117 results on '"Dorta, Miriam Leandro"'
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2. Promastigote parasites cultured from the lesions of patients with mucosal leishmaniasis are more resistant to oxidative stress than promastigotes from a cutaneous lesion
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Ávila, Lucilla Ribeiro, Gomes, Clayson Moura, Oliveira, Pollyana Guimarães, Gomes, Rodrigo Saar, Vinaud, Marina Clare, Dorta, Miriam Leandro, Uliana, Silvia Reni Bortolin, Ribeiro-Dias, Fátima, and Oliveira, Milton Adriano Pelli
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- 2018
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3. Heterologous Booster with BNT162b2 Induced High Specific Antibody Levels in CoronaVac Vaccinees
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Masson, Letícia Carrijo, primary, Servian, Carolina do Prado, additional, Jardim, Vitor Hugo, additional, dos Anjos, Déborah, additional, Dorta, Miriam Leandro, additional, Batalha-Carvalho, João Victor, additional, Moro, Ana Maria, additional, Romão, Pedro Roosevelt Torres, additional, Souza, Menira, additional, Fiaccadori, Fabiola Souza, additional, and Fonseca, Simone Gonçalves, additional
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- 2023
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4. Leishmania (Viannia) braziliensis amastigotes induces the expression of TNFα and IL-10 by human peripheral blood mononuclear cells in vitro in a TLR4-dependent manner
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Galdino, Hélio, Jr., Saar Gomes, Rodrigo, dos Santos, Jessica Cristina, Pessoni, Lívia Lara, Maldaner, Anetícia Eduarda, Marques, Stéfanne Madalena, Gomes, Clayson Moura, Dorta, Miriam Leandro, de Oliveira, Milton Adriano Pelli, Joosten, Leo A.B., and Ribeiro-Dias, Fátima
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- 2016
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5. Performance of two immunochromatographic tests for diagnosis of visceral leishmaniasis in patients coinfected with HIV
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da Silva, Mauro Roberto Biá, Brandão, Natália Alberto Alves, Colovati, Marco, de Sousa, Margella Marconcine Pinheiro, de Lima, Larissa Coelho, Dorta, Miriam Leandro, Ribeiro-Dias, Fátima, Costa, Dorcas Lamounier, Costa, Carlos Henrique Nery, and de Oliveira, Milton Adriano Pelli
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- 2017
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6. AVALIAÇÃO DE FATORES QUE IMPACTAM NA INCIDÊNCIA DE RECIDIVAS DE LEISHMANIOSE TEGUMENTAR OU LEISHMANIOSE VISCERAL EM PACIENTES CO-INFECTADOS COM O VÍRUS DA IMUNODEFICIÊNCIA HUMANA
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Araújo, Camila Freire, Oliveira, Iara Barreto Neves, Silva, Muriel Vilela Teodoro, Pereira, Ledice Inácia de Araújo, Pinto, Sebastião Alves, Silveira, Murilo Barros, Dorta, Miriam Leandro, Fonseca, Simone Gonçalves, Gomes, Rodrigo Saar, and Ribeiro-Dias, Fátima
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- 2022
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7. Plasma membrane rigidity effects of 4-hydroxy-2-nonenal in Leishmania, erythrocyte and macrophage
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Alonso, Lais, primary, Menegatti, Ricardo, additional, Dorta, Miriam Leandro, additional, and Alonso, Antonio, additional
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- 2022
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8. New world Leishmania spp. infection in people living with HIV: Concerns about relapses and secondary prophylaxis
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Araújo, Camila Freire, primary, Oliveira, Iara Barreto Neves, additional, Silva, Muriel Vilela Teodoro, additional, Pereira, Ledice Inácia de Araújo, additional, Pinto, Sebastião Alves, additional, Silveira, Murilo Barros, additional, Dorta, Miriam Leandro, additional, Fonseca, Simone Gonçalves, additional, Gomes, Rodrigo Saar, additional, and Ribeiro-Dias, Fátima, additional
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- 2021
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9. Comparative EPR spectroscopy analysis of amphotericin B and miltefosine interactions with Leishmania, erythrocyte and macrophage membranes
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Alonso, Lais, primary, Mendanha, Sebastião Antônio, additional, Gomes, Rodrigo Saar, additional, Dorta, Miriam Leandro, additional, and Alonso, Antonio, additional
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- 2021
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10. Analysis of the Interactions of Amphotericin B with the Leishmania Plasma Membrane Using EPR Spectroscopy
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Alonso, Lais, primary, Mendanha, Sebastião Antônio, additional, Dorta, Miriam Leandro, additional, and Alonso, Antonio, additional
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- 2020
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11. Antileishmanial activity of the chalcone derivative LQFM064 associated with reduced fluidity in the parasite membrane as assessed by EPR spectroscopy
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Alonso, Lais, primary, Menegatti, Ricardo, additional, Gomes, Rodrigo Saar, additional, Dorta, Miriam Leandro, additional, Luzin, Rangel Magalhães, additional, Lião, Luciano Morais, additional, and Alonso, Antonio, additional
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- 2020
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12. Genetic variation in Interleukin-32 influence the immune response against New World Leishmania species and susceptibility to American Tegumentary Leishmaniasis
