. Apart from the highly conserved multidrug resistance gene family known from higher eukaryotes, Leishmania spp. also possess genus-specific resistance marker genes. One of them, ARM58, was first identified in Leishmania braziliensis using a functional cloning approach, and its domain structure was characterized in L. infantum Here we report that L. infantum ARM58 is part of a gene cluster at the telomeric end of chromosome 34 also comprising the neighboring genes ARM56 and HSP23. We show that overexpression of all three genes can confer antimony resistance to intracellular amastigotes. Upon overexpression in L. donovani, ARM58 and ARM56 are secreted via exosomes, suggesting a scavenger/secretion mechanism of action. Using a combination of functional cloning and next-generation sequencing, we found that the gene cluster was selected only under antimonyl tartrate challenge and weakly under Cu(2+) challenge but not under sodium arsenite, Cd(2+), or miltefosine challenge. The selective advantage is less pronounced in intracellular amastigotes treated with the sodium stibogluconate, possibly due to the known macrophage-stimulatory activity of this drug, against which these resistance markers may not be active. Our data point to the specificity of these three genes for antimony resistance.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Resistência a Medicamentos/genética , Leishmania infantum/efeitos dos fármacos , Proteínas de Protozoários/genética , Telômero/química , Gluconato de Antimônio e Sódio/farmacologia , Cádmio/farmacologia , Clonagem Molecular , Cobre/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Exossomos/química , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Leishmania infantum/genética , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/metabolismo , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/genética , Família Multigênica , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Proteínas de Protozoários/metabolismo , Telômero/metabolismo
11.
Rev Soc Bras Med Trop ; 49(2): 196-203, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27192589

RESUMO

INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9µg/mL, whereas that of MA was 60µg/mL. Sb-FEL showed an IC50 value of 0.2µg/mL, whereas that of free Sb was 9µg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.


Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Fosfatidilserinas/farmacologia , Animais , Gluconato de Antimônio e Sódio/química , Antiprotozoários/química , Cricetinae , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Lipossomos , Meglumina/química , Antimoniato de Meglumina , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organometálicos/química , Testes de Sensibilidade Parasitária , Fosfatidilserinas/química
12.
Exp Parasitol ; 154: 93-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25911243

RESUMO

In this study, in vitro anti-leishmanial activity of buparvaquone was evaluated against promastigotes and intracellular amastigotes of Pakistani Leishmania tropica isolate KWH23 in relation to the current standard chemotherapy for leishmaniasis (sodium stibogluconate, sodium stibogluconate, amphotericin B and miltefosine). For buparvaquone, mean % inhibition in intracellular amastigotes at four different concentrations (1.35 µM, 0.51 µM, 0.17 µM and 0.057 µM) was 78%, 44%, 20% and 14% respectively, whereas, against promastigotes it was 89%, 77%, 45% and 35% respectively. IC50 values calculated to estimate the anti-leishmanial activity of buparvaquone against intra-cellular amastigotes and promastigotes was 0.53 µM (95% C.I. = 0.32-0.89) and 0.15 µM (95% C.I. = 0.01-1.84) respectively. Amphotericin B was the most potent in-vitro drug tested, with an IC50 of 0.075 µM (95% C.I. = 0.006-0.907) against promastigotes, and 0.065 µM (95% C.I. = 0.048-0.089) against intra-cellular amastigotes. Amphotericin B was more cytotoxic against THP1 cells, with an IC50 of 0.15 µM (95% C.I. = 0.01-0.95) and an apparent in-vitro therapeutic index of 2.0, than was buparvaquone, with an IC50 of 12.03 µM (95% C.I. = 5.36-26.96) against THP1 cells and a therapeutic index of 80.2. The study proposes that buparvaquone may be further investigated as a candidate drug for treatment of cutaneous leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Leishmania tropica/efeitos dos fármacos , Naftoquinonas/farmacologia , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Gluconato de Antimônio e Sódio/farmacologia , Gluconato de Antimônio e Sódio/toxicidade , Antiprotozoários/toxicidade , Linhagem Celular Tumoral/efeitos dos fármacos , Criança , Humanos , Concentração Inibidora 50 , Leishmania tropica/crescimento & desenvolvimento , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Macrófagos/parasitologia , Masculino , Meglumina/farmacologia , Meglumina/toxicidade , Antimoniato de Meglumina , Naftoquinonas/toxicidade , Compostos Organometálicos/farmacologia , Compostos Organometálicos/toxicidade , Paquistão , Testes de Sensibilidade Parasitária
13.
Int J Antimicrob Agents ; 45(3): 268-77, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25600891

