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1.
Exp Parasitol ; 205: 107738, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31442456

RESUMO

Leishmaniases, caused by Leishmania spp., are among the most prevalent infectious diseases in the world and their treatment may present high toxicity and side/adverse effects. This study evaluated the antileishmanial activity of the Hexanic Eluate subfraction from Maytenus guianensis bark (HEMg) incorporated in microparticles of PLGA. One batch of microparticles produced contained HEMg (HEMgP) and another contained the PLGA polymer alone (PCTE). The microparticles were characterized in regards to diameter, Zeta potential, encapsulation rate and morphology and their cytotoxicity was evaluated against J774 macrophages. The infection assay employing peritoneal macrophages witth L. amazonensis and cytokine dosages were performed on the cell supernatants. The groups of infected BALB/C mice were treated, euthanized and the parasite load and cytokine production were evaluated. The diameters and zeta potential were: 4 µm and -11.6 mV (PCTE) and 7.8 µm and -26.7 mV (HEMgP). The encapsulation rate was ≅ 15% and the morphology of the particles was spherical and homogeneous. In the infection assay, HEMgP inhibited the amastigotes by 70% (24 h) and 59% (48 h) and induced IL-12 and TNF-α production. HEMg in solution reduced the number of parasites in the lymph nodes by 50% and HEMgP administration increased the levels of IL-12 and TNF-α cytokines in lymph nodes and in the lesion site. When encapsulated, HEMg maintained its antileishmanial activity, but in a more attenuated and sustained form over time, showing promise as complementary/alternative therapy against cutaneous leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Leishmania mexicana/efeitos dos fármacos , Maytenus/química , Casca de Planta/química , Extratos Vegetais/farmacologia , Animais , Biodegradação Ambiental , Linhagem Celular , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/ultraestrutura , Concentração Inibidora 50 , Linfonodos/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura
2.
Artigo em Inglês | MEDLINE | ID: mdl-31355149

RESUMO

Lipophosphoglycan (LPG) is the major Leishmania surface glycoconjugate having importance during the host-parasite interface. Leishmania (Viannia) braziliensis displays a spectrum of clinical forms including: typical cutaneous leishmaniasis (TL), mucocutaneous (ML), and atypical lesions (AL). Those variations in the immunopathology may be a result of intraspecies polymorphisms in the parasite's virulence factors. In this context, we evaluated the role of LPG of strains originated from patients with different clinical manifestations and the sandfly vector. Six isolates of L. braziliensis were used: M2903, RR051 and RR418 (TL), RR410 (AL), M15991 (ML), and M8401 (vector). LPGs were extracted and purified by hydrophobic interaction. Peritoneal macrophages from C57BL/6 and respective knock-outs (TLR2-/- and TLR-4-/-) were primed with IFN-γ and exposed to different LPGs for nitric oxide (NO) and cytokine production (IL-1ß, IL-6, IL-12, and TNF-α). LPGs differentially activated the production of NO and cytokines via TLR4. In order to ascertain if such functional variations were related to intraspecies polymorphisms in the LPG, the purified glycoconjugates were subjected to western blot with specific LPG antibodies (CA7AE and LT22). Based on antibody reactivity preliminary variations in the repeat units were detected. To confirm these findings, LPGs were depolymerized for purification of repeat units. After thin layer chromatography, intraspecies polymorphisms were confirmed especially in the type and/size of sugars branching-off the repeat units motif. In conclusion, different isolates of L. braziliensis from different clinical forms and hosts possess polymorphisms in their LPGs that functionally affected macrophage responses.


Assuntos
Glicoesfingolipídeos/química , Glicoesfingolipídeos/imunologia , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmaniose Cutânea/imunologia , Ativação de Macrófagos , Receptor 4 Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Técnicas de Inativação de Genes , Glicoesfingolipídeos/isolamento & purificação , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Macrófagos/imunologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico , Psychodidae/parasitologia , Receptor 4 Toll-Like/genética , Fatores de Virulência
3.
Artigo em Inglês | MEDLINE | ID: mdl-31316919

RESUMO

Concurrently, leishmaniasis and AIDS are global public health issues and the overlap between these diseases adds additional treats to the management of co-infected patients. Lopinavir (LPV) has a well characterized anti-HIV and leishmanicidal action, and to analyze its combined action with miltefosine (MFS) could help to envisage strategies to the management of co-infected patients. Here, we evaluate the interaction between LPV and MFS against Leishmania infantum infection by in vitro and in vivo approaches. The effect of the compounds alone or in association was assessed for 72 h in mouse peritoneal macrophages infected with L. infantum by the determination of the IC50s and FICIs. Subsequently, mice were orally treated twice daily during 5 days with the compounds alone or in association and evaluated after 30 days. The in vitro assays revealed an IC50 of 0.24 µM and 9.89 µM of MFS and LPV, respectively, and an additive effect of the compounds (FICI 1.28). The in vivo assays revealed that LPV alone reduced the parasite load in the spleen and liver by 52 and 40%, respectively. The combined treatment of infected BALB/c mice revealed that the compounds alone required at least two times higher doses than when administered in association to virtually eliminate the parasite. Mice plasma biochemical parameters assessed revealed that the combined therapy did not present any relevant hepatotoxicity. In conclusion, the association of MFS with LPV allowed a reduction in each compound concentration to achieve the same outcome in the treatment of visceral leishmaniasis. Although a pronounced synergistic effect was not evidenced, it does not discard that such combination could be useful in humans co-infected with HIV and Leishmania parasites.