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dos Santos, Jéssica Cristina, primary, Leite Quixabeira, Valéria Bernadete, additional, Teodoro Silva, Muriel Vilela, additional, Damen, Michelle S. M. A., additional, Schraa, Kiki, additional, Jaeger, Martin, additional, Oosting, Marije, additional, Keating, Samuel T., additional, Dorta, Miriam Leandro, additional, Alves Pinto, Sebastião, additional, Bugalho Duarte, Fernanda, additional, de Araújo Pereira, Ledice Inácia, additional, Netea, Mihai G., additional, Ribeiro-Dias, Fátima, additional, and Joosten, Leo A. B., additional
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- 2020
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13. Antileishmanial and cytotoxic activities of ionic surfactants compared to those of miltefosine
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Alonso, Lais, primary, Cardoso, Éder Jeferson Souza, additional, Gomes, Rodrigo Saar, additional, Mendanha, Sebastião Antônio, additional, Dorta, Miriam Leandro, additional, and Alonso, Antonio, additional
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- 2019
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14. In vitro antileishmanial and cytotoxic activities of nerolidol are associated with changes in plasma membrane dynamics
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Alonso, Lais, primary, Fernandes, Kelly Souza, additional, Mendanha, Sebastião Antônio, additional, Gonçalves, Pablo José, additional, Gomes, Rodrigo Saar, additional, Dorta, Miriam Leandro, additional, and Alonso, Antonio, additional
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- 2019
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15. Alterations in monocyte subsets and cytokine production after TLR activation in American Cutaneous Leishmaniasis
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Quixabeira, Valéria Bernadete Leite, primary, Pereira, Ledice Inacia de Araújo, additional, Veras, Poliana Ribeiro Valadares, additional, da Costa, Ana Carolina Vieira, additional, Fonseca, Larissa Gomides, additional, Galdino Jr., Hélio, additional, da Silva Jr., Ildefonso Alves, additional, Morato, Camila Imai, additional, Pinto, Sebastião Alves, additional, Pereira, Ana Joaquina Cohen Serique, additional, Dorta, Miriam Leandro, additional, Oliveira, Milton Adriano Pelli de, additional, Gomes, Rodrigo Saar, additional, and Ribeiro‐Dias, Fátima, additional
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- 2019
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16. Case Report: Atypical Cutaneous Leishmaniasis in a Patient with Mixed Leishmania guyanensis and Leishmania amazonensis Infection
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Gosch, Carina Scolari, primary, Uliana, Silvia Reni Bortolin, additional, Amorim, Célia Bastos, additional, Dorta, Miriam Leandro, additional, Pinto, Sebastião Alves, additional, Resende, Bruna Silva, additional, Ribeiro-Dias, Fátima, additional, Pereira, Ledice Inacia de Araújo, additional, Coelho, Adriano Cappellazzo, additional, and Marques, Cálita Pollyanna, additional
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- 2018
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17. Human Interleukin-32γ Plays a Protective Role in an Experimental Model of Visceral Leishmaniasis in Mice
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Gomes, Rodrigo Saar, primary, Silva, Muriel Vilela Teodoro, additional, dos Santos, Jéssica Cristina, additional, van Linge, Christine, additional, Reis, Juliana Machado, additional, Teixeira, Mauro Martins, additional, Pinto, Sebastião Alves, additional, Dorta, Miriam Leandro, additional, Bai, Xiyuan, additional, Chan, Edward D., additional, Dinarello, Charles A., additional, Oliveira, Milton Adriano Pelli, additional, Joosten, Leo A. B., additional, and Ribeiro-Dias, Fátima, additional
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- 2018
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18. American tegumentary leishmaniasis: epidemiological and molecular characterization of prevalent Leishmania species in the State of Tocantins, Brazil, 2011-2015
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Gosch, Carina Scolari, primary, Marques, Cálita Pollyanna, additional, Resende, Bruna Silva, additional, Souza, Joandson dos Santos, additional, Rocha, Ray Ameida da Silva, additional, Lopes, Deyse Sabrinne Souza, additional, Gosch, Marcelo Scolari, additional, Dias, Fátima Ribeiro, additional, and Dorta, Miriam Leandro, additional
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- 2017
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19. TLR4 and TLR2 activation is differentially associated with age during Parkinson’s disease
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Rocha Sobrinho, Hermínio Maurício da, primary, Silva, Delson José da, additional, Gomides, Larissa Fonseca, additional, Dorta, Miriam Leandro, additional, Oliveira, Milton Adriano Pelli de, additional, and Ribeiro-Dias, Fátima, additional
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- 2017
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20. Analysis of the Interactions of Amphotericin B with the LeishmaniaPlasma Membrane Using EPR Spectroscopy
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Alonso, Lais, Mendanha, Sebastião Antônio, Dorta, Miriam Leandro, and Alonso, Antonio
- Abstract
Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania amazonensispromastigotes, 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.
