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1.
Mol Immunol ; 125: 104-114, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32659595

RESUMO

Leishmania are obligate protozoan parasites responsible for substantial public health problems in tropical and subtropical regions around the world, with L. braziliensis being one of the causative agents of American Tegumentary Leishmaniasis. Macrophages, fundamental cells in the innate inflammatory response against Leishmania, constitute a heterogeneous group with multiple activation phenotypes and functions. The outcome of this infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. The importance of lipids, the major components of cell membranes, goes beyond their basic structural functions. Lipid bioactive molecules have been described in Leishmania spp., and in the recent years the knowledge about the biological relevance of lipids in particular and their relationship with the immune response is expanding. The present work analyzes the biological effects of L. braziliensis lipids from lysed promastigotes (PRO) to mimic rapid modulatory processes that could occur in the initial steps of infection or the effects of lipids from lysed and incubated promastigotes (PROinc), simulating the parasite lipid degradation processes triggered after parasite lysis that might occur in the mammalian host. To perform these studies, lipid profiles of PRO and PROinc were compared with lipids from amastigotes under similar conditions (AMA and AMAinc), and the effect of these lipid extracts were analyzed on the induction of an inflammatory response in murine peritoneal macrophages: LB induction, COX-2, iNOS and Arginase expression, TNF-α, IL-10 and NO production, Arginase activity and M1/M2 markers mRNA induction.


Assuntos
Antígenos de Protozoários/imunologia , Leishmania braziliensis/imunologia , Leishmaniose/imunologia , Lipídeos/imunologia , Ativação de Macrófagos/imunologia , Animais , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C
2.
Rev Bras Parasitol Vet ; 29(2): e003520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520088

RESUMO

Blood samples and swabs from ocular conjunctiva and mouth were obtained from 64 cats. Of 64 serum samples, 19 were positive for Leishmania antibodies by ELISA (29.80%). Eight cats were positive by PCR (12.5%) in swab samples from mouth and/or ocular mucosa. Poor kappa agreement between serological and molecular results (k = 0.16) was obtained. From five positive PCR samples one was L. braziliensis and four were L. infantum. Phylogenetic analysis performed with the five isolates of Leishmania, showed that samples of L. infantum isolated from the cats were phylogenetically close to those isolated from domestic dogs in Brazil, while the L. braziliensis is very similar to the one described in humans in Venezuela. The study demonstrated that, despite high seropositivity for Leishmania in cats living in the study region, poor agreement between serological and molecular results indicate that positive serology is not indicative of Leishmania infection in cats. Parasite DNA can be detected in ocular conjunctiva and oral swabs from cats, indicating that such samples could be used for diagnosis. Results of phylogenetic analyzes show that L. infantum circulating in Brazil is capable of infecting different hosts, demonstrating the parasite's ability to overcome the interspecies barrier.


Assuntos
Doenças do Gato/parasitologia , Leishmania braziliensis/isolamento & purificação , Leishmania infantum/isolamento & purificação , Leishmaniose/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Doenças do Gato/diagnóstico , Gatos , DNA de Protozoário/análise , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose/diagnóstico , Filogenia , Reação em Cadeia da Polimerase/veterinária
3.
PLoS One ; 15(5): e0232829, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379842

RESUMO

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


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Membrana Celular/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Afinidade de Anticorpos , Especificidade de Anticorpos , Argentina/epidemiologia , Doadores de Sangue , Doenças Endêmicas , Humanos , Leishmania braziliensis/crescimento & desenvolvimento , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Mucocutânea/sangue , Leishmaniose Mucocutânea/diagnóstico , Leishmaniose Mucocutânea/parasitologia , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Trypanosoma cruzi/imunologia
4.
Mem Inst Oswaldo Cruz ; 115: e190408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32321156