Assuntos
Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/complicações , Leishmaniose Visceral/tratamento farmacológico , Lopinavir/uso terapêutico , Fosforilcolina/análogos & derivados , Administração Oral , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/uso terapêutico , Coinfecção/tratamento farmacológico , Modelos Animais de Doenças , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Concentração Inibidora 50 , Leishmaniose Visceral/sangue , Fígado/parasitologia , Lopinavir/administração & dosagem , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Fosforilcolina/administração & dosagem , Fosforilcolina/uso terapêutico , Baço/parasitologia , Resultado do Tratamento
4.
Fitoterapia ; 137: 104251, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31271783

RESUMO

The MeOH extract from leaves of Saururus cernuus L. (Saururaceae) displayed in vitro activity against trypomastigote forms of T. cruzi (100% of parasite death at 200 µg/mL), suggesting the presence of bioactive compounds. Thus, the bioactivity-guided fractionation was carried out, leading to the isolation of three related neolignan derivatives, identified as threo-austrobailignan-5 (1), threo-austrobailignan-6 (2), and threo-dihydroguaiaretic acid (3). Anti-T. cruzi activity of compounds 1-3 was performed against cell-derived trypomastigotes and intracellular amastigotes. Additionally, the mammalian cytotoxicity was investigated using NCTC cells. Compound 2 was the most effective against extracellular trypomastigotes with IC50 of 3.7 µM, while compound 3 showed activity in both clinically relevant forms of the parasite, trypomastigotes and amastigotes, with IC50 values of 7.0 and 16.2 µM, respectively. However, the structurally related compound 1 was inactive. Based on these results, compounds 2 and 3 were selected to evaluate the mechanism of cellular death. Compound 2 induced alteration in the plasma membrane permeability and consequently in the ROS levels after 120 min of incubation. By using flow cytometry and fluorescence microscopy, compound 3 showed alterations in the mitochondrial membrane potential (ΔΨm) of trypomastigotes. Considering the promising chemical and biological properties of neolignans 2 and 3, these compounds could be used as starting points to develop new lead compounds for Chagas disease.


Assuntos
Lignanas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Saururaceae/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Brasil , Células Cultivadas , Guaiacol/análogos & derivados , Lignanas/isolamento & purificação , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Folhas de Planta/química , Espécies Reativas de Oxigênio/metabolismo , Tripanossomicidas/isolamento & purificação
5.
Parasitol Int ; 71: 163-166, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30991111

RESUMO

Leishmaniasis is a poverty-related disease, the chemotherapy of which is based on few drugs. The in vitro macrophage-amastigote model using mouse peritoneal cells, human-monocyte transformed macrophages and immortalized cell lines have been used to test new and safe antileishmanial drugs. Considering the differences for drug sensitivities between these Leishmania infected cells, the efficacy of amphotericin B, pentavalent antimonial, miltefosine and resveratrol was evaluated in a recently developed ex vivo culture of macrophages isolated from mouse lesion induced by L. amazonensis (CD11b+F4/80+CD68+CD14+) compared with infected peritoneal macrophages (CD11b+F4/80+CD68+CD14+). The results show that IC50 values of amphotericin B, miltefosine and pentavalent antimonial for parasites in lesional and peritoneal macrophages were similar, although high doses of these compounds did not result in total clearance of parasites in lesional cells (amphotericin B), peritoneal cells (miltefosine) and both cell cultures (pentavalent antimonial). Amastigotes infecting lesional macrophages were more resistant to resveratrol as compared to parasites in peritoneal macrophages. The cytoxicity of miltefosine and resveratrol was higher in infected peritoneal macrophages than in lesional cells. These data suggest that the antileishmanial effect and citotoxicity of some anti leishmanial compounds are dependent of macrophage source and mouse peritoneal macrophages loaded with amastigotes do not represent the lesion cell.