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- 2020
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21. Platelet-activating factor increases reactive oxygen species-mediated microbicidal activity of human macrophages infected with Leishmania (Viannia) braziliensis
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Borges, Arissa Felipe, primary, Morato, Camila Imai, additional, Gomes, Rodrigo Saar, additional, Dorta, Miriam Leandro, additional, de Oliveira, Milton Adriano Pelli, additional, and Ribeiro-Dias, Fátima, additional
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- 2017
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22. IL-32γ promotes the healing of murine cutaneous lesions caused by Leishmania braziliensis infection in contrast to Leishmania amazonensis
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Gomes, Rodrigo Saar, primary, Silva, Muriel Vilela Teodoro, additional, dos Santos, Jéssica Cristina, additional, de Lima Silva, Lucas Luiz, additional, Batista, Aline Carvalho, additional, Machado, Juliana Reis, additional, Teixeira, Mauro Martins, additional, Dorta, Miriam Leandro, additional, de Oliveira, Milton Adriano Pelli, additional, Dinarello, Charles A, additional, Joosten, Leo A. B., additional, and Ribeiro-Dias, Fátima, additional
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- 2017
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23. The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins
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Fernandes, Kelly Souza, primary, de Souza, Paulo Eduardo Narcizo, additional, Dorta, Miriam Leandro, additional, and Alonso, Antonio, additional
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- 2017
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24. Decreased Toll-Like Receptor 2 and Toll-Like Receptor 7/8-Induced Cytokines in Parkinson's Disease Patients
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da Silva, Delson José, primary, Borges, Arissa Felipe, additional, Souza, Priscila Oliveira, additional, Reis de Souza, Patrícia, additional, Ribeiro de Barros Cardoso, Cristina, additional, Dorta, Miriam Leandro, additional, Pelli de Oliveira, Milton Adriano, additional, Teixeira, Antônio Lúcio, additional, and Ribeiro-Dias, Fátima, additional
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- 2016
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25. Performance of two immunochromatographic tests for diagnosis of visceral leishmaniasis in patients coinfected with HIV.
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da Silva, Mauro Roberto Biá, Brandão, Natália Alberto Alves, Colovati, Marco, de Sousa, Margella Marconcine Pinheiro, de Lima, Larissa Coelho, Dorta, Miriam Leandro, Ribeiro-Dias, Fátima, Costa, Dorcas Lamounier, Costa, Carlos Henrique Nery, and de Oliveira, Milton Adriano Pelli
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LEISHMANIASIS ,HIV-positive persons ,ANTIGENS ,SENSITIVITY analysis ,VIRAL antibodies ,IMMUNOLOGY ,DISEASES - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2018
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26. TLR4 and TLR2 activation is differentially associated with age during Parkinson’s disease.
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Rocha Sobrinho, Hermínio Maurício da, Silva, Delson José da, Gomides, Larissa Fonseca, Dorta, Miriam Leandro, Oliveira, Milton Adriano Pelli de, and Ribeiro-Dias, Fátima
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PARKINSON'S disease ,TOLL-like receptors ,LEUCOCYTES ,LIPOPOLYSACCHARIDES ,NEUROGLIA ,TUMOR necrosis factors - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2018
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27. Biofilms of black tooth stains: PCR analysis reveals presence of Streptococcus mutans
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Costa,Marília Teixeira, Dorta,Miriam Leandro, Ribeiro-Dias,Fátima, and Pimenta,Fabiana Cristina
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stomatognathic diseases ,PCR ,stomatognathic system ,dental caries ,black-pigmented bacteria ,black tooth stain ,mutans streptococci - Abstract
This study investigated the presence of the black-pigmented bacteria Prevotella nigrescens and Prevotella intermedia, the non-black-pigmented bacteria Actinomyces spp and particularly the cariogenic pathogen Streptococcus mutans in the dental biofilms of patients with or without black extrinsic tooth stains, using the multiplex polymerase chain reaction (PCR) technique. Analysis of the dental biofilms of patients with (n=26) or without (n=26) black tooth stains was performed using duplex PCR for the 16S ribosomal RNA gene (P. nigrescens, P. intermedia, Actinomyces spp) and glucosyltransferase-I gene for S. mutans. P. nigrescens and S. mutans were the most frequent bacteria detected in both groups. The least frequently detected were P. intermedia and Actinomyces spp. The similar bacterial composition of dental biofilms of black tooth stains and healthy tooth surfaces indicates that black tooth stains are not free of cariogenic bacteria. O objetivo deste estudo foi investigar a presença das bactérias pigmentadoras de negro Prevotella nigrescens e Prevotella intermedia, da não pigmentadora de negro Actinomyces spp e particularmente a bactéria cariogênica Streptococcus mutans, no biofilme dentário de pacientes com ou sem manchas dentárias extrínsecas negras, utilizando a técnica multiplex PCR (reação em cadeia da polimerase). Análises do biofilme dentário de pacientes com manchas (n=26) e sem manchas (n=26) foram realizadas utilizando a multiplex PCR para o gene 16S RNA ribosomal (P. nigrescens, P. intermedia, Actinomyces spp) e o gene glucosiltransferase-I para S. mutans. P. nigrescens e S. mutans foram as bactérias mais frequentemente detectadas em ambos os grupos. As menos frequentemente detectadas foram P. intermedia e Actinomyces spp. A similaridade entre a composição bacteriana dos biofilmes dentários das manchas dentárias extrínsecas negras e das superfícies dentárias sem manchas indicam que as manchas dentárias extrínsecas negras não estão livres de bactérias cariogênicas.