RESUMO

BACKGROUND: The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES: In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS: Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS: The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(ß1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS: Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania braziliensis/imunologia , Leishmania infantum/imunologia , Monócitos/parasitologia , Óxido Nítrico/biossíntese , Fagocitose/imunologia , Adulto , Resistência a Medicamentos , Feminino , Citometria de Fluxo , Humanos , Imunidade Inata , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Masculino , Monócitos/imunologia , Adulto Jovem
5.
Rev Soc Bras Med Trop ; 53: e20190433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348430

RESUMO

INTRODUCTION: As highly specific molecular biology-based techniques may not be sensitive enough for the diagnosis of American tegumentary leishmaniasis (ATL), clinicians frequently rely on immunological tests before treatment initiation. Hence, the correct combination of diagnostic tests is imperative for ATL diagnosis. We aimed to evaluate the accuracy of the Montenegro (Leishmanin) skin test (MST) in polymerase chain reaction (PCR)-negative patients to accurately detect ATL. METHODS: Patients with a clinical picture compatible with ATL were divided into ATL (confirmed by lesion smear, culture indirect immunofluorescence, and/or histopathology) and no-ATL (diseases that can mimic leishmaniasis) groups. Conventional PCR for the minicircle kDNA of Leishmania was performed, and the MST was carried out for PCR-negative patients. RESULTS: Ninety-nine patients were included in this study, including 79 diagnosed with ATL (6 with mucocutaneous leishmaniasis) and 20 without ATL (no-ATL group). The MST showed a high sensitivity of 90.0% (95% confidence interval [CI] = 69.90-97.21) in PCR-negative patients that was 10% higher than the sensitivity reported in PCR-positive population (79.66%; 95% CI = 67.73-87.96). CONCLUSIONS: One of the most important reasons for PCR negativity among patients with active ATL is the presence of a strong cellular immunological response, especially in chronic and mucocutaneous leishmaniasis. This reinforces the considerable utility of the tests that detect cellular responses against Leishmania antigens such as the MST in PCR-negative patients when the performance in screening situations is questionable.


Assuntos
Testes Intradérmicos/métodos , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Adulto , Idoso , Doença Crônica , Estudos Transversais , DNA de Protozoário/genética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Leishmania braziliensis/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade
6.
Immunology ; 159(4): 355-356, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32182636

RESUMO

Both CD8+ T cells and NK cells contribute to the immune response against the protozoan Leishmania parasite. Both are able to generate IFN-γ and both display cytotoxic features. These features may enable them to not only contribute to parasite clearance but also to cause immune-mediated pathology. This pathology is evident, for example, in the Leismania-induced skin lesions found in patients with cutaneous leismaniasis (CL). Here we highlight new data demonstrating that CD8+ T cells and NK cells in CL display a highly cytotoxic senescent phenotype, and that the senescent T cells play a major role in mediating skin pathology. This is the first demonstration that senescent CD8 T cells contribute to immunopathology in vivo.


Assuntos
Citotoxicidade Imunológica , Leishmania braziliensis/patogenicidade , Leishmaniose Cutânea/patologia , Pele/patologia , Linfócitos T Citotóxicos/patologia , Antígenos CD57/genética , Antígenos CD57/imunologia , Senescência Celular/imunologia , Expressão Gênica , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/parasitologia , Células Matadoras Naturais/patologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Oligossacarídeos/genética , Oligossacarídeos/imunologia , Antígeno Sialil Lewis X/análogos & derivados , Antígeno Sialil Lewis X/genética , Antígeno Sialil Lewis X/imunologia , Pele/imunologia , Pele/parasitologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/parasitologia
7.
PLoS One ; 15(3): e0229400, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32203546

