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1.
Eur J Pharm Sci ; 163: 105859, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33894283

RESUMO

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania (L.) amazonensis promastigotes, human erythrocytes and J774.A1 murine macrophages, in comparison with reported and novel data for miltefosine (MIL). One of the objectives of this work is to look for the relationships between the activities of these two drugs in the Leishmania parasite with their changes in the cell membrane. A spin-labeled stearic acid inserted into the cell membranes showed strong interactions with putative AmB/sterol complexes, characterized by reductions in molecular dynamics. The concentration of the drugs in the plasma membrane that reduced the cell population by 50%, and the membrane-water partition coefficient of the drugs, were assessed. These biophysical parameters enabled estimates of possible therapeutic concentrations of these two drugs in the interstitial fluids of the tissues to be made. AmB displayed higher affinity for the plasma membrane of L. amazonensis than for that of the macrophage and erythrocyte, denoting a preference for a membrane that contains ergosterol. AmB also demonstrated higher hemolytic potential than MIL for measurements on erythrocytes in both PBS and whole blood. For MIL, the EPR technique detected membrane changes induced by the drug in the same concentration range that inhibited the growth of parasites, but in the case of AmB, an 8-fold higher concentration of the IC50 was necessary to observe a reduction in membrane fluidity, suggesting a better localized effect of AmB on the membrane. Taken together, the results demonstrate that the antiproliferative and cytotoxic effects of both drugs are associated with changes in cell membranes.

2.
J Phys Chem B ; 124(45): 10157-10165, 2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33119317

RESUMO

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania amazonensis promastigotes, erythrocytes, and J774 macrophages. Spin labels embedded into the cell membranes detected strong interactions with putative AmB/sterol complexes that resulted in pronounced changes in the EPR spectra, which can be interpreted as a reduction in membrane fluidity or an increase in the polarity assessed by the spin probe. The EPR spectra of spin-labeled lipids corroborated the findings that AmB does not enter phospholipid membrane-sterol models and probably forms extramembranous aggregates, as predicted by the sterol sponge model. Furthermore, these aggregates were shown to extract the spin probe androstanol from the lipid bilayer. However, in contrast to the results for the model membrane, EPR spectroscopy suggested that AmB easily enters the membranes of the studied cells, implying that the entry process is dependent on interactions with the membrane proteins.

3.
Eur J Pharm Sci ; 151: 105407, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504805

RESUMO

A novel chalcone derivative, LQFM064, demonstrated antileishmanial activity against Leishmania (L.) amazonensis, with an IC50 value of ~10 µM for the promastigote form. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled stearic acid incorporated in the plasma membrane of L. amazonensis promastigotes revealed that after 2 h of treatment with LQFM064, the parasite showed remarkable reductions in membrane fluidity. The features of the altered EPR spectra were similar to those reported for the erythrocyte membrane, which was suggested to be due to the cross-linking of oxidized hemoglobin with the cytoskeleton spectrin. In comparison to miltefosine (MIL), LQFM064 demonstrated a much lower hemolytic potential against both erythrocytes in PBS and whole blood, less cytotoxicity in J774.A1 macrophages and equivalent ability to kill parasites internalized in J774.A1 macrophages. Measurements of the IC50 values for assays with different cell concentrations enabled the estimation of the membrane-water partition coefficient (KM/W), as well as the concentrations of LQFM064 in membrane (cm50) and aqueous phase (cw50) that reduces the cell population by 50%. From the KM/W and cm50 values it was deduced that LQFM064 has a greater affinity than MIL for the parasite membrane, but the antiproliferative activity of both substances is exerted at a similar concentration in the plasma membrane.