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- 2012
28. Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice
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Oliveira, Milton Adriano Pelli de, Pires, Alause da Silva, Bastos, Rosidete Pereira de, Lima, Glória Maria Collet de Araujo, Pinto, Sebastião Alves, Pereira, Ledice Inácia de Araujo, Pereira, Ana Joaquina Cohen Serique, Abrahamsohn, Ises de Almeida, Dorta, Miriam Leandro, and Ribeiro-Dias, Fátima
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Interferon gamma deficient mice ,Leishmania isolation ,parasitic diseases ,Experimental leishmaniasis ,Leishmaniasis ,Leishmania identification ,Leishmania (Viannia) subgenus - Abstract
Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species. O isolamento e a identificação da espécie de parasito do gênero Leishmania são importantes para a confirmação e auxiliam na epidemiologia da leishmaniose. Os camundongos são freqüentemente utilizados para isolar patógenos, porém, as linhagens mais comuns de camundongos são resistentes à infecção por parasitos do subgênero Leishmania (Viannia). Neste estudo, avaliamos a inoculação de macerados de biópsias de pacientes infectados em camundongos deficientes do gene do interferon gama (IFNγ KO) como um método para aumentar a possibilidade de isolar Leishmania spp. Biópsias de 25 pacientes infectados com Leishmania sp. foram avaliadas para a presença de parasitos pelos métodos de imunohistoquímica (IHC) e histopatologia convencional. Os parasitos foram observados, respectivamente, em 88% e 83% das biópsias. Leishmania sp. foi isolada de macerados de biópsia de 52% dos pacientes infectados, quando cultivados em meio Grace, porém, 13% destes isolados foram perdidos devido a contaminações. Inoculação dos macerados em camundongos IFNγ KO proporcionou o isolamento de parasitos oriundos de 31,8% dos pacientes. A maioria dos isolados pertence ao subgênero L. (Viannia), exceto um que pertence ao subgênero L. (Leishmania), como confirmado pela reação da polimerase em cadeia. Nossos resultados preliminares sugerem que o uso de camundongos IFNγ KO pode ser útil para aumentar a possibilidade de isolamento de leishmânias encontradas nas Américas.
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- 2010
29. Identification and Biological Characterization ofLeishmania (Viannia) guyanensisIsolated from a Patient with Tegumentary Leishmaniasis in Goiás, a Nonendemic Area for This Species in Brazil
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Pires, Alause da Silva, primary, Borges, Arissa Felipe, additional, Cappellazzo Coelho, Adriano, additional, Dorta, Miriam Leandro, additional, Lino Junior, Ruy de Souza, additional, Pereira, Ledice Inacia de Araújo, additional, Pinto, Sebastião Alves, additional, Pelli de Oliveira, Milton Adriano, additional, de Matos, Grazzielle Guimarães, additional, Abrahamsohn, Ises A., additional, Uliana, Silvia Reni B., additional, Lima, Glória Maria Collet de Araújo, additional, and Ribeiro-Dias, Fátima, additional
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- 2015
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30. In VitroMetacyclogenesis ofLeishmania (Viannia) braziliensisandLeishmania (Leishmania) amazonensisClinical Field Isolates, as Evaluated by Morphology, Complement Resistance, and Infectivity to Human Macrophages
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da Silva, Ildefonso Alves, primary, Morato, Camila Imai, additional, Quixabeira, Valéria Bernadete Leite, additional, Pereira, Ledice Inácia de Araújo, additional, Dorta, Miriam Leandro, additional, de Oliveira, Milton Adriano Pelli, additional, Horta, Maria Fátima, additional, and Ribeiro-Dias, Fátima, additional
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- 2015
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31. IL-32γ promotes the healing of murine cutaneous lesions caused by Leishmania braziliensis infection in contrast to Leishmania amazonensis.
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Saar Gomes, Rodrigo, Teodoro Silva, Muriel Vilela, dos Santos, Jéssica Cristina, de Lima Silva, Lucas Luiz, Carvalho Batista, Aline, Reis Machado, Juliana, Martins Teixeira, Mauro, Dorta, Miriam Leandro, de Oliveira, Milton Adriano Pelli, Dinarello, Charles A., Joosten, Leo A. B., and Ribeiro-Dias, Fátima
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INTERLEUKINS ,CUTANEOUS leishmaniasis ,WOUND healing ,IMMUNE response ,LABORATORY mice ,THERAPEUTICS - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2017
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32. Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values
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Camargos, Heverton Silva, primary, Moreira, Rodrigo Alves, additional, Mendanha, Sebastião Antonio, additional, Fernandes, Kelly Souza, additional, Dorta, Miriam Leandro, additional, and Alonso, Antonio, additional
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- 2014
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33. Miltefosine Increases Lipid and Protein Dynamics in Leishmania amazonensis Membranes at Concentrations Similar to Those Needed for Cytotoxicity Activity
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Moreira, Rodrigo Alves, primary, Mendanha, Sebastião Antonio, additional, Fernandes, Kelly Souza, additional, Matos, Grazzielle Guimaraes, additional, Alonso, Lais, additional, Dorta, Miriam Leandro, additional, and Alonso, Antonio, additional
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- 2014
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34. Interleukin 32γ (IL-32γ) is highly expressed in cutaneous and mucosal lesions of American Tegumentary Leishmaniasis patients: association with tumor necrosis factor (TNF) and IL-10
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Galdino, Hélio, primary, Maldaner, Anetícia Eduarda, additional, Pessoni, Lívia Lara, additional, Soriani, Frederico M, additional, Pereira, Ledice Inácia de Araújo, additional, Pinto, Sebastião Alves, additional, Duarte, Fernanda Bugalho, additional, Gomes, Clayson Moura, additional, Fleuri, Anna Karoline Aguiar, additional, Dorta, Miriam Leandro, additional, de Oliveira, Milton Adriano Pelli, additional, Teixeira, Mauro Martins, additional, Batista, Aline Carvalho, additional, Joosten, Leo A B, additional, Vieira, Leda Quercia, additional, and Ribeiro-Dias, Fátima, additional
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- 2014
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35. Biofilms of black tooth stains: PCR analysis reveals presence of Streptococcus mutans
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Costa, Marília Teixeira, primary, Dorta, Miriam Leandro, additional, Ribeiro-Dias, Fátima, additional, and Pimenta, Fabiana Cristina, additional
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- 2012
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36. Leishmania major: Recruitment of Gr-1+ cells into draining lymph nodes during infection is important for early IL-12 and IFNγ production
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dos Santos, Milla Schmaltz Tatico, primary, Vaz Cardoso, Ludimila Paula, additional, Nascimento, Gustavo Rios, additional, Lino, Ruy de Sousa, additional, Dorta, Miriam Leandro, additional, de Oliveira, Milton Adriano Pelli, additional, and Ribeiro-Dias, Fátima, additional
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- 2008
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37. In Vitro Metacyclogenesis of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Clinical Field Isolates, as Evaluated by Morphology, Complement Resistance, and Infectivity to Human Macrophages.