RESUMO

The pathogenesis of cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis is dictated mainly by the immune-mediated-tissue inflammation developed. The understanding of the immunological mechanisms that generate tissue damage or resolution of lesions is the key to the development of effective vaccine protocols and proper therapeutic schemes. It is clear that the specific immune response mediated by T cells is responsible for the beneficial outcome of the disease, however, the roles of CD4+ T, CD8+ T, NK and NKT cell subpopulations in immunopathogenesis of CL need to be elucidated. Peripheral blood cells from patients before, during and after the antimonial therapy, as well as healthy individuals (HI) were cultured with (LbAgS) or without (NS) L. braziliensis antigens (LbAg). Afterwards, the frequencies of LbAg-specific-cytotoxic CD8+ T, CD4+ T, NK and CD3+CD56+ NKT cells, as well as their activation and exhaustion profiles, were defined by flow cytometry. We observed higher frequencies of CD8+ T, NK and CD3+CD56+ NKT cells and lower frequencies of CD4+ T lymphocytes in LbAgS cell cultures from patients before treatment. The specific response to LbAg resulted in an expansion of cytotoxic-activated CD4+ T, CD8+ T, and NK cells, before and during treatment, indicating specificity in the response by these cells against L. braziliensis. Furthermore, comparing the differences of frequencies of cytotoxic-activated CD4+T, CD8+T, and NK cells, among before and during treatment patients and HI groups, we conclude that these cell populations are in charge of immune response elicited by antimonial therapy. Interestingly, we also observed that NK cells were induced by LbAg to an exhaustion profile during all clinical stages of the disease. The increased antigen-specific activation and cytotoxic activity are in line with the strong inflammatory response described in this disease, a likely cause of tissue damage. These findings reinforce the involvement of these distinct cytotoxic-activated cell populations in the immunopathogenesis of CL, showing a character of specificity in this immune response.


Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Adulto , Idoso , Complexo CD3/metabolismo , Antígeno CD56/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Citotóxicos , Adulto Jovem
8.
Immunology ; 159(4): 429-440, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31925782

RESUMO

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim  CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+  CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.


Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/patologia , Leishmania braziliensis/patogenicidade , Leishmaniose Cutânea/patologia , Pele/patologia , Linfócitos T Citotóxicos/patologia , Antígeno CD56/genética , Antígeno CD56/imunologia , Antígenos CD57/genética , Antígenos CD57/imunologia , Estudos de Casos e Controles , Senescência Celular/imunologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/parasitologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Masculino , Oligossacarídeos/genética , Oligossacarídeos/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Índice de Gravidade de Doença , Antígeno Sialil Lewis X/análogos & derivados , Antígeno Sialil Lewis X/genética , Antígeno Sialil Lewis X/imunologia , Transdução de Sinais , Pele/imunologia , Pele/parasitologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/parasitologia
9.
BMC Infect Dis ; 19(1): 1015, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783798

RESUMO

BACKGROUND: The present study aimed to demonstrate the applicability of a flow cytometry-based serology approach to identify spontaneous cure by the detection of immunoglobulin G, and also, the diagnosis and cure criterion by the IgG1 isotype in American Tegumentary Leishmaniasis - ATL caused by L. (V.) braziliensis. Also, a comparison between flow cytometry with the serological conventional technique was performed. METHODS: Forty five individuals were included in study. They were assessed in two moments: First, 8 subjects spontaneously cured of ATL, 8 healthy individuals and 15 patients who had a positive diagnosis for ATL were selected before treatment to identify spontaneous cure by immunoglobulin G detection. Secondly, 14 patients who were positive for ATL were selected and had their blood collected before and 1, 2 and 5 years after treatment, respectively, for the diagnostic tests (ELISA and flow cytometry) and cure criterion evaluation using the IgG1 isotype. RESULTS: The analysis of the mean percentage of positive fluorescent parasites (PPFP) along with the titration curves of IgG anti-fixed promastigotes of L.(V.)braziliensis, confirmed the applicability of this method for monitoring spontaneous cure in ATL with outstanding co-positivity (100%) and co-negativity (100%) performance indexes. Regarding the results of the comparison between flow cytometry and ELISA it was seen that there was a better accuracy of the first one in relation to the other. When IgG1 applicability was evaluated, it was observed that before treatment, 36.8% of the patients were negative; in patients 1 year post-treatment, 82.3%; 2 years post-treatment, 27.2% and in patients 5 years post-treatment, 87.5%. The overall analysis of the results suggests that flow cytometry can be applied to ATL detection, and that the use of IgG1 isotype has possibilities to contribute as a more specific diagnostic method. CONCLUSIONS: Therefore, this area has great perspectives use for the diagnosis and cure criterion, and also it can be scaled up with the possibility to characterize the different clinical stages of the disease. Together, these findings demonstrate the applicability of a flow cytometry-based serology approach and opens up new avenues of research with this technique, such as the understanding the humoral response in ATL patients.