4.
PLoS Negl Trop Dis ; 14(2): e0008029, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32023240

RESUMO

Interleukin-32 is a novel inflammatory mediator that has been described to be important in the immunopathogenesis and control of infections caused by Leishmania parasites. By performing experiments with primary human cells in vitro, we demonstrate that the expression of IL-32 isoforms is dependent on the time exposed to L. amazonensis and L. braziliensis antigens. Moreover, for the first time we show the functional consequences of three different genetic variations in the IL32 (rs4786370, rs4349147, rs1555001) modulating IL-32γ expression, influencing innate and adaptive cytokine production after Leishmania exposure. Using a Brazilian cohort of 107 American Tegumentary Leishmaniasis patients and a control cohort of 245 healthy individuals, the IL32 rs4786370 genetic variant was associated with protection against ATL, whereas the IL32 rs4349147 was associated with susceptibility to the development of localized cutaneous and mucosal leishmaniasis. These novel insights may help improve therapeutic strategies and lead to benefits for patients suffering from Leishmania infections.


Assuntos
Predisposição Genética para Doença , Variação Genética , Interleucinas/genética , Leishmania/classificação , Leishmaniose Cutânea/genética , Adulto , Idoso , Brasil/epidemiologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Isoformas de Proteínas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
5.
Colloids Surf B Biointerfaces ; 183: 110421, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31401463

RESUMO

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


Assuntos
Antiprotozoários/farmacologia , Cetrimônio/farmacologia , Citotoxinas/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Antiprotozoários/química , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cetrimônio/química , Citotoxinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Concentração Inibidora 50 , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Simulação de Dinâmica Molecular , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Compostos de Amônio Quaternário/química , Dodecilsulfato de Sódio/química , Marcadores de Spin , Ácidos Esteáricos/química , Tensoativos/química
6.
Biochim Biophys Acta Biomembr ; 1861(6): 1049-1056, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30890467

RESUMO

The sesquiterpene nerolidol is a membrane-active compound that has demonstrated antitumor, antibacterial, antifungal and antiparasitic activities. In this study, we used electron paramagnetic resonance (EPR) spectroscopy and biophysical parameters determined via cell culture assays to study the mechanisms underlying the in vitro antileishmanial activity of nerolidol. The EPR spectra of a spin-labeled stearic acid indicated notable interactions of nerolidol with the cell membrane of Leishmania amazonensis amastigotes. The nerolidol IC50 values in L. amazonensis amastigotes and promastigotes were found to depend on the cell concentration used in the assay. This dependence was described by an equation that considers various cell suspension parameters, such as the 50% inhibitory concentrations of nerolidol in the cell membrane (cm50) and the aqueous phase (cw50) and the membrane-water partition coefficient of nerolidol (KM/W). Via cytotoxicity (CC50) and hemolytic potential (HC50) data, these parameters were also determined for nerolidol in macrophages and erythrocytes. With a cw50 of 125 µM, macrophages were less sensitive to nerolidol than amastigotes and promastigotes, which had mean cw50 values of 56 and 74 µM, respectively. The estimated cm50 values of nerolidol for amastigotes and promastigotes and macrophages were between 2.6 and 3.0 M, indicating substantial accumulation of nerolidol in the cell membrane. In addition, the spin-label EPR data indicated that membrane dynamic changes occurred in L. amazonensis amastigotes at concentrations similar to the nerolidol IC50 value.


Assuntos
Antineoplásicos/farmacologia , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , Sesquiterpenos/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Hemólise/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C
7.
Parasite Immunol ; 41(5): e12623, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30870579

RESUMO

Phenotypic and functional aspects of monocytes from Localized Cutaneous Leishmaniasis (LCL) patients were evaluated. The frequencies of monocyte subsets and TLR2/TLR4 expression were evaluated in fresh peripheral blood whereas cytokine production was evaluated in whole blood cell cultures stimulated with TLR agonists or Leishmania braziliensis antigen (Ag). CD16+ monocytes frequency was increased in patients compared with controls. A TLR4 agonist (LPS) induced expression of TNF and IL-10 in monocyte subsets of patients and controls. The CD14+ CD16+ monocytes expressed higher levels of these cytokines than CD14+ CD16- cells. The levels of secreted TNF were higher in whole blood cell cultures from patients than controls after LPS/TLR4 or Ag stimulation. Whereas in controls there was a positive correlation between TNF and IL-10 levels, this was not observed in stimulated cell cultures from patients. The high levels of LPS-induced TNF were associated with the number of lesions and the percentages of CD14hi CD16+ monocytes. The levels of TLR2-induced TNF were also associated with number of lesions. All monocyte subsets from patients expressed higher levels of TLR2 and TLR4 than controls. Data suggest that systemically activated monocytes contribute for an imbalance in pro- and anti-inflammatory cytokine production during LCL, participating in the immunopathogenesis of the disease.