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da Silva, Ildefonso Alves, Morato, Camila Imai, Quixabeira, Valéria Bernadete Leite, Pereira, Ledice Inácia de Araújo, Dorta, Miriam Leandro, de Oliveira, Milton Adriano Pelli, Horta, Maria Fátima, and Ribeiro-Dias, Fátima
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REACTIVE oxygen species ,FLOW cytometry ,LEISHMANIASIS ,MACROPHAGES ,RESEARCH funding ,STATISTICS ,T-test (Statistics) ,DATA analysis ,IN vitro studies ,MANN Whitney U Test - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2015
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38. Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values.
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Camargos, Heverton Silva, Moreira, Rodrigo Alves, Mendanha, Sebastião Antonio, Fernandes, Kelly Souza, Dorta, Miriam Leandro, and Alonso, Antonio
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LEISHMANIASIS treatment ,TERPENES ,LIPIDS ,CELL membranes ,ANTIPARASITIC agents ,BIOCHEMICAL mechanism of action ,DRUG delivery systems - Abstract
Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10
6 parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4–9%) at their respective IC50 values. For assays with high cell concentrations (2×109 parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis. [ABSTRACT FROM AUTHOR]- Published
- 2014
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39. Leishmania spp. PARASITE ISOLATION THROUGH INOCULATION OF PATIENT BIOPSY MACERATES IN INTERFERON GAMMA KNOCKOUT MICE.
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de Oliveira, Milton Adriano Pelli, da Silva Pires, Alause, de Bastos, Rosidete Pereira, de Araujo Lima, Glória Maria Collet, Pinto, Sebastião Alves, de Araujo Pereira, Ledice Inácia, Pereira, Ana Joaquina Cohen Serique, de Almeida Abrahamsohn, Ises, Dorta, Miriam Leandro, and Ribeiro-Dias, Fátima
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LEISHMANIA ,LEISHMANIASIS ,BIOPSY ,INJECTIONS ,INTERFERONS ,EPIDEMIOLOGY ,LABORATORY mice ,PATHOGENIC microorganisms ,HISTOPATHOLOGY - Abstract
Copyright of Revista do Instituto de Medicina Tropical de São Paulo is the property of Instituto de Medicina Tropical de Sao Paulo and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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- 2010
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40. Miltefosine Increases Lipid and Protein Dynamics in Leishmania amazonensisMembranes at Concentrations Similar to Those Needed for Cytotoxicity Activity
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Moreira, Rodrigo Alves, Mendanha, Sebastião Antonio, Fernandes, Kelly Souza, Matos, Grazzielle Guimaraes, Alonso, Lais, Dorta, Miriam Leandro, and Alonso, Antonio
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ABSTRACTMiltefosine (MT) is a membrane-active alkylphospholipid licensed for the topical treatment of breast cancer skin metastases and the oral treatment of leishmaniasis, although its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid and a thiol-specific spin label in the plasma membrane of Leishmaniapromastigotes showed that MT causes dramatic increases in membrane dynamics. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.
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- 2014
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41. Leishmania (Viannia) braziliensis amastigotes induces the expression of TNFα and IL-10 by human peripheral blood mononuclear cells in vitro in a TLR4-dependent manner.
