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1.
Front Microbiol ; 10: 96, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30804901

RESUMO

Although members of the Paracoccidioides complex are not obligate intracellular pathogens, they present the ability to survive and multiply inside epithelial cells and phagocytes of mammals, which may favor the spread of the fungus in host tissues. Macrophages resident in the lung are the first line of defense against paracoccidioidomycosis (PCM), presenting mechanisms to control the pathogen dissemination through the granuloma formation or eliminating the fungus through phagocytosis. Phagocytosis triggers an oxidative burst, in which there is an increase in the production of toxic elements, derived from oxygen and nitrogen. The interior of the phagolysosome is a harsh environment to the internalized pathogens, since in addition to the oxygen and nitrogen reactive species, microorganisms face nutrient shortages and proteases activity. Through the NanoUPLC-MS E technology, we analyzed the proteomic response of Paracoccidioides brasiliensis during the infection of alveolar macrophages primed or not by interferon gamma (IFN-γ). At 6 hs post-infection, only (IFN-γ)-primed macrophages were able to kill the fungus. We observed the regulation of amino acids degradation, tricarboxylic acid cycle, respiratory chain, ATP synthesis, glyoxylate cycle, as well as an increase in the expression of defense proteins related to oxidative stress, heat shock, and virulence factors under both conditions analyzed. However, some pathways described as essential for the survival of pathogens inside macrophages were observed only or with higher intensity in yeast cells recovered from non-primed macrophages, as phosphate pentoses pathway, methylcitrate cycle, synthesis of cell wall components, and mitochondrial activity. The data indicate that the intracellular environment of non-primed macrophages could be more permissive to the survival and multiplication of P. brasiliensis. The identification of key molecules for the establishment of infection can help the understanding of the nature of the parasite-host relationship and pathogenesis of PCM.

2.
Cell Immunol ; 337: 54-61, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30773217

RESUMO

Dendritic cells (DC) have the unique ability to capture microorganisms and activate naive T lymphocytes. Obtaining DC derived from progenitors demands high cost and prolonged cultivation. Different immortalized DC has been isolated but most of them have immature phenotype and depending on growing factors or other stimuli to be used. In this study we characterized the cell line AP284 as a DC. AP284 cells express high levels of CD11b, MHC class II, 33D1 and CD209b. They also express high amounts of CD80 costimulatory molecule and different toll like receptors (TLR). After stimuli with TLR agonist they produce surprising amount of IL-12p40 related to IL-23 formation but not IL-12p70. They are also able to produce IL-6 and favor amplification of a Th17 but not Th1 profile. This DC line may be useful for a better understanding of factors and cellular interactions responsible for the induction of IL-12p40, IL-23 and Th17 generation.


Assuntos
Técnicas de Cultura de Células/métodos , Células Dendríticas/imunologia , Células Th17/metabolismo , Animais , Diferenciação Celular , Linhagem Celular/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Interleucina-12/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Th1/imunologia , Células Th17/imunologia , Receptor 7 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
BMC Infect Dis ; 14: 249, 2014 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-24884781

RESUMO

BACKGROUND: The interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis. METHODS: IL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, ß, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP. RESULTS: We showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals. CONCLUSIONS: These data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection.


Assuntos
Interleucina-10/biossíntese , Interleucinas/biossíntese , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Interleucinas/genética , Leishmaniose Cutânea/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Membrana Mucosa/metabolismo , Pele/metabolismo , Regulação para Cima , Adulto Jovem
10.
Microbes Infect ; 15(5): 347-64, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23421979

RESUMO

An efficient oxidative stress response is important to the fungal pathogen Paracoccidioides to survive within the human host. In this study, oxidative stress was mimicked by exposure of yeast cells to hydrogen peroxide (2 mM H2O2). To investigate the effect of H2O2 on the proteome of Paracoccidioides, we used a large scale 2-DE protein gel electrophoresis approach to analyze differentially expressed proteins/isoforms that were detected in early (2 h) and in late (6 h) oxidative stress treatments. All proteins/isoforms were grouped based on their functional categories that revealed a global activation of antioxidant enzymes, such as catalase, superoxide dismutase, cytochrome C peroxidase and thioredoxin. A view of the metabolic cell profile, as determined by proteomics, depicted a shift in the yeast cells metabolism as suggested by the activation of the pentose phosphate pathway, a great source of cellular reducing power in the form of NADPH. Additionally, in silico analyzes depicted 34 oxidoreductases proteins/isoforms putatively involved with defense against oxidative stress. Confirmatory assays of enzymatic activity, flow cytometry, transcript levels and NADPH measurements, produced data in agreement with proteomic analysis.


Assuntos
Proteínas Fúngicas/análise , Oxidantes/toxicidade , Estresse Oxidativo , Paracoccidioides/química , Paracoccidioides/efeitos dos fármacos , Proteoma/análise , Eletroforese em Gel Bidimensional , Peróxido de Hidrogênio/toxicidade , Espectrometria de Massas , Redes e Vias Metabólicas , Fatores de Tempo
11.
Exp Parasitol ; 119(3): 403-10, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18501355

RESUMO

The production of interleukin-12 and interferon-gamma is a key event for controlling leishmaniasis. Here, we tested the hypothesis that after murine infection with Leishmania major, cell migration into draining lymph nodes is crucial for early production of those cytokines. We showed that inflammatory cells carrying the marker of recently migrated cells, the Gr-1 antigen, including polymorphonuclear and mononuclear cells, migrate rapidly into the site of promastigote infection and, subsequently, into draining lymph nodes. Treatment with RB6-8C5 monoclonal antibody reduced local inflammation and migration of Gr-1+ cells into the draining lymph nodes. This reduction was associated with a decrease of interleukin-12 production by draining lymph node cells from BALB/c mice but not C57BL/6 mice. Additionally, interferon-gamma was also reduced in both mouse strains after depletion of Gr-1+ cells, suggesting that these cells are important for early interleukin-12 and interferon-gamma production. Our findings suggest that recently migrated myeloid cells, more than resident cells, are the major source of the early IL-12 production after L. major infection.


Assuntos
Interferon gama/biossíntese , Interleucina-12/biossíntese , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Linfonodos/citologia , Animais , Anticorpos Monoclonais/imunologia , Movimento Celular/imunologia , Hibridomas , Imuno-Histoquímica , Leishmaniose Cutânea/patologia , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/imunologia , Neutrófilos/citologia , Neutrófilos/imunologia , Receptores de Quimiocinas/imunologia , Organismos Livres de Patógenos Específicos
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