RESUMO

Since there are very few affordable antileishmanial drugs available, antimonial resistance has crippled antileishmanial therapy, thereby emphasising the need for development of novel therapeutic strategies. This study aimed to evaluate the antileishmanial role of combined therapy with sodium antimony gluconate (SAG) and the triterpenoid glycyrrhizic acid (GA) against infection with SAG-resistant Leishmania (GE1F8R). Combination therapy with GA and SAG successfully limited infection with SAG-resistant Leishmania in a synergistic manner (fractional inhibitory concentration index <1.0). At the same time, mice infected with SAG-resistant Leishmania and co-treated with GA and SAG exhibited a significant reduction in hepatic and splenic parasite burden. In probing the mechanism, it was observed that GA treatment suppressed the expression and efflux activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1), two host ABC transporters responsible for antimony efflux from host cells infected with SAG-resistant parasites. This suppression correlated with greater intracellular antimony retention during SAG therapy both in vitro and in vivo, which was reflected in the reduced parasite load. Furthermore, co-administration of GA and SAG induced a shift in the cytokine balance towards a Th1 phenotype by augmenting pro-inflammatory cytokines (such as IL-12, IFNγ and TNFα) and inducing nitric oxide generation in GE1F8R-infected macrophages as well as GE1F8R-infected mice. This study aims to provide an affordable leishmanicidal alternative to expensive antileishmanial drugs such as miltefosine and amphotericin B. Furthermore, this report explores the role of GA as a resistance modulator in MRP1- and P-gp-overexpressing conditions.


Assuntos
Gluconato de Antimônio e Sódio/administração & dosagem , Antiprotozoários/administração & dosagem , Resistência a Medicamentos , Ácido Glicirrízico/administração & dosagem , Leishmania/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Animais , Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Modelos Animais de Doenças , Quimioterapia Combinada/métodos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Ácido Glicirrízico/farmacologia , Leishmania/isolamento & purificação , Leishmaniose Visceral/parasitologia , Fígado/parasitologia , Camundongos Endogâmicos BALB C , Carga Parasitária , Baço/parasitologia , Resultado do Tratamento
14.
Antimicrob Agents Chemother ; 59(1): 344-55, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25367907

RESUMO

Pentavalent antimonials have been the first-line treatment for leishmaniasis for decades. However, the development of resistance to sodium stibogluconate (SSG) has limited its use, especially for treating visceral leishmaniasis (VL). The present work aims to optimize a cationic liposomal formulation of SSG for the treatment of both SSG-sensitive (AG83) and SSG-resistant (GE1F8R and CK1R) Leishmania donovani infections. Parasite killing was determined by the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic counting of Giemsa-stained macrophages. Macrophage uptake studies were carried out by confocal microscopic imaging. Parasite-liposome interactions were visualized through transmission electron microscopy. Toxicity tests were performed using assay kits. Organ parasite burdens were determined by microscopic counting and limiting dilution assays. Cytokines were measured by enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Although all cationic liposomes studied demonstrated leishmanicidal activity, phosphatidylcholine (PC)-dimethyldioctadecylammonium bromide (DDAB) vesicles were most effective, followed by PC-stearylamine (SA) liposomes. Since entrapment of SSG in PC-DDAB liposomes demonstrated enhanced ultrastructural alterations in promastigotes, PC-DDAB-SSG vesicles were further investigated in vitro and in vivo. PC-DDAB-SSG could effectively alleviate SSG-sensitive and SSG-resistant L. donovani infections in the liver, spleen, and bone marrow of BALB/c mice at a dose of SSG (3 mg/kg body weight) not reported previously. The parasiticidal activity of these vesicles was attributed to better interactions with the parasite membranes, resulting in direct killing, and generation of a strong host-protective environment, necessitating a very low dose of SSG for effective cures.