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Jr.Galdino, Hélio, Saar Gomes, Rodrigo, dos Santos, Jessica Cristina, Pessoni, Lívia Lara, Maldaner, Anetícia Eduarda, Marques, Stéfanne Madalena, Gomes, Clayson Moura, Dorta, Miriam Leandro, de Oliveira, Milton Adriano Pelli, Joosten, Leo A.B., and Ribeiro-Dias, Fátima
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LEISHMANIA , *TOLL-like receptors , *TUMOR necrosis factors , *IN vitro studies , *LABORATORY mice - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2016
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42. Diagnosis of leishmaniasis in Felis catus domesticus from endemic urban area of northern region of Brazil
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Sousa, Sebastiana Adriana Pereira, Jayme, Valéria de Sá, Santos, Helcileia Dias, Dorta, Miriam Leandro, Galvão, Samara Rocha, Silveira Neto, Osvaldo José da, and Lopes, Welber Daniel Zanetti
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Anticorpo ,Cat ,Amastigota ,Leishmania spp ,Gato ,Antibody ,Reação em cadeia pela polimerase amastigote ,Polymerase chain reaction ,CLINICA VETERINARIA [CLINICA E CIRURGIA ANIMAL] - Abstract
Tendo em vista que o Tocantins mostra-se um dos principais estados brasileiros com maior número de casos de leishmaniose visceral humana e canina e que o gato pode atuar como possível reservatório da doença, objetivou-se detectar a presença de Leishmania spp. em felinos domésticos do município endêmico de Araguaína, estado do Tocantins, abordando as possíveis associações com a infecção (características individuais dos animais, presença de sinais clínicos e co-infecções). Para tal, amostras de sangue, medula óssea, linfonodo, fígado, baço e pele foram analisadas por meio do exame direto, Reação de Imunofluorescência indireta (RIFI) e Reação em cadeia pela Polimerase (PCR). Amostras de soro também foram testadas para o Vírus da Imunodeficiência Felina (FIV) e Vírus da Leucemia Felina (FeLV) utilizando o teste SNAPshot Dx, IDEXX®. Anticorpos anti- Leishmania spp. foram detectados em 26,2% (22/84) (IC: 17,2% - 36,9%) dos animais. Desses, 22,7% (5/22) (IC: 0,74% - 10, 12%) foram reativos para FeLV, sendo que um deles também mostrou positividade para FIV. Não houve associação entre essas infecções e a leishmaniose (p=0,1189). Variáveis como sexo, comprimento do pelo, cor da pelagem, estado corporal, idade e raça não tiveram associação com a soropositividade (p>0,05). A frequência de L. (L.) infantum chagasi obtida pela PCR foi de 4,4% (5/113) (IC: 1,5% - 10%) e o resultado obtido por meio do exame parasitológico direto, foi de 5,3% (3/57) (IC: 1,1% - 14,6%). Dentre os animais positivos no diagnóstico molecular (5/113), 75% (3/4) das amostras de baço, linfonodo submandibular e pele, 60% (3/5) das amostras de sangue e 33,33% (1/3) da amostras de medula mostraram positividade. Dos animais que apresentaram formas amastigotas no exame direto (3/57), todos os tecidos testados foram positivos (3/3). A concordância entre PCR e exame parasitológico foi considerada moderada (valor de Kappa = 0,544, IC: 0,2874 – 0,8006). Houve associação entre positividade e animais que apresentaram pelo menos um sinal clínico (p=0,0231). Dentre estes sinais, esplenomegalia (p=0,0069) e hepatomegalia (p=0,0008) foram estatisticamente significativos. Os resultados obtidos aqui representam considerável impacto do ponto de vista de saúde pública, uma vez que a leishmaniose é grave uma zoonose presente principalmente regiões subdesenvolvidas e em processo de urbanização, como é o caso do Tocantins. Assim, recomenda-se o desenvolvimento e a intensificação de medidas de controle direcionadas à espécie felina, tais como conscientização de órgãos e profissionais de saúde, orientação da população quanto à importância da guarda responsável, assistência veterinária e cuidados ambientais, e capacitação dos profissionais veterinários de forma a atuarem no diagnóstico preciso da infecção. Considering that Tocantins is one of the main brazilian states with the greatest number of cases of human and canine visceral leishmaniasis and that the cat can act as a possible reservoir for the disease, it was aimed to detect the presence of Leishmania spp. in domestic cats of the endemic municipality of Araguaína, state of Tocantins, approaching the possible associations with the infection (individual characteristics of the animals, presence of clinical signs and co-infections). For this, blood, bone marrow, lymph node, liver, spleen and skin samples were analyzed by direct examination, Immunofluorescence Antibody Test (IFAT) and Polymerase Chain Reaction (PCR). Serum samples were also tested for Feline immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) using the SNAPshot Dx, IDEXX® test. Anti-Leishmania spp. antibodies were detected in 26.2% (22/84) (IC: 172% - 36.9%) of the animals. Of these, 22.7% (5/22) (CI: 0.74% - 10.12%) were reactive for FeLV, one of which also showed positivity for FIV. There was no association between these infections and leishmaniasis (p = 0.1189). Variables such as sex, hair length, coat color, body condition, age and race had no association with seropositivity (p> 0.05). The frequency of L. (L.) infantum chagasi obtained by PCR was 4.4% (5/113) (CI: 1.5% - 10%) and the result obtained through direct parasitological examination was 5.3% (3/57) (CI: 1.1% - 14.6%). Among the positive animals in the molecular diagnosis (5/113), 75% (3/4) of the spleen, submandibular lymph nodes and skin samples, 60% (3/5) of the blood samples and 33.33% (1/3) of the bone marrow samples showed positivity. Of the animals that presented amastigote forms in the direct examination (3/57), all tested tissues were positive (3/3). The agreement between PCR and parasitological examination was considered moderate (Kappa = 0.544, CI: 0.2874 - 0.8006). There was an association between positivity and animals presenting at least one clinical sign (p = 0.0231). Among these signs, splenomegaly (p =0.0069) and hepatomegaly (p = 0.0008) were statistically significant. The results obtained here represent a considerable impact from the point of view of public health, since the leishmaniasis is serious a zoonosis present mainly undeveloped regions and in the process of urbanization, as is the case of Tocantins. Thus, it is recommended the development and intensification of control measures directed to the feline species, such as the awareness of organs and health professionals, the orientation of the population regarding the importance of responsible care, veterinary assistance and environmental care, and training of veterinary professionals in order to act in the precise diagnosis of the infection. Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq Outro