Assuntos
Leishmaniose Cutânea/imunologia , Adulto , Idoso , Citocinas/imunologia , Feminino , Humanos , Interleucina-10/imunologia , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Adulto Jovem
8.
Am J Trop Med Hyg ; 99(5): 1165-1169, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30203744

RESUMO

The disseminated form of leishmaniasis is a serious and rare disease, being diagnosed in 2% of the cutaneous cases registered per year in Brazil. The main characteristic is the appearance of multiple pleomorphic lesions on the cutaneous surface. A 68-year-old male from the rural area of Tocantins, Brazil, presented atypical disseminated cutaneous leishmaniasis (ACL). The clinical course and histopathological and immunological findings presented a mixed pattern that hindered diagnosis and therapeutic management. Molecular typing revealed a mixed infection with Leishmania (V.) guyanensis and Leishmania (L.) amazonensis. Molecular identification of the agents responsible for ACL is important for adequate therapeutic planning, minimizing the possibility of sequellae that impact the quality of life of the patient.


Assuntos
Coinfecção/diagnóstico , Coinfecção/parasitologia , Leishmania guyanensis/genética , Leishmania mexicana/genética , Leishmaniose Cutânea/diagnóstico , Idoso , Anfotericina B/uso terapêutico , Antiprotozoários/uso terapêutico , Brasil , DNA de Protozoário/genética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Leishmania guyanensis/isolamento & purificação , Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Masculino , Tipagem Molecular , População Rural , Pele/parasitologia , Pele/patologia
9.
Free Radic Biol Med ; 129: 35-45, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30196081

RESUMO

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


Assuntos
Antiprotozoários/farmacologia , Peróxido de Hidrogênio/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Estágios do Ciclo de Vida/efeitos dos fármacos , Óxido Nítrico/farmacologia , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Meios de Cultura/química , Feminino , Interações Hospedeiro-Parasita , Humanos , Imunidade Inata , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/isolamento & purificação , Leishmania braziliensis/metabolismo , Leishmaniose Tegumentar Difusa/imunologia , Leishmaniose Tegumentar Difusa/metabolismo , Leishmaniose Tegumentar Difusa/parasitologia , Leishmaniose Mucocutânea/imunologia , Leishmaniose Mucocutânea/metabolismo , Leishmaniose Mucocutânea/parasitologia , Estágios do Ciclo de Vida/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
10.
Infect Immun ; 86(5)2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29483288

RESUMO

Visceral leishmaniasis (VL) is a chronic parasitic disease caused by Leishmania infantum in the Americas. During VL, several proinflammatory cytokines are produced in spleen, liver, and bone marrow. However, the role of interleukin-32 (IL-32) has not been explored in this disease. IL-32 can induce production of proinflammatory cytokines in innate immune cells and polarize the adaptive immune response. Herein, we discovered that L. infantum antigens induced expression of mRNA mainly for the IL-32γ isoform but also induced low levels of the IL-32ß transcript in human peripheral blood mononuclear cells. Furthermore, infection of human IL-32γ transgenic mice (IL-32γTg mice) with L. infantum promastigote forms increased IL-32γ expression in the spleen and liver. Interestingly, IL-32γTg mice harbored less parasitism in the spleen and liver than wild-type (WT) mice. In addition, IL-32γTg mice showed increased granuloma formation in the liver compared to WT mice. The protection against VL was associated with increased production of nitric oxide (NO), interferon gamma (IFN-γ), IL-17A, and tumor necrosis factor alpha by splenic cells restimulated ex vivo with L. infantum antigens. In parallel, there was an increase in the number of Th1 and Th17 T cells in the spleens of IL-32γTg mice infected with L. infantum IL-32γ induction of IFN-γ and IL-17A expression was found to be essential for NO production by splenic cells of infected animals. These data indicate that IL-32γ potentiates the Th1/Th17 immune response during experimental VL, thus contributing to the control of L. infantum infection.