Assuntos
Antiprotozoários/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Macrófagos/parasitologia , Anfotericina B/farmacologia , Animais , Técnicas de Cultura de Células , Feminino , Concentração Inibidora 50 , Leishmaniose/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia
6.
Vet Parasitol ; 268: 16-20, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30981301

RESUMO

Neospora caninum is an intracellular parasite that causes neosporosis in cattle. Bovine neosporosis is considered a major cause of bovine abortion worldwide. Rapid replication of N. caninum tachyzoites within host cells is responsible for the acute phase of N. caninum infection. Evidence shows that the host immune response plays an essential role in recognizing and regulating the replication of invading pathogens. Nucleotide-binding oligomerization domain receptors (NLRs) are a class of cytoplasmic sensors that can sense pathogens and induce the formation of the inflammasome complex. Activation of the inflammasome promotes restriction of microbial replication. Our previous study revealed NLRP3 inflammasome activation in N. caninum-infected murine macrophages. However, the role of inflammasome activity in N. caninum-infected bovine cells is unknown. To address this question, a bovine peritoneal macrophage cell line was used to investigate the role of inflammasome activation in regulating intracellular N. caninum replication. The results showed that inflammasome mediated activation of caspase-1 occurs in N. caninum-infected bovine macrophages, and caspase-1-dependent cell death was considered to be induced in N. caninum-infected bovine macrophages because N. caninum induced cell death decreased following pretreatment with zVAD-fmk and VX765. Meanwhile, the inhibition of caspase-1 in N. caninum-infected bovine macrophages led to the presence of more parasites in the parasitophorous vacuole. In contrast, inflammasome activation induced by ATP treatment in N. caninum-infected bovine macrophages contributed to the clearance of N. caninum. In addition, pyroptotic cell supernatant collected from ATP-stimulated bovine macrophages also impaired the ability of this parasite to infect new cells. In conclusion, this study is the first report on the role of the bovine inflammasome in restraining intracellular N. caninum replication and suggests that the bovine inflammasome may be a potential target for future development of drugs or vaccines against N. caninum infection in cattle.


Assuntos
Doenças dos Bovinos/imunologia , Coccidiose/imunologia , Inflamassomos/imunologia , Macrófagos Peritoneais/parasitologia , Trifosfato de Adenosina/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Caspase 1/metabolismo , Bovinos , Proliferação de Células , Células Cultivadas , Citoplasma/parasitologia , Dipeptídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Neospora , para-Aminobenzoatos/farmacologia
7.
Exp Parasitol ; 199: 30-37, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30817917

RESUMO

The treatment against leishmaniasis presents problems, since the currently used drugs are toxic and/or have high costs. In addition, parasite resistance has increased. As a consequence, in this study, a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine or GF1059, was in vitro and in vivo tested against Leishmania parasites. Experiments were performed to evaluate in vitro antileishmanial activity and cytotoxicity, as well as the treatment of infected macrophages and the inhibition of infection using pre-treated parasites. This study also investigated the GF1059 mechanism of action in L. amazonensis. Results showed that the compound was highly effective against L. infantum and L. amazonensis, presenting a selectivity index of 154.6 and 86.4, respectively, against promastigotes and of 137.6 and 74.3, respectively, against amastigotes. GF1059 was also effective in the treatment of infected macrophages and inhibited the infection of these cells when parasites were pre-incubated with it. The molecule also induced changes in the parasites' mitochondrial membrane potential and cell integrity, and caused an increase in the reactive oxygen species production in L. amazonensis. Experiments performed in BALB/c mice, which had been previously infected with L. amazonensis promastigotes, and thus treated with GF1059, showed that these animals presented significant reductions in the parasite load when the infected tissue, spleen, liver, and draining lymph node were evaluated. GF1059-treated mice presented both lower parasitism and low levels of enzymatic markers, as compared to those receiving amphotericin B, which was used as control. In conclusion, data suggested that GF1059 can be considered a possible therapeutic target to be tested against leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Cloroquinolinóis/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Anfotericina B/toxicidade , Animais , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Cloroquinolinóis/uso terapêutico , Cloroquinolinóis/toxicidade , Modelos Animais de Doenças , Eritrócitos/efeitos dos fármacos , Feminino , Concentração Inibidora 50 , Leishmania infantum/crescimento & desenvolvimento , Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Visceral/tratamento farmacológico , Fígado/parasitologia , Linfonodos/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Espécies Reativas de Oxigênio/metabolismo , Baço/parasitologia
8.
PLoS One ; 14(2): e0210737, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30721235