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- 2017
43. Standardization of PCR / RFLP product analysis that amplifies the Ribosomal Internal Transcribed spacer (ITS) and Glucose- 6-phosphate dehydrogenase (G6PD) genes for identification of Leishmania spp. in polyacrylamide gel
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Marques, Cálita Pollyanna, Dorta, Miriam Leandro, Soares, Joanna Darc A. Herzog, and Gomes, Rodrigo Saars
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Padronização ,PARASITOLOGIA [CIENCIAS BIOLOGICAS] ,Polyacrylamide ,Validação ,Molecular analysis ,Discriminate species ,Validation ,Poliacrilamida ,Discriminar espécies ,Standardization ,Análise molecular - Abstract
A Leishmaniose Tegumentar Americana (LTA) é causada por protozoários do gênero Leishmania e acomete pele e/ou mucosas. É uma zoonose endêmica no Brasil sendo notificada em todas as Regiões. Há uma relação entre a espécie de parasito e as manifestações clínicas, contribuindo à complexidade diagnóstica, que até o presente, não existi o diagnóstico espécie-específico. No presente trabalho, a proposta foi padronizar a análise dos produtos da PCR/RFLP aos genes ITS e G6PD em gel de poliacrilamida. Para padronizar as análises dos produtos da PCR/RFLP aos genes ITS e G6PD, foram utilizadas DNA da amostra de Leishmania spp., de referência, sendo testada a poliacrilamida na concentração de 5%, 6%, 7% 8% e 10%. O delineamento do protocolo considerou o tamanho dos amplicons gerados pela PCR/RFLP, correlacionando-o a concentração da poliacrilamida. Para padronizar o tempo da eletroforese foi monitorada a migração dos corantes usados no tampão da amostra de DNA. Para validar a técnica, foram selecionadas 521 amostras de pacientes tocantinenses fixadas em lâminas e 43 amostras de isolados de parasitos armazenados no Leishbank. As amostras dos pacientes tocantinenses foram analisadas pela PCR/RFLP ao kDNA e foram selecionadas àquelas positivas para a validação. A concentração da matriz para analise dos produtos da PCR e RFLP ao gene ITS ficou padronizada respectivamente em 6% e 8% e distinguiu a espécie L.(L.) amazonensis, porém as espécies L. (V.) braziliensis, L. (V.) guyanensis e L. (V.) lainsoni não foram distinguidas entre si, ocorrendo o mesmo entre L. (V.) naiffi e L. (V.) shawi. A concentração da matriz para analise dos produtos da PCR ao gene G6PD ficou padronizada em 6% e distinguiu L. (V.) braziliensis das demais. A validação da análise em gel de poliacrilamida dos produtos da PCR ao alvo ITS a partir de amostras de pacientes fixadas em lâminas não ser realizada, pois das 256 amostras analisadas somente 4 amplificaram. A validação da análise dos produtos da PCR ao alvo G6PD em gel de poliacrilamida a partir de 48 amostras fixadas em lâminas caracterizou 81% como L. (V.) braziliensis e, caracterizou 63% dos isolados de parasitos como L. (V.) braziliensis. As análises pela PCR/RFLP realizadas para discriminar Leishmania spp. se complementam e, assim, contribuem para o diagnóstico espécie-específico da LTA. American Cutaneous Leishmaniasis (LTA) is caused by protozoa of the genus Leishmania and it attacks the skin and/or mucous membranes. It is an endemic zoonosis in Brazil being notified in all the Regions. There is a relation between the species of parasite and the clinical manifestations, contributing to the diagnostic complexity, which up to the present, did not exist species-specific diagnosis. In the present work, the proposal was to standardize the analysis of the PCR/RFLP products to the ITS and G6PD genes in polyacrylamide gel. In order to standardize PCR/RFLP analysis on the ITS and G6PD genes, DNA from the reference Leishmania spp. Sample was used and 5%, 6%, 7% 8% and 10% polyacrylamide were tested. The protocol design considered the size of the amplicons generated by the PCR / RFLP, correlating the concentration of the polyacrylamide. To standardize the electrophoresis time the migration of the dyes used in the DNA sample buffer was monitored. To validate the technique, we selected 521 samples from Tocantins patients fixed on slides and 43 samples of parasite isolates stored in Leishbank. The samples from the patients from Tocantins were analyzed by PCR/RFLP to the kDNA and were selected to those positive for the validation. The concentration of the matrix for analysis of the PCR and RFLP products to the ITS gene was standardized in 6% and 8%, respectively, and distinguished the species L. (L.) amazonensis, but the species L. (V.) braziliensis, L. (V.) guyanensis and L. (V.) lainsoni were not distinguished among themselves, occurring the same between L. (V.) naiffi and L. (V.) shawi. The concentration of the matrix for analysis of the PCR products to the G6PD gene was standardized in 6% and distinguished L. (V.) braziliensis from the others. The validation of the polyacrylamide gel analysis of the PCR products to the ITS target from samples of patients fixed in slides was not performed, since of the 256 samples analyzed only 4 amplified. The validation of the analysis of the products of the PCR to the G6PD target in polyacrylamide gel from 48 samples fixed in slides characterized 81% as L. (V.) braziliensis and, characterized 63% of the isolates of parasites as L. (V.) braziliensis. The PCR/RFLP analyzes performed to discriminate Leishmania spp. are complementary and thus contribute to the species-specific diagnosis of LTA. Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