Assuntos
Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/uso terapêutico , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Lipossomos/uso terapêutico , Animais , Gluconato de Antimônio e Sódio/química , Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Medula Óssea/parasitologia , Cricetinae , Resistência a Medicamentos , Humanos , Leishmaniose Visceral/parasitologia , Lipossomos/farmacologia , Fígado/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Parasitária , Baço/parasitologia
15.
Bioorg Med Chem Lett ; 25(2): 410-3, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25475205

RESUMO

Some novel heteroretinoid-bisbenzylidine ketone hybrids have been synthesized and evaluated for their in vitro antileishmanial activity against intramacrophagic amastigotes of Leishmania donovani. Among all the nine synthetic compounds, five compounds (7c, 7d and 7f-h) have shown significant (less than 7µM) activity against intramacrophagic amastigotes. The IC50 values of these compounds were found better than the reference drugs sodium stibogluconate (SSG) and miltefosine. This study helped us in identifying the new class of compounds that could be exploited as potent antileishmanial agents.


Assuntos
Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Cetonas/síntese química , Leishmania donovani/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Retinoides/química , Animais , Gluconato de Antimônio e Sódio/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Concentração Inibidora 50 , Leishmaniose/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Relação Estrutura-Atividade , Células Vero
16.
J Immunol ; 193(8): 4083-94, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25217162

RESUMO

The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kala-azar patients infected with antimony-resistant Leishmania donovani (Sb(R)LD). Previously we showed that MDR-1 upregulation in Sb(R)LD infection is IL-10-dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from Sb(R)LD-infected macrophages (Sb(R)LD-Mϕs) and favors accumulation of surrogates of antimonials. It inhibits IL-10-driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from Sb(R)LD-Mϕs. It abrogates Sb(R)LD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in Sb(R)LD-Mϕs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in Sb(R)LD-Mϕs. Furthermore, exogenous rIL-12 inhibits intracellular Sb(R)LD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in Sb(R)LD-Mϕs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ Sb(R)LD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on Sb(R)LD clearance from infected hosts.


Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Imipramina/uso terapêutico , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Leishmania donovani/efeitos dos fármacos , Tripanossomicidas/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Acetilação/efeitos dos fármacos , Animais , Anticorpos/imunologia , Antimônio/farmacologia , Células Cultivadas , Cricetinae , Resistência a Medicamentos , Desacetilase 6 de Histona , Histona Desacetilases/biossíntese , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-12/farmacologia , Subunidade p35 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/genética , Leishmania donovani/imunologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Subunidade p50 de NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Linfócitos T/imunologia , Fator de Transcrição RelA/metabolismo
17.
Int J Clin Pharmacol Ther ; 52(10): 880-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25109414

RESUMO

BACKGROUND: Leishmania is a unicellular protozoan parasite causing a wide range of human diseases ranging from localized self-healing cutaneous lesions to fatal visceral infections. OBJECTIVE: The aim of the present study is to assess the cytotoxic, anti-proliferative, and apoptotic effects of oleuropein on Leishmania major promastigotes (MHOM/SA/84/JISH) and to compare its effects with the reference drug sodium stibogluconate (pentostam). METHODS: Cytotoxicity and promastigote proliferation were measured using MTT colorimetric assay. Furthermore, the Annexin V/propidium iodide staining technique followed by flow cytometry was used for studying the cell death properties of oleuropein. RESULTS: In the present report we have shown that oleuropein, a pharmacologically safe, natural product of olive leaf, has a potent leishmanicidal effect. Indeed, oleuropein exhibits cytotoxic and anti-proliferative effects against Leishmania major promastigotes. Moreover, oleuropein triggers death through apoptosis, whereas pentostam induces death mainly via necrosis on Leishmania major promastigotes. CONCLUSION: Here we demonstrate for the first time that the non-toxic, natural product oleuropein has apoptotic properties against Leishmania major promastigotes. Further studies are needed to investigate its molecular pathway.