RESUMO

The arsenic contamination of ground water in visceral leishmaniasis (VL) endemic areas in Bihar, India leads to human exposure through drinking water. Possibly, the consumed arsenic (As) accumulates in the tissues of VL patients, who subsequently internalize intracellular amastigotes to confer resistance against chemotherapy to the parasite, leading to modulation in the host's immune response. This hypothesis appears to be consistent with the in vitro findings that in arsenic-exposed parasites, the mitochondrial membrane potential became depolarized, whereas the reduced thiol and lactate production was overexpressed with enhanced glucose consumption; therefore, the reduced thiol possibly supports an immunosuppressive state in the host cells. This observation was well supported by the down-regulated expression of pro-inflammatory cytokines (IL-2, IL-12, IFN-γ, and TNF-α) with a suppressed anti-leishmanial function of macrophage (NO, ROS). In contrast, the pathophysiological mechanism of VL has received ample support by the promotion of Th2 cytokines (IL-4 and IL-10) in the presence of arsenic-exposed Leishmania parasites (LdAS). Dysfunction of mitochondria and the overexpression of lactate production raise the possibility of the Warburg effect being operative through the up-regulation of glucose consumption by parasites to enhance the energy production, possibly augmenting virulence. Therefore, we surmise from our data that arsenic exposure to Leishmania donovani modulates the immune response and infection pattern by impairing parasite function, which may affect the anti-leishmanial effect in VL.


Assuntos
Arsênico/farmacologia , Leishmania donovani/imunologia , Leishmaniose Visceral , Macrófagos Peritoneais , Animais , Citocinas/imunologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Macrófagos Peritoneais/patologia , Camundongos , Óxido Nítrico/imunologia , Espécies Reativas de Oxigênio/imunologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-30805314

RESUMO

Host- as well as parasite-specific factors are equally crucial in allowing either the Leishmania parasites to dominate, or host macrophages to resist infection. To identify such factors, we infected murine peritoneal macrophages with either the virulent (vAG83) or the non-virulent (nvAG83) parasites of L. donovani. Then, through dual RNA-seq, we simultaneously elucidated the transcriptomic changes occurring both in the host and the parasites. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed (DE) genes, we showed that the vAG83-infected macrophages exhibit biased anti-inflammatory responses compared to the macrophages infected with the nvAG83. Moreover, the vAG83-infected macrophages displayed suppression of many important cellular processes, including protein synthesis. Further, through protein-protein interaction study, we showed significant downregulation in the expression of many hubs and hub-bottleneck genes in macrophages infected with vAG83 as compared to nvAG83. Cell signaling study showed that these two parasites activated the MAPK and PI3K-AKT signaling pathways differentially in the host cells. Through gene ontology analyses of the parasite-specific genes, we discovered that the genes for virulent factors and parasite survival were significantly upregulated in the intracellular amastigotes of vAG83. In contrast, genes involved in the immune stimulations, and those involved in negative regulation of the cell cycle and transcriptional regulation, were upregulated in the nvAG83. Collectively, these results depicted a differential regulation in the host and the parasite-specific molecules during in vitro persistence and clearance of the parasites.


Assuntos
Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Análise de Sequência de RNA , Animais , Células Cultivadas , Biologia Computacional , Camundongos , Anotação de Sequência Molecular
10.
Phytomedicine ; 57: 262-270, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30802712

RESUMO

BACKGROUND: Leishmaniasis is a disease caused by protozoan parasites of the Leishmania genus whose current treatment has high cost, highly toxic, and difficult administration, which makes it very important to find alternative natural compounds of high efficiency and low cost. PURPOSE: This study assessed the in vitro effect of caffeic acid (CA) on promastigotes and L. amazonensis-infected macrophages. METHODS: Evaluation of the in vitro leishmanicidal activity of CA against promastigotes and L. amazonensis infected peritoneal macrophages, as well its microbicide mechanisms. RESULTS: CA 12.5-100 µg/ml were able to inhibit promastigotes proliferation at all tested periods. The IC50, 12.5 µg/ml, also altered promastigote cell morphology and cell volume accompanied by loss of mitochondrial integrity, increase in reactive oxygen species (ROS) production, phosphatidylserine exposure, and loss of plasma membrane integrity - characterizing the apoptosis-like process. Moreover, CA reduced the percentage of infected macrophages and the number of amastigotes per macrophages increasing TNF-α, ROS, NO and reducing IL-10 levels as well as iron availability. CONCLUSION: CA showed in vitro antipromastigote and antiamostigote by increasing oxidant and inflammatory response important to eliminate the parasite.


Assuntos
Antiprotozoários/farmacologia , Ácidos Cafeicos/farmacologia , Leishmania/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Ferro/metabolismo , Leishmania/patogenicidade , Leishmania/fisiologia , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
Int Immunopharmacol ; 69: 178-183, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30716588