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- 2017
44. Evaluation of monocytes from patients with tegumentary leishmaniasis before and after treatment
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Veras, Poliana Ribeiro Valadares, Dias, Fátima Ribeiro, Dorta, Miriam Leandro, and Gomes, Rodrigo Saar
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Monócitos ,IMUNOLOGIA APLICADA [IMUNOLOGIA] ,Leishmanioses ,Leishmaniasis ,Monocytes - Abstract
A leishmaniose tegumentar americana (LTA) é uma doença infecto-parasitária causada por protozoários do gênero Leishmania. Esta doença apresenta-se como leishmaniose cutânea (LC) e leishmaniose mucosa (LM). Os mecanismos da imunopatogenia e do controle da infecção ainda não estão completamente esclarecidos, sendo que os monócitos podem participar destes mecanismos. Os monócitos são subdivididos em três populações (CD14hiCD16-, os clássicos; CD14hiCD16+, os intermediários; e CD14loCD16+, os não clássicos) e são as principais células produtoras de citocinas no sangue periférico. O objetivo deste estudo foi avaliar se existem alterações nas proporções das três subpopulações dos monócitos e das citocinas fator de necrose tumoral (TNF) e interleucina 10 (IL-10), em pacientes com LC ou LM, antes e após o tratamento. Sangue periférico de pacientes (n = 54; 31 LC e 23 LM) e controles (n = 54) foi utilizado para identificar os monócitos por citometria de fluxo e para hemoculturas. As hemoculturas foram incubadas na ausência (Meio) ou presença de agonistas (LPS, Pam3Cys) de receptores similares a toll dos tipos dois e quatro (TLR2 e TLR4) ou de antígenos (AG) de L. (Viannia) braziliensis, para avaliar a produção de TNF e IL-10. As citocinas foram quantificadas por ensaio imunoenzimático (ELISA). Os resultados mostraram um aumento da porcentagem dos monócitos CD16+, especialmente da subpopulação CD14loCD16+, em pacientes com LC, mas não com LM, antes do tratamento (p < 0,05). Após o tratamento, as porcentagens destes monócitos, em pacientes com LC, retornaram para níveis similares àqueles de indivíduos sadios. Em pacientes com LM, também ocorreu uma redução das porcentagens dos monócitos CD16+ após o tratamento. A produção de TNF e a de IL-10 não estavam significantemente alteradas nas hemoculturas de pacientes, quando comparadas com as de controles sadios. Entre os estímulos usados, somente o AG não induziu quantidades significantes de IL-10 nas hemoculturas dos pacientes. Após o tratamento, as concentrações de TNF diminuíram nas hemoculturas de pacientes com LC, exceto quando o estímulo foi Pam3Cys (TLR2), o qual induziu aumento na produção de TNF (p < 0,05). Em hemoculturas de pacientes com LM não foram detectadas diferenças significantes entre as concentrações de TNF e IL-10 produzidas antes e após o tratamento. As concentrações de IL-10 também não foram significantemente alteradas após o tratamento dos pacientes com LC. Os dados indicam que as porcentagens de monócitos CD16+ estão aumentadas na LC. Ainda, sugerem que monócitos de pacientes com LC ou LM apresentam uma diminuída capacidade de produção de IL-10 em resposta ao AG, o que pode dificultar o controle da resposta inflamatória. Os dados também sugerem que a capacidade de resposta dos monócitos via TLR2 pode estar suprimida na LC, sendo elevada após o tratamento. Uma análise de TLR2 e TLR4 em monócitos de pacientes com LTA pode auxiliar a entender melhor a indução de citocinas nestas células. American tegumentary leishmaniasis (ATL) is an infectious parasitic disease caused by Leishmania protozoa. The disease presents as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms of the immunopathogenesis and infection control are not yet fully understood, and monocytes can be involved in these mechanisms. The monocytes are divided into three subsets (CD14hiCD16-, classical monocytes; CD14hiCD16+, the intermediates; and CD14loCD16+ non- classical) and are the major cell producing cytokines in peripheral blood.The objective of this study was to assess possible alterations in the percentages of monocytes subsets; and in the tumor necrosis factor (TNF) and interleukin 10 (IL -10) production in whole blood cultures from patients with CL and ML, before and after treatment. Peripheral blood from patients (n = 54; 31 CL and ML 23) and controls (n = 54) was used to identify monocytes by flow cytometry and for whole blood cultures. The blood was incubated in the absence (medium) or presence of toll-like receptor agonists (Pam3Cys and LPS for TLR2 and TLR4, respectively) or L. (V.) braziliensisantigens (AG) to assess TNF and IL-10. Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA).The results showed an increase in the percentage of CD16+ monocytes, especially CD14loCD16+monocyte subset in CL patients, but not in ML, before treatment (p
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- 2015
45. Evaluation of the use of immunoglobulin E (IgE) anti - leishmania as marker of prognosis in american Tegumentary leishamaniasis
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Leão, Natallia Moreira Lopes, Dorta, Miriam Leandro, Ametila, Roqueline, Ferreira, Glória Martins de Freitas Aversi, and Dias, Fátima Ribeiro
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Tratamento ,PARASITOLOGIA [CIENCIAS BIOLOGICAS] ,treatment ,LTA ,ELISA ,IgE ,L. (V.) braziliensis - Abstract
As leishmanioses compreendem um grupo de doenças, com grande diversidade epidemiológica e clínica. A detecção de IgE pode constituir uma ferramenta valiosa para o prognóstico da Leishmaniose Tegumentar Americana (LTA). O objetivo do presente trabalho foi avaliar se Imunoglobulina E (IgE) anti - Leishmania podem ser utilizados como biomarcadores de prognóstico na LTA. Foi realizada a padronização do ensaio imunoenzimático (ELISA) utilizando extrato antigênico de L. (V.) braziliensis para detecção de IgE específica para leishmânia, nos plasmas de pacientes com LTA. Foi determinado que a diluição que discriminava amostras positivas de negativas era de 1/20 para o plasma e 1/100 para o anticorpo anti-IgE marcado com peroxidase. Dos 40 pacientes com LTA atendidos no ambulatório de endemias do