Assuntos
Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Anticorpos Antiprotozoários/sangue , Antiprotozoários/uso terapêutico , Área Sob a Curva , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoglobulina G/sangue , Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/tratamento farmacológico , Curva ROC , Remissão Espontânea
10.
Vaccine ; 37(49): 7269-7279, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31575491

RESUMO

The poor immunogenicity displayed by some antigens has encouraged the development of strategies to improve the immune response and safety of vaccine candidates, resulting in an intense search for substances that potentiate vaccine response. Adjuvants have these properties helping vaccine candidates to induce a strong, durable, and fast immune response. In this study, we evaluated the specific immune response of adjuvants alone, Saponin (SAP), Incomplete Freund's Adjuvant (IFA) and Monophosphoryl lipid-A SE (MPL-SE®) and in combination with total antigen of L. braziliensis (LB): LBSAP, LBIFA and LBMPL. The specific immune response induced by these compositions demonstrated that they were powerfully immunogenic, increasing cellular infiltration in the skin. Draining lymph nodes cultures showed that LBIFA and LBMPL have higher ability to increase the capacity of APCs to present antigens, with increased frequency of CD11c+CD86+ cells. SAP, MPL, LBSAP, LBIFA and LBMPL could activate lymphocytes increasing expression of CD69 and CD25. LBSAP group was an excellent inducer of pro-inflammatory cytokines at 24 h. At 48 h, higher cytokines production was observed in IFA, LBIFA, MPL and LBMPL groups. Our data demonstrate that LBSAP and LBMPL are potential formulations to be tested in other experimental models. Also, the data obtained could expand the knowledge about immune response after sensitization and also contribute to the development of safe, immunogenic and effective vaccines.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos de Protozoários/imunologia , Leishmania braziliensis/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Animais , Anticorpos Antiprotozoários/imunologia , Formação de Anticorpos/imunologia , Adjuvante de Freund/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Lipídeos/imunologia , Masculino , Camundongos , Saponinas/imunologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-31555609

RESUMO

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


Assuntos
Glicoesfingolipídeos/metabolismo , Interações Hospedeiro-Patógeno , Leishmania braziliensis/imunologia , Leishmaniose Mucocutânea/imunologia , Leishmaniose Mucocutânea/parasitologia , Metaloendopeptidases/metabolismo , Fagossomos/metabolismo , Animais , Células Cultivadas , Evasão da Resposta Imune , Leishmania braziliensis/crescimento & desenvolvimento , Proteína 1 de Membrana Associada ao Lisossomo/antagonistas & inibidores , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos Endogâmicos C57BL , Biogênese de Organelas
12.
BMC Infect Dis ; 19(1): 747, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455227