Assuntos
Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Interleucinas/imunologia , Interleucinas/fisiologia , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Fatores de Proteção , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Animais
11.
Immunol Invest ; 47(1): 71-88, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29077524

RESUMO

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by loss of dopaminergic neurons associated with neuroinflammation. Toll-like receptors (TLRs) are expressed in peripheral blood leukocytes and also in neurons and glial cells mediating inflammation. This study aimed to investigate the peripheral blood leukocyte response to TLR2 and TLR4 agonists in young and elderly PD patients. Two groups of patients with PD were evaluated (≤ 55 years old and ≥ 65 years old), age-matched with healthy controls (n = 26). Severity of PD was evaluated by Unified Parkinson's Disease Rating Scale (UPDRS). Whole blood cultures were stimulated with lipopolysaccharide (LPS), a TLR4 agonist or Pam3Cys (Pam), a TLR2 agonist. Tumor necrosis factor alpha (TNFα) and interleukin 10 (IL-10) were measured by immunoenzimatic assay. 6 h-TNFα production was increased after TLR4 stimulation, mainly in young PD patients, whereas TLR2-induced TNFα and IL-10 levels were decreased in PD patients independent of age (p < 0.05). A reverse correlation between LPS-induced TNFα production and age was observed in PD patients and controls, but TNFα induced by TLR2 agonist was not associated with age of PD patients or controls. TNFα production induced by TLR4 but not by TLR2 was reversely associated with the age at PD onset and disease duration. No associations between UPDRS scores and cytokine levels were detected. In conclusion, TLR4 and TLR2 responses seem to be differentially affected during PD. Data suggest that TLR2 deficiency in periphery is independent of age of the patients, age at PD onset, or PD duration.


Assuntos
Fatores Etários , Envelhecimento/imunologia , Doença de Parkinson/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto , Idoso , Células Cultivadas , Humanos , Interleucina-10/metabolismo , Lipopolissacarídeos/imunologia , Lipoproteínas/imunologia , Pessoa de Meia-Idade , Risco , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistas , Fator de Necrose Tumoral alfa/metabolismo
12.
Parasitol Res ; 117(2): 419-427, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29270768

RESUMO

Because of visceral leishmaniasis (VL) urbanization and spreading of the human immunodeficiency virus (HIV) infection to rural areas, coinfection has become more common. Here, we compared the accuracy of Kalazar Detect® (KD), an rK39-based immunochromatographic (IC) test, and OrangeLife® (OL), an rK39 + rK28 IC test, for diagnosing VL in patients coinfected with HIV in an endemic area in Brazil. Seventy-six VL patients and 40 patients with other diseases, of which 31 and 21 patients, respectively, were infected with HIV, were examined. The sensitivity of OL and KD tests was 88.89 and 95.45%, respectively, in patients without HIV. The sensitivity dropped to 67.74 and 61.29%, respectively, in coinfected patients. The decrease in sensitivity was not related to a decrease in the production of Leishmania-specific IgG. Because of the low sensitivity of rk39 test in HIV-infected patients, we suggest that patients with negative rK39 results should undergo further investigation with additional serological tests that are not based only on the rK39 antigen and examination of bone marrow aspirates.


Assuntos
Cromatografia de Afinidade/métodos , Infecções por HIV/complicações , Leishmaniose Visceral/diagnóstico , Adulto , Antígenos de Protozoários/imunologia , Brasil , Coinfecção , Feminino , Humanos , Leishmaniose Visceral/complicações , Masculino , Proteínas de Protozoários/imunologia , Sensibilidade e Especificidade
13.
Rev Inst Med Trop Sao Paulo ; 59: e91, 2017 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-29267599

RESUMO

Determination of the epidemiological profile of the American tegumentary leishmaniasis (ATL) and identification of Leishmania species that are prevalent in the State of Tocantins were carried out through a retrospective and descriptive study based on data reported in SINAN, in the period from 2011 to 2015. Molecular techniques such as PCR-RFLP and PCR-G6PD to amplify Leishmania DNA were performed on stored on Giemsa-stained slides from lesion scarifications of ATL patients who were amastigote-positive by the direct microscopic examination. There were 1,434 ATL cases in Tocantins reported in this period. The highest incidence was reported in men aged over 60 years, rural residents, the most affected ethnic group was mixed ethnicity (mixed black and white) and the ones with lower education. The predominant clinical form was cutaneous, being diagnosed mainly by laboratory methods. Pentavalent antimonial was effective in resolving cases. The predominant species found in 271 analyzed samples from 32 municipalities located in 8 different health regions of Tocantins was Leishmania (Viannia) braziliensis. Identifying the epidemiological profile and characterizing the Leishmania spp species on regional level is essential to establish control and prevention behaviors, minimizing the number of cases and treatment resistance, recurrence and evolution to mucosal forms.


Assuntos
Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Adolescente , Adulto , Distribuição por Idade , Brasil/epidemiologia , Criança , Pré-Escolar , DNA de Protozoário , Feminino , Humanos , Lactente , Leishmania braziliensis/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Prevalência , Estudos Retrospectivos , Distribuição por Sexo , Fatores Socioeconômicos , Fatores de Tempo , Adulto Jovem
14.
Pathog Dis ; 75(7)2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28830073

RESUMO

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


Assuntos
Leishmania braziliensis/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Espécies Reativas de Oxigênio/agonistas , Acetofenonas/farmacologia , Di-Hidropiridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Humanos , Leishmania braziliensis/crescimento & desenvolvimento , Macrófagos/imunologia , Macrófagos/parasitologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fator de Ativação de Plaquetas/antagonistas & inibidores , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/efeitos dos fármacos
15.
Parasit Vectors ; 10(1): 336, 2017 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-28709468

RESUMO

BACKGROUND: Interleukin 32 (IL-32) is a pro-inflammatory cytokine induced in patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. Here, we investigated whether IL-32 is also expressed in patient lesions caused by L. amazonensis. In addition, we evaluated experimental L. amazonensis and L. braziliensis infections in C57BL/6 transgenic mice for human IL-32γ (IL-32γTg) in comparison with wild-type (WT) mice that do not express the IL-32 gene. RESULTS: Human cutaneous lesions caused by L. amazonensis express higher levels of IL-32 than healthy control skin. In mice, the presence of IL-32γ promoted the control of cutaneous lesions caused by L. braziliensis, but not lesions caused by L. amazonensis in an ear dermis infection model. In addition, IL-32γTg mice displayed less tissue parasitism and inflammation in IL-32γTg than WT mice during the healing phase of L. braziliensis infection. Production of antigen-specific pro-inflammatory cytokines was higher in IL-32γTg mice than in WT mice during L. braziliensis infection but not during L. amazonensis infection. CONCLUSIONS: Human cutaneous lesions caused by L. amazonensis express high levels of IL-32. In mice, the presence of IL-32γ contributes to the lesion healing caused by L. braziliensis but not by L. amazonensis. Data suggest that despite the ability for both species to induce IL-32 in humans, the connections between this cytokine and other immune players induced by related species of parasites can lead to distinct outcomes of the murine infections.


Assuntos
Interleucinas/metabolismo , Leishmania braziliensis/imunologia , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Cicatrização , Animais , Modelos Animais de Doenças , Humanos , Interleucinas/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/parasitologia , Pele/patologia
16.
Biochim Biophys Acta Biomembr ; 1859(1): 1-9, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27773565

RESUMO

In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (cw50) and the cell membrane (cm50) and the membrane-aqueous medium partition coefficient of MT (K). With a cw50 of 8.7µM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had cw50 values of 2.4-3.1µM. The estimated cm50 of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays.


Assuntos
Antiprotozoários/farmacologia , Membrana Celular/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Estágios do Ciclo de Vida/efeitos dos fármacos , Proteínas de Membrana/química , Fosforilcolina/análogos & derivados , Proteínas de Protozoários/química , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Concentração Inibidora 50 , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/metabolismo , Estágios do Ciclo de Vida/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosforilcolina/farmacologia , Cultura Primária de Células , Proteínas de Protozoários/metabolismo , Marcadores de Spin
17.
Cytokine ; 88: 184-192, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27649507

RESUMO

While the role of Toll-like receptors (TLRs) has been investigated in murine models of tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis, the interaction between TLRs and Leishmania sp. has not been investigated in human cells. The aim of this study was to evaluate the involvement of TLR4 in cytokine production of human peripheral blood mononuclear cells (PBMCs) induced by L. braziliensis, and whether the parasite alters the expression of TLR4 on monocytes/macrophages. Amastigote forms were obtained from mice lesions and PBMCs were isolated from healthy donors. PBMCs were cultured in absence or presence of IFNγ, TLR4 neutralizing antibodies, natural antagonist of TLR4 (Bartonella LPS), TLR4 agonist (E. coli LPS), and amastigote forms. The concentrations of tumor necrosis factor (TNFα) and interleukin 10 (IL-10) were assayed by ELISA and TLR4 expression by flow cytometry. Amastigotes forms of L. braziliensis induced TNFα and IL-10 production only in IFNγ-primed PBMCs. The TNFα and IL-10 production was inhibited by TLR4 neutralization, both with anti-TLR4 antibodies and Bartonella LPS. Interestingly, addition of E. coli LPS further increased TNFα but not IL-10 production induced by L. braziliensis amastigotes. Amastigotes of L. braziliensis strongly reduced membrane TLR4 expression on monocytes/macrophages, apparently by internalization after the infection. The present study reveals that TLR4 drives the production of TNFα and IL-10 induced by L. braziliensis amastigotes and that the parasites decrease TLR4 expression on monocyte surface.


Assuntos
Interleucina-10/imunologia , Leishmania braziliensis/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adolescente , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade
18.
Neuroimmunomodulation ; 23(1): 58-66, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26886382

RESUMO

OBJECTIVES: Toll-like receptors (TLRs) are expressed in several immune cells including blood monocytes and resident macrophages, such as microglia in the central nervous system. TLRs recognize pathogen- or damage-associated molecular patterns, leading to the release of inflammatory and toxic molecules, which can contribute to neuroinflammation associated with Parkinson's disease (PD). The aim of this study was to compare the potential of peripheral blood cells from PD patients or healthy subjects to produce cytokines after exposure to TLR agonists, and to investigate TLR2 and TLR4 expression on monocyte subsets. METHODS: Twenty-one patients and 21 healthy controls were recruited. Patients were evaluated according to the Unified Parkinson's Disease Rating Scale, and Hoehn and Yahr stage. Cytokines were measured in supernatants of whole blood cultures after incubation with TLR2, TLR4, or TLR7/8 agonists, by cytometric bead array. Expression of CD14, CD16, TLR2, and TLR4 was analyzed by cytometry. RESULTS: Patient blood cells produced lower levels of cytokines in response to TLR2 and also after TLR7/8/R848 activation than controls. Percentages of CD14+CD16+ or CD14+CD16- monocytes and TLR2 and TLR4 expression were similar between patients and controls. CONCLUSIONS: Blood leukocyte TLR2 and TLR7/8 responses are impaired in PD. This was neither associated with imbalance in monocyte subsets nor with TLR2/TLR4 expression on these cells. The association between a decreased TLR response in periphery and damage of brain in PD must be further investigated.


Assuntos
Células Sanguíneas/metabolismo , Citocinas/metabolismo , Doença de Parkinson/sangue , Receptor 2 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Idoso , Células Sanguíneas/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Humanos , Imidazóis/farmacologia , Lipopolissacarídeos/farmacologia , Lipoproteínas/farmacologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Estatística como Assunto
19.
Biomed Res Int ; 2015: 350764, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26583102

RESUMO

The aim of this study was to characterize clinical field isolates of Leishmania spp. obtained from patients with American Tegumentary Leishmaniasis (ATL) who live in Goiás state, Brazil. The presumed areas of infection were in Goiás, Tocantins, and Pará states. Three isolates of parasites were identified as L. (Viannia) braziliensis and one as L. (V.) guyanensis. The in vitro growth profiles were found to be similar for all parasites. Nevertheless, in C57BL/6 mice, L. (V.) guyanensis infection was better controlled than L. (V.) braziliensis. Yet in C57BL/6 mice deficient in interferon gamma, L. (V.) guyanensis lesions developed faster than those caused by L. (V.) braziliensis isolates. In BALB/c mice, the development of lesions was similar for isolates from both species; however, on the 11th week of infection, amastigotes could not be observed in macrophages from L. (V.) guyanensis-infected mice. Thus, L. (V.) guyanensis can be circulating in Goiás, a state where autochthonous cases of this species had not yet been reported. Considering the difficulties to differentiate L. (V.) guyanensis from L. (V.) braziliensis at the molecular, morphological, and clinical (human and murine models) levels, the presence of L. (V.) guyanensis infections is possibly underestimated in several regions of Brazil.


Assuntos
Leishmania braziliensis/patogenicidade , Leishmania guyanensis/patogenicidade , Leishmaniose Cutânea/parasitologia , Animais , Brasil , Humanos , Leishmania braziliensis/isolamento & purificação , Leishmania guyanensis/isolamento & purificação , Leishmaniose Cutânea/patologia , Camundongos
20.
Biomed Res Int ; 2015: 393049, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25695070

RESUMO

This study was designed to assess in vitro metacyclogenesis of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis clinical field isolates obtained from patient lesions (L. braziliensis IMG3 and PPS6m; L. amazonensis MAB6). Metacyclogenesis was evaluated by different criteria, namely, promastigote size (morphometric analysis and flow cytometry), surface modifications (loss of lectin or monoclonal antibody (mAb) binding, complement resistance), and infectivity to human macrophages. Growth curves were similar for all parasites evaluated. The various features analyzed were expressed in a high percentage of promastigotes at 6th and 10th days of culture and a low percentage at the 2nd day. However, in most isolates, these features, considered as markers of metacyclogenesis, seemed to develop with different time courses, since the percentages of metacyclic forms detected with each technique were usually different. Parasites from 6th or 10th day and those negatively selected with lectin or mAb similarly infected human macrophages. From all isolates analyzed, L. amazonensis PH8 and MAB6 showed the highest and the lowest levels of susceptibility, respectively, to leishmanicidal activity of IFN-γ/LPS-activated macrophages. Our results showed that by using different techniques to evaluate different aspects of metacyclogenesis (morphological and biochemical modifications) different percentages of metacyclic promastigotes can be detected in each isolate culture.


Assuntos
Proteínas do Sistema Complemento/imunologia , Leishmania braziliensis/citologia , Leishmania braziliensis/imunologia , Estágios do Ciclo de Vida/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Anticorpos Monoclonais/imunologia , Células Cultivadas , Humanos , Técnicas In Vitro , Interferon gama/imunologia , Lectinas/imunologia , Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Lipopolissacarídeos/imunologia
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