RESUMO

Glucose-6-phosphate dehydrogenase (G6PDH) ultimately plays a critical role in macrophage functions used against infectious agents. The present study investigated whether changes in G6PDH activity could influence the resistance of infected macrophages against Leishmania major infection. Mouse peritoneal and J774 macrophages were infected, respectively, ex vivo and in vitro, with L. major and then exposed to an inhibitor (6-aminonicotinamide) or activator (LPS + melatonin) of G6PDH activity for 24 h. Cell viability [using MTT assay] was measured to assess any direct toxicity from the doses of inhibitor/activator used for the macrophage treatments. Nitric oxide (NO) produced by the cells and released into culture supernatants was measured (Griess method) and cell G6PDH activity was also determined. Moreover, the number of amastigotes form Leishmania in macrophages that developed over a 7-d period was evaluated. The results showed that an increase in G6PDH activity after treatment of both types of macrophages with a combination of LPS + melatonin caused significant increases in NO production and cell resistance against L. major amastigote formation/survival. However, exposure to 6-aminonicotinamide led to remarkable suppression of G6PDH activity and NO production, events that were associated with a deterioration in cell resistance against (and an increase in cell levels of) the parasites. The results suggested that activation or suppression of G6PDH activity could affect leishmanicidal function of both mouse peritoneal and J774 macrophages. Thus, regulation of macrophages via modulation of G6PDH activity appears to provide a novel window for those seeking to develop alternative therapies for the treatment of leishmaniasis.


Assuntos
Glucosefosfato Desidrogenase/metabolismo , Leishmania major/fisiologia , Macrófagos Peritoneais/fisiologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Imunomodulação , Leishmaniose Cutânea , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Oxirredução
12.
Artigo em Inglês | MEDLINE | ID: mdl-30478164

RESUMO

Conventional chemotherapy against leishmaniasis includes agents exhibiting considerable toxicity. In addition, reports of drug resistance are not uncommon. Thus, safe and effective therapies are urgently needed. Isoselenocyanate compounds have recently been identified with potential antitumor activity. It is well known that some antitumor agents demonstrate effects against Leishmania In this study, the in vitro leishmanicidal activities of several organo-selenium and organo-sulfur compounds were tested against Leishmania major and Leishmania amazonensis parasites, using promastigotes and intracellular amastigote forms. The cytotoxicity of these agents was measured in murine peritoneal macrophages and their selectivity indexes were calculated. One of the tested compounds, the isoselenocyanate derivative NISC-6, showed selectivity indexes 2- and 10-fold higher than those of the reference drug amphotericin B when evaluated in L. amazonensis and L. major, respectively. The American strain (L. amazonensis) was less sensitive to NISC-6 than L. major, showing a trend similar to that observed previously for amphotericin B. In addition, we also observed that NISC-6 significantly reduced the number of amastigotes per infected macrophage. On the other hand, we showed that NISC-6 decreases expression levels of Leishmania genes involved in the cell cycle, such as topoisomerase-2 (TOP-2), PCNA, and MCM4, therefore contributing to its leishmanicidal activity. The effect of this compound on cell cycle progression was confirmed by flow cytometry. We observed a significant increase of cells in the G1 phase and a dramatic reduction of cells in the S phase compared to untreated cells. Altogether, our data suggest that the isoselenocyanate NISC-6 may be a promising candidate for new drug development against leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Leishmania major/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Compostos Organosselênicos/farmacologia , Compostos de Enxofre/farmacologia , Anfotericina B/farmacologia , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Leishmaniose Cutânea/parasitologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C
13.
Artigo em Inglês | MEDLINE | ID: mdl-30297370

RESUMO

Drug repurposing affords the implementation of new treatments at a moderate cost and under a faster time-scale. Most of the clinical drugs against Leishmania share this origin. The antidepressant sertraline has been successfully assayed in a murine model of visceral leishmaniasis. Nevertheless, sertraline targets in Leishmania were poorly defined. In order to get a detailed insight into the leishmanicidal mechanism of sertraline on Leishmania infantum, unbiased multiplatform metabolomics and transmission electron microscopy were combined with a focused insight into the sertraline effects on the bioenergetics metabolism of the parasite. Sertraline induced respiration uncoupling, a significant decrease of intracellular ATP level, and oxidative stress in L. infantum promastigotes. Metabolomics evidenced an extended metabolic disarray caused by sertraline. This encompasses a remarkable variation of the levels of thiol-redox and polyamine biosynthetic intermediates, as well as a shortage of intracellular amino acids used as metabolic fuel by Leishmania Sertraline killed Leishmania through a multitarget mechanism of action, tackling essential metabolic pathways of the parasite. As such, sertraline is a valuable candidate for visceral leishmaniasis treatment under a drug repurposing strategy.