RESUMO

BACKGROUND: Leishmaniasis caused by different species of Leishmania affect 98 countries worldwide. Visceral Leishmaniasis (VL) is the mortal clinical presentation of the disease that causes the dead to more than 90% of the patients who suffer it. The diagnosis of VL is made by the direct observation of the parasite in bone marrow, spleen and/or liver aspirates that requires complex proceedings. Therefore, serum samples are submitted to Indirect Immunofluorescence to identify the presence of anti-Leishmania antibodies. Despite the variability in the diagnostic performance of the Immunochromatographic Tests (ICTs), there are many evidences that suggest that ICTs can be used for epidemiological screening. However, in Colombia there are not any evidence about the performance of the ICTs for VL diagnosis, both for human and canine serum samples. Therefore, this study evaluated the diagnostic performance of 4 ICTs for VL (2 ICTs in human sera and 2 ICTs in canine sera) in samples from endemic areas of Colombia. METHODS: We selected a total of 156 human serum samples (82 positive and 74 negative for VL) and 126 canine serum samples (71 positive and 54 negative) diagnosed by in house Indirect Immunofluorescence (IIF). The samples were submitted to the ICTs following the manufacturers' instructions. Statistical analysis was performed to evaluate the diagnostic performance of each ICT in comparison with the IIF. PCR for HSP70 gene and sanger sequencing was performed in samples with negative results for both ICTs. RESULTS: The sensitivity (S) of both ICTs for human samples (Ad-bio Leishmania IgG/IgM Combo Rapid Test and Kalazar Detect™) was 91.5% and specificity (E) were 93.2 and 89.2% respectively, while for the ICTs tested on canine samples (Kalazar Detect™ Rapid Test, Canine and DPP® CVL rapid test) we found S values between 82.9 and 85.7% and E values between 79.6 and 92.6%. We found L. infantum by PCR and sequencing in 2 human samples, and L. braziliensis and L. amazonensis in canine serum samples that were negative by both ICTs. CONCLUSIONS: We conclude that both tests evaluated on human samples have a similar diagnostic performance, while the Kalazar Detect™ Rapid Test, Canine showed a better diagnostic performance than the DPP® CVL rapid test evaluated on canine samples. Also, we suggest that it is necessary to design tests with antigens of the circulating strains to increase its diagnostic utility.


Assuntos
Doenças do Cão/diagnóstico , Imunoensaio/métodos , Leishmaniose Visceral/diagnóstico , Animais , Colômbia , Testes Diagnósticos de Rotina , Doenças do Cão/parasitologia , Cães , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
13.
Rev Inst Med Trop Sao Paulo ; 61: e42, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31432991

RESUMO

American Tegumentary leishmaniasis (ATL) is an infectious disease caused by several species of Leishmania . Even though the direct detection of parasites has low sensitivity, it is still the gold standard for the laboratory diagnosis of ATL. Recent studies have shown promising results of Enzyme-Linked Immunosorbent Assays ( ELISAs) using recombinant antigens. The aim of this study is to compare the accuracy of ELISAs using novel antigens with the standard ELISA based on soluble antigens of Leishmania (SLA) to diagnose ATL. Studies that analyzed patients with ATL and studies that evaluated the diagnostic accuracy of ELISAs using novel antigens and SLA were included. The Fourteen studies from PubMed, Regional Portal of the Virtual Health Library (BVS), Brazilian Society of Dermatology, Virtual Health Library (IBECS), Literature in the Health Sciences in Latin America and the Caribbean (LILACS), Medical Literature Analysis and Retrieval System Online (Medline), Elsevier Embase, Cochrane Library, The National Institute for Health and Care Excellence (NICE), and Cumulative Index to Nursing and Allied Health Literature (CINAHL) were included. The novel ELISA antigens showed a high sensitivity (93.8%-100%) and specificity (82.5-100%), a better diagnostic performance than SLA-based ELISAs (1-97.4% and 57.5-100%, respectively). Only 10 studies analyzed cross-reactions in serum samples from patients with Chagas disease, and only two studies reported a percentage of cross-reactivity. In this systematic review, the novel ELISA antigens showed better sensitivity and specificity with respect to SLA-based ELISAs. However, a meta-analysis should be performed to confirm this finding.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Humanos , Sensibilidade e Especificidade
14.
Cytokine ; 123: 154784, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31344596

RESUMO

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is the most spread clinical form of leishmaniasis in Brazil. However, only a few part of the people infected develop clinically perceptive disease, suggesting the influence of human genetic components in the CL pathogeny. The rs2275913 SNP is the nucleotide variant of the IL17A gene. The A allele is associated with a vast number of infectious and non-infectious diseases. Here, we investigated the association of the rs2275913 SNP (G/A) from IL-17A and two forms of susceptibility to CL in Brazil by case-control study. Furthermore, we evaluated the functional relevance of this SNP during the immune response of the host and analyzed its impact in the parasite elimination. Weak associations of A allele with susceptibility to L. braziliensis infection or to symptomatic CL were observed, and a tendency of A allele carriers to be more susceptible to infection and cutaneous disease. Functional analysis of the Th17 cell phenotypes revealed lower frequencies of CD4+ IL-17+ cells in samples of infected people with AA/AG genotypes. Furthermore, people carrying the A allele maintain higher parasite loads, reinforcing the genetic susceptibility findings. This study adds knowledge about the influence of a significant genetic variation on IL-17 promoter on CL pathogenesis, and may contribute to enhance the knowledge about the role of IL-17 in the L. braziliensis infections.