Assuntos
Antiprotozoários/farmacologia , Apoptose/efeitos dos fármacos , Iridoides/farmacologia , Leishmania major/efeitos dos fármacos , Gluconato de Antimônio e Sódio/farmacologia , Relação Dose-Resposta a Droga
18.
Parasitology ; 141(4): 554-62, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24618257

RESUMO

It is well established that visceral leishmaniasis (VL; also known as Kala azar) causes immunosuppression, and a successful drug treatment is associated with the development of cell-mediated immunity. Therefore combining a drug with an immune enhancer can provide a better approach for the treatment of the disease. Keeping this in mind, the in vivo antileishmanial efficacy of immunochemotherapy was evaluated with the use of a 78 kDa antigen with or without monophosphoryl lipid A (MPL-A) along with a traditional drug sodium stibogluconate (SSG) in Leishmania donovani infected BALB/c mice. Mice were infected intracardially with promastigotes of L. donovani, and 30 days after infection, these animals were given specific immunotherapy (78 kDa/78 kDa+MPL-A) or chemotherapy (SSG) or immunochemotherapy (SSG+78 kDa/SSG+78 kDa+MPL-A). Animals were euthanased on 1, 15 and 30 post-treatment days. The antileishmanial potential of the immunochemotherapy was revealed by significant reduction in the parasite burden (P<0·001). These animals were also found to exhibit increased delayed type hypersensitivity (DTH) responses, higher IgG2a levels, lower IgG1 levels and greater cytokine (IFN-γ and IL-2) concentrations compared with chemotherapy or immunotherapy alone, pointing towards the generation of a strong protective (Th1) type of immune response. Immunochemotherapy with SSG+78 kDa+MPL-A was found to be most effective in protecting mice against VL and therefore can be an alternative option for treatment of VL.


Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Antiprotozoários/uso terapêutico , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/farmacologia , Leishmaniose Visceral/imunologia , Animais , Gluconato de Antimônio e Sódio/farmacologia , Citocinas/imunologia , Quimioterapia Combinada , Feminino , Imunidade Celular , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária
19.
Antimicrob Agents Chemother ; 58(6): 2997-3007, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24614385

RESUMO

Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥ 3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ~5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (Sb(V)) and trivalent antimonial (Sb(III)) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of Sb(V) (from 41.2 ± 0.6 µg/ml to 66.5 ± 3.9 µg/ml) and Sb(III) (from 24.0 ± 0.3 µg/ml to 43.4 ± 1.8 µg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon Sb(III) treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism.


Assuntos
Antígenos de Protozoários/genética , Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Leishmania donovani/imunologia , Leishmaniose Visceral/tratamento farmacológico , Antígeno Nuclear de Célula em Proliferação/genética , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/metabolismo , Sequência de Bases , Linhagem Celular , Cricetinae , Resistência a Medicamentos , Expressão Gênica , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteômica , Análise de Sequência de DNA
20.
PLoS Negl Trop Dis ; 7(12): e2527, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24340105

RESUMO

BACKGROUND: Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. METHODOLOGY AND PRINCIPAL FINDINGS: The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode. CONCLUSION/SIGNIFICANCE: This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.


Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Proliferação de Células , Resistência a Medicamentos , Expressão Gênica , Leishmania donovani/efeitos dos fármacos , Proteínas Ribossômicas/biossíntese , Animais , Western Blotting , Citosol/química , Perfilação da Expressão Gênica , Humanos , Leishmania donovani/genética , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/isolamento & purificação , Mesocricetus , Camundongos , Reação em Cadeia da Polimerase em Tempo Real
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