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/metabolismo , Sertralina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antidepressivos/farmacologia , Membrana Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos
14.
Artigo em Inglês | MEDLINE | ID: mdl-30214897

RESUMO

The current long-term treatment for leishmaniasis causes severe side effects and resistance in some cases. An evaluation of the anti-leishmanial potential of an HSP90-inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), demonstrated its potent effect against Leishmania spp. in vitro and in vivo. We have previously shown that 17-AAG can kill L. (L) amazonensis promastigotes with an IC50 of 65 nM and intracellular amastigote at concentrations as low as 125 nM. As this compound presents low solubility and high toxicity in human clinical trials, we prepared an inclusion complex containing hydroxypropyl-ß-cyclodextrin and 17-AAG (17-AAG:HPßCD) to improve its solubility. This complex was characterized by scanning electron microscopy, and X-ray diffraction. Liposomes-containing 17-AAG:HPßCD was prepared and evaluated for encapsulation efficiency (EE%), particle size, polydispersity index (PDI), pH, and zeta potential, before and after accelerated and long-term stability testing. An evaluation of leishmanicidal activity against promastigotes and intracellular amastigotes of L. (L) amazonensis was also performed. The characterization techniques utilized confirmed the formation of the inclusion complex, HPßCD:17-AAG, with a resulting 33-fold-enhancement in compound water solubility. Stability studies revealed that 17-AAG:HPßCD-loaded liposomes were smaller than 200 nm, with 99% EE. Stability testing detected no alterations in PDI that was 0.295, pH 7.63, and zeta potential +22.6, suggesting liposome stability, and suitability for evaluating leishmanicidal activity. Treatment of infected macrophages with 0.006 nM of 17-AAG:HPßCD or 17-AAG:HPßCD-loaded liposomes resulted in almost complete amastigote clearance inside macrophages after 48 h. This reduction is similar to the one observed in infected macrophages treated with 2 µM amphotericin B. Our results showed that nanotechnology and drug delivery systems could be used to increase the antileishmanial efficacy and potency of 17-AAG in vitro, while also resulting in reduced toxicity that indicates these formulations may represent a potential therapeutic strategy against leishmaniasis.


Assuntos
Benzoquinonas/farmacologia , Portadores de Fármacos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Leishmania mexicana/efeitos dos fármacos , Lipossomos/metabolismo , Macrófagos Peritoneais/parasitologia , Animais , Benzoquinonas/química , Células Cultivadas , Portadores de Fármacos/química , Composição de Medicamentos , Estabilidade de Medicamentos , Inibidores Enzimáticos/química , Lactamas Macrocíclicas/química , Lipossomos/química , Camundongos Endogâmicos CBA , Viabilidade Microbiana/efeitos dos fármacos , Solubilidade , Índice Terapêutico
15.
Phytomedicine ; 46: 11-20, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30097110

RESUMO

BACKGROUND: American tegumentary leishmaniasis (ATL) is a zoonotic disease caused by protozoa of the genus Leishmania. The high toxicity, high costs and resistance of some strains to current drugs has prompted the search for therapeutic alternatives for the management of this disease. Sphagneticola trilobata is a plant that has diterpenes as main constituents, including grandiflorenic acid (GFA) that has antiinflammatory, antiprotozoal, antibacterial and antinociceptive activity. PURPOSE: The aim of our study was to determine the effect of GFA on both the promastigotes and the amastigotes of Leishmania amazonensis. METHODS: Isolation by chromatographic methods and chemical identification of GFA, then evaluation of the in vitro leishmanicidal activity of this compound against Leishmania amazonensis promastigotes and L. amazonensis infected peritoneal Balb/c macrophages, as well its action and microbicide mechanisms. RESULTS: GFA treatment significantly inhibited the proliferation of promastigotes. This antiproliferative effect was accompanied by morphological changes in the parasite with 25 nM GFA. Afterwards, we investigated the mechanisms involved in the death of the protozoan; there was an increase in the production of reactive oxygen species (ROS), phosphatidylserine exposure, permeabilization of the plasma membrane and decreased mitochondrial depolarization. In addition, we observed that the treatment caused a reduction in the percentage of infected cells and the number of amastigotes per macrophage, without showing cytotoxicity in low doses to peritoneal macrophages and sheep erythrocytes. GFA increased IL-10 and total iron bound to transferrin in infected macrophages. Our results showed that GFA treatment acts on promastigote forms through an apoptosis-like mechanism and on intracellular amastigote forms, dependent of regulatory cytokine IL-10 modulation with increase in total iron bound to transferrin. CONCLUSION: GFA showed in vitro antileishmanial activity on L. amazonensis promastigotes forms and on L. amazonensis-infected macrophages.


Assuntos
Antiprotozoários/farmacologia , Diterpenos/farmacologia , Leishmania/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Interleucina-10/metabolismo , Ferro/metabolismo , Leishmaniose Cutânea/tratamento farmacológico , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Ovinos
16.
Biomed Pharmacother ; 106: 1082-1090, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30119174

RESUMO

The search for new drugs for the treatment of leishmaniasis is an important strategy for improving the current therapeutic arsenal for the disease. There are several limitations to the available drugs including high toxicity, low efficacy, prolonged parenteral administration, and high costs. Steroids are a diverse group of compounds with various applications in pharmacology. However, the antileishmanial activity of this class of molecules has not yet been explored. Therefore, in the present study, we investigated the antileishmanial activity and cytotoxicity of novel steroids against murine macrophages with a focus on the derivatives of cholesterol (CD), cholic acid (CA), and deoxycholic acid (DA). Furthermore, the mechanism of action of the best compound was assessed, and in silico studies to evaluate the physicochemical and pharmacokinetic properties were also conducted. Among the sixteen derivatives, schiffbase2, CD2 and deoxycholic acid derivatives (DOCADs) were effective against promastigotes of Leishmania species. Despite their low toxicity to macrophages, the majority of DOCADs were active against intracellular amastigotes of L. amazonensis, and DOCAD5 exhibited the best biological effect against these parasitic stages (IC50 = 15.34 µM). Neither the CA derivatives (CAD) nor DA alone inhibited the intracellular parasites. Thus, the absence of hydroxyl in the C-7 position of the steroid nucleus, as well as the modification of the acid group in DOCADs were considered important for antileishmanial activity. The treatment of L. amazonensis promastigote forms with DOCAD5 induced biochemical changes such as depolarization of the mitochondrial membrane potential, increased ROS production and cell cycle arrest. No alterations in parasite plasma membrane integrity were observed. In silico physicochemical and pharmacokinetic studies suggest that DOCAD5 could be a good candidate for an oral drug. The data demonstrate the potential antileishmanial effect of certain steroid derivatives and encourage new in vivo studies.


Assuntos
Colesterol/farmacologia , Ácido Desoxicólico/farmacologia , Descoberta de Drogas/métodos , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Macrófagos Peritoneais/efeitos dos fármacos , Tripanossomicidas/farmacologia , Administração Oral , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Colesterol/análogos & derivados , Colesterol/síntese química , Colesterol/farmacocinética , Ácido Cólico/síntese química , Ácido Cólico/farmacocinética , Ácido Cólico/farmacologia , Ácido Desoxicólico/análogos & derivados , Ácido Desoxicólico/síntese química , Ácido Desoxicólico/farmacocinética , Relação Dose-Resposta a Droga , Leishmania/crescimento & desenvolvimento , Leishmania/metabolismo , Leishmaniose/parasitologia , Macrófagos Peritoneais/parasitologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/farmacocinética
17.
Exp Parasitol ; 192: 12-18, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30026113

RESUMO

Cutaneous leishmaniasis is the most common form of leishmaniasis and the available chemotherapy causes serious side effects, justifying the search for new therapies. This study investigated the antileishmanial activity of bovine serum albumin (BSA) nanoparticles containing amphotericin B (AmB) against Leishmania amazonensis. The antiproliferative activity against promastigotes and amastigotes was assessed and the cytotoxicity was determined and compared to commercial AmB-deoxycholate (AmB-D). In vivo antileishmania activity was evaluated in murine cutaneous leishmaniasis model. BSA nanoparticles showed spherical shape, mean size about 180 nm, zeta potential of ≈ -45 mV and AmB encapsulation efficiency >95%. AmB-D was effective in promastigote and amastigote forms, while AmB-loaded BSA nanoparticles were more effective against amastigotes than promastigotes. AmB-D was more effective than AmB-loaded BSA nanoparticles in both forms, however, the lowest cytotoxicity against macrophages was achieved by AmB-nanoparticles. BALB/c mice treated with AmB-D or AmB-loaded BSA nanoparticles showed a significant decrease in the lesion thickness at the infected footpad. Histopathological analysis after 3 weeks of treatment revealed AmB-D-related toxicity in heart, spleen, lung, liver and kidneys, while treatment with AmB-loaded BSA nanoparticles did not reveal tissue toxicity. The antileishmanial efficacy and the reduced toxicity become BSA nanoparticles containing AmB a potential candidate for treating cutaneous leishmaniasis.


Assuntos
Anfotericina B/administração & dosagem , Antiprotozoários/administração & dosagem , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Nanopartículas/uso terapêutico , Soroalbumina Bovina , Análise de Variância , Animais , Linhagem Celular , Portadores de Fármacos , Concentração Inibidora 50 , Rim/patologia , Fígado/patologia , Pulmão/patologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Miocárdio/patologia , Nanopartículas/ultraestrutura , Tamanho da Partícula , Baço/patologia
18.
J Mater Sci Mater Med ; 29(8): 125, 2018 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-30056571

RESUMO

Amphotericin B (A) as an antileishmanial drug has limited clinical application owing to severe side-effects and low-water solubility. This is the first study reported using Anionic Linear Globular Dendrimer (ALGD) as A carrier for the increase of A solubility rate, decrease its toxicity, and improve its therapeutic effects. ALGD was synthesized and A was loaded into nanoparticles for the first time with the drug-loading efficiency of 82%. Drug loading was confirmed using characterization methods. The drug solubility rate was increased by 478-folds. The results of the study showed that the A toxicity was significantly decreased by 95% in vitro and in vivo environments, which was confirmed by pathology findings and enzymatic evaluation. Furthermore, the nanodrug caused that mortality rate was reached to zero. Moreover, the nanodrug was as potent as the free drug and glucantime (GUL) in reducing the parasite burden and parasite number. These findings indicated the potency of ALGD to decrease the drug side-effects, increase the drug solubility rate, and improve the drug efficacy. Moreover, the nanoformulation was a non-toxic and cost-effective formulation. The conformity between in vitro and in vivo results suggested that the A-loaded ALGD could be considered as a promising candidate in reducing the side-effects of A in leishmaniasis treatment.


Assuntos
Anfotericina B/farmacologia , Sistemas de Liberação de Medicamentos , Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Nanoestruturas , Anfotericina B/administração & dosagem , Anfotericina B/efeitos adversos , Anfotericina B/química , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dendrímeros , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C
19.
Biomed Pharmacother ; 103: 1294-1301, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29864911

RESUMO

Activity, mechanisms of action, and toxicity of natural compounds have been investigated in a context in which knowledge on which pathway is activated remains crucial to understand the action mechanism of these bioactive substances when treating an infected host. Herein, we showed an ability of copaiba oil and kaurenoic acid to eliminate Trypanosoma cruzi forms by infected macrophages through other mechanisms in addition to nitric oxide, reactive oxygen species, iron metabolism, and antioxidant defense. Both compounds induced an anti-inflammatory response with an increase in IL-10 and TGF-ß as well as a decrease in IL-12 production. Despite being able to modulate the immune response in host cells, the antimicrobial activity of copaiba oil and kaurenoic acid seems to be a direct action of the compounds on the parasites, causing their death.


Assuntos
Antiprotozoários/farmacologia , Diterpenos/farmacologia , Fabaceae/química , Macrófagos Peritoneais/metabolismo , Óleos Voláteis/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Ferritinas/genética , Ferritinas/metabolismo , Células HeLa , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos Endogâmicos BALB C , Modelos Biológicos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
20.
Front Immunol ; 9: 1222, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29910815

RESUMO

Toxoplasma gondii infection evokes a strong Th1-type response with interleukin (IL)-12 and interferon (IFN)-γ secretion. Recent studies suggest that the infection of pregnant mice with T. gondii may lead to adverse pregnancy results caused by subversion of physiological immune tolerance at maternofetal interface rather than direct invasion of the parasite. Genotype-associated dense granule protein GRA15II tends to induce classically activated macrophage (M1) differentiation and subsequently activating NK, Th1, and Th17 cells whereas rhoptry protein ROP16I/III drives macrophages to alternatively activated macrophage (M2) polarization and elicits Th2 immune response. Unlike the archetypal strains of types I, II, and III, type Chinese 1 strains possess both GRA15II and ROP16I/III, suggesting a distinct pathogenesis of Toxoplasma-involved adverse pregnancies. We constructed T. gondii type Chinese 1 strain of WH3Δrop16 based on CRISPR/Cas9 technology to explore the ROP16I/III-deficient/GRA15II-dominant parasites in induction of trophoblast apoptosis in vitro and abnormal pregnant outcomes of mice in vivo. Our study showed that Toxoplasma WH3Δrop16 remarkably induced apoptosis of trophoblasts. C57BL/6 pregnant mice injected with the tachyzoites of WH3Δrop16 presented increased absorptivity of fetuses in comparison with the mice infected with WH3 wild type (WH3 WT) parasites although no remarkable difference of virulence to mice was seen between the two strains. Additionally, the mice inoculated with WH3Δrop16 tachyzoites exhibited a notable expression of both IL-17A and IFN-γ, while the percentage of CD4+CD25+FoxP3 [T regulatory cells (Tregs)] were diminished in splenocytes and placenta tissues compared to those infected with WH3 WT parasites. Accordingly, expressions of IL-4, IL-10, and transforming growth factor beta 1, the pivotal cytokines of Th2 and Tregs response, were significantly dampened whereas IFN-γ and IL-12 expressions were upregulated in WH3Δrop16-infected mice, which gave rise to more prominent outcomes of abnormal pregnancies. Our results indicated that the WH3Δrop16 parasites with gra15II background of T. gondii type Chinese 1 strains may cause miscarriage and stillbirth due to subversion of the maternal immune tolerance and system immunity of the animals and the GRA15II effector contributes to the process of adverse pregnant consequences.


Assuntos
Patrimônio Genético , Predisposição Genética para Doença , Complicações Infecciosas na Gravidez/parasitologia , Resultado da Gravidez , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasmose Animal/parasitologia , Animais , Apoptose , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Feminino , Marcação de Genes , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos , Placenta , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Baço/imunologia , Baço/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Toxoplasmose Animal/imunologia , Trofoblastos/metabolismo
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