Assuntos
Predisposição Genética para Doença , Genótipo , Interleucina-17 , Leishmania braziliensis/imunologia , Leishmaniose Cutânea , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Masculino , Pessoa de Meia-Idade , Células Th17/imunologia , Células Th17/patologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-31119102

RESUMO

Cutaneous leishmaniasis (CL) caused by infection with Leishmania braziliensis is characterized by an exaggerated inflammatory response that controls the parasite burden, but also contributes to pathology. While myeloid cells are required to eliminate the parasite, recent studies indicate that they may also participate in the inflammatory response driving disease progression. The innate immune response to leishmania is driven in part by the Toll-like receptors (TLRs) TLR2, TLR4, and TLR9. In this study, we used flow cytometric analysis to compare TLR2 and TLR4 expression in monocyte subsets (classical, intermediate, and non-classical) from CL patients and healthy subjects (HS). We also determined if there was an association of either the pro-inflammatory cytokine TNF or the anti-inflammatory cytokine IL-10 with TLR2 or TLR4 expression levels after L. braziliensis infection. In vitro infection with L. braziliensis caused CL monocytes to up-regulate TLR2 and TLR4 expression. We also found that intermediate monocytes expressed the highest levels of TLR2 and TLR4 and that infected monocytes produced more TNF and IL-10 than uninfected monocytes. Finally, while classical and intermediate monocytes were mainly responsible for TNF production, classical monocytes were the main source of IL-10. Collectively, our studies revealed that up-regulated TLR2/4 expression and TNF production by intermediate/inflammatory subsets of monocytes from patients correlates with detrimental outcome of cutaneous leishmaniasis.


Assuntos
Interleucina-10/biossíntese , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/patologia , Monócitos/imunologia , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Leishmaniose Cutânea/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/parasitologia , Adulto Jovem
16.
Cell Immunol ; 341: 103920, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31078283

RESUMO

Localized cutaneous leishmaniasis (LCL) can ultimately progress to chronic ulcerated lesions with strong local inflammatory reactions. The functional role of certain inflammasomes in mediating inflammation caused by Leishmania braziliensis needs to be addressed. By combining PCR-array, quantitative real-time PCR and immunohistochemical analysis, we identified inflammasome genes, such as IL-1ß, NLRP3, NLRP1, NLRC5, AIM2 and P2RX7, that were upregulated in LCL patients. Temporal gene expression studies showed that the early phase of LCL displayed increased NLRP3 and reduced AIM2 and NLRP1 expression, while the late stages showed increased AIM2 and NLRP1 and lower NLRP3 expression. Our findings also showed that AIM2, NLRP1, and P2RX7 promoted susceptibility to experimental L. braziliensis infection. These results highlight the importance of inflammasome machinery in human LCL and suggest that inflammasome machinery plays a role in the acute and chronic phases of the disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação a DNA/genética , Inflamassomos/genética , Leishmaniose Cutânea/genética , Receptores Purinérgicos P2X7/genética , Pele/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Adulto , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas de Ligação a DNA/imunologia , Progressão da Doença , Suscetibilidade a Doenças , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inflamassomos/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Leishmania braziliensis/imunologia , Leishmania braziliensis/patogenicidade , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Receptores Purinérgicos P2X7/imunologia , Transdução de Sinais , Pele/parasitologia , Pele/patologia
17.
Front Immunol ; 10: 724, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024556

RESUMO

Leishmania (V.) braziliensis is the etiological agent of Cutaneous (CL) and Mucocutaneous leishmaniasis (ML) in the New World. CL can be more benign but ML can be severe and disfiguring. Immunity to these diseases include hypersensitivity, an enhanced inflammatory response with strong IFN-γ and TNF-α secretion. Additionally, the production of IL-10 which down modulates the immune response is reduced. The Nucleoside hydrolase (NH36) of Leishmania (L.) donovani is the main antigen of the Leishmune veterinary vaccine and its F3 domain induces a CD4+ T cell-mediated protection against L. (L.) infantum chagasi infection. Prevention of L. (L.) amazonensis infection requires in contrast an additional CD8+ T cell mediated response induced by the F1 domain. Consequently, the F1F3 recombinant chimera, which contains both domains cloned in tandem, optimized the vaccine efficacy against L. (L.) amazonensis mouse infection. We compared the efficacies of NH36, F1, F3, and the FIF3 chimera against L. (V.) braziliensis mouse infection. The F1F3 chimera increased the NH36 specific IgA and response before and after infection and the IgG and IgG3 levels after challenge. It also induced a 49% stronger intradermal response to leishmanial antigen (IDR) than NH36 that was positively correlated to the levels of IFN-γ and TNF-α, IgG, IgG2a, IgG2b, and IgG3 anti-NH36 antibodies. However, stronger Th1 responses with elevated IFN-γ/IL-10 and TNF-α/IL-10 ratios were promoted by the F3 and F1 vaccines and detected in infected controls while the F1F3 chimera promoted the highest IL-10 secretion, which reduced the pathological Th1 response, and characterized the induction of a mixed and/or T-cell regulatory response. We identified the epitopes responsible for these immune responses. The F3 vaccine induced the earliest immunity and after challenge, the F1F3 chimera promoted the highest CD4+ and CD8+ cytokine-secreting T cell responses, and the predominant frequencies of multifunctional CD4+ and CD8+IL-2+TNF-α+IFN-γ+ T cells. Also as observed against L. (L.) amazonensis infection, the F1F3 chimera showed the strongest reduction of the ear lesions sizes induced by L. (V.) braziliensis. Our results confirm the potential use of the F1F3 chimera in a multi-species cross-protective vaccine against L. (V.) braziliensis.


Assuntos
Quimera/imunologia , Proteção Cruzada/imunologia , Epitopos/imunologia , Leishmania braziliensis/imunologia , Leishmania donovani/imunologia , Leishmaniose Cutânea/imunologia , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Citocinas/imunologia , Feminino , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C
18.
Parasite Immunol ; 41(5): e12620, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30815888

RESUMO

AIMS: The polymorphism observed in Leishmania braziliensis is associated with different clinical forms of leishmaniasis. Neutrophils (PMNs) participate in the pathogenesis of leishmania infection, and here, we evaluate neutrophil function after infection with isolates of L. braziliensis from cutaneous leishmaniasis (CL) or disseminated leishmaniasis (DL) patients. METHODS AND RESULTS: Neutrophils from 30 healthy subjects (HS) were infected with isolates of L. (V.) braziliensis obtained from three CL and three DL patients. They were infected at the ratio of 3:1 parasites per neutrophil, and leishmania uptake was evaluated by microscopy. The neutrophil activation markers and oxidative burst by expression of dihidrorhodamine (DHR) were evaluated by flow cytometry and cytokine production by ELISA. The frequency of infected cells and the number of amastigotes were higher in neutrophils infected with CL isolates compared to DL isolates (P < 0.05). The DHR and CD66b expression after infection with DL isolate was lower than with CL isolates. There was no difference regarding chemokine production. CONCLUSION: The L. (V.) braziliensis isolates of DL induced lower respiratory burst and neutrophils activation markers compared with CL isolates which may contribute to parasite survival and dissemination in DL patients.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Adolescente , Adulto , Animais , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/parasitologia , Masculino , Ativação de Neutrófilo , Neutrófilos/imunologia , Adulto Jovem
19.
Acta Trop ; 193: 12-17, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30772331

RESUMO

In Brazil, the visceral leishmaniasis (VL) is caused by Leishmania infantum, while the tegumentary leishmaniasis (TL) etiological agents are mainly Leishmania braziliensis and Leishmania amazonensis. The canine visceral leishmaniasis (CVL) diagnosis is an important step of the VL control program in Brazil, which involves the elimination of infected dogs, the main urban VL reservoirs. The current serology-based diagnostic tests have shown cross-reactivity between these three species, whereas molecular diagnosis allows high sensitivity and specie identification. In the present study, 349 dogs of the metropolitan region of Belo Horizonte (Minas Gerais state) were screened by conjunctival swab and the samples analyzed by ITS-1 nested PCR. Thirty dogs (8.5%) tested positive. The RFLP of amplicons using HaeIII demonstrated that 17/30 samples presented a banding pattern compatible with L. infantum, 4/30 matched with L. amazonenis, 1/30 with L. braziliensis and 8/30 showed a mixed infection pattern. The samples that were distinct of L. infantum or presented a mixed pattern were submitted to RFPL with HaeIII and RsaI enzymes that confirmed the mixed pattern. Such patterns were also confirmed by Sanger Sequencing. The results pointed eight dogs with mixed infections and the establishment of TL causing species in the Belo Horizonte dog population. These findings highlight the need for more comprehensive epidemiological studies, since the TL transmission profile might be changing. This study also shows the potential of the ITS1-nPCR associated with RFLP for the proper Leishmania diagnosis and typing in the dog population.


Assuntos
Coinfecção/veterinária , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Leishmaniose Cutânea/veterinária , Leishmaniose Visceral/veterinária , Leishmaniose/veterinária , Animais , Brasil , Coinfecção/diagnóstico , Coinfecção/parasitologia , Cães , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Testes Sorológicos
20.
BMC Genomics ; 20(1): 118, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30732584

RESUMO

BACKGROUND: The leishmaniasis are parasitic diseases caused by protozoans of the genus Leishmania, highly divergent eukaryotes, characterized by unique biological features. To survive in both the mammalian hosts and insect vectors, these pathogens make use of a number of mechanisms, many of which are associated with parasite specific proteases. The metalloprotease GP63, the major Leishmania surface antigen, has been found to have multiple functions required for the parasite's survival. GP63 is encoded by multiple genes and their copy numbers vary considerably between different species and are increased in those from the subgenus Viannia, including L. braziliensis. RESULTS: By comparing multiple sequences from Leishmania and related organisms this study sought to characterize paralogs in silico, evaluating their differences and similarities and the implications for the GP63 function. The Leishmania GP63 genes are encoded on chromosomes 10, 28 and 31, with the genes from the latter two chromosomes more related to genes found in insect or plant parasites. Those from chromosome 10 have experienced independent expansions in numbers in Leishmania, especially in L. braziliensis. These could be clustered in three groups associated with different mRNA 3' untranslated regions as well as distinct C-terminal ends for the encoded proteins, with presumably distinct expression patterns and subcellular localizations. Sequence variations between the chromosome 10 genes were linked to intragenic recombination events, mapped to the external surface of the proteins and predicted to be immunogenic, implying a role against the host immune response. CONCLUSIONS: Our results suggest a greater role for the sequence variation found among the chromosome 10 GP63 genes, possibly related to the pathogenesis of L. braziliensis and closely related species within the mammalian host. They also indicate different functions associated to genes mapped to different chromosomes. For the chromosome 10 genes, variable subcellular localizations were found to be most likely associated with multiple functions and target substrates for this versatile protease.


Assuntos
Simulação por Computador , Variação Genética , Evasão da Resposta Imune/genética , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Metaloendopeptidases/genética , Sequência de Aminoácidos , Cromossomos/genética , Epitopos de Linfócito B/imunologia , Evolução Molecular , Leishmania braziliensis/patogenicidade , Metaloendopeptidases/química , Recombinação Genética , Homologia de Sequência do Ácido Nucleico , Virulência/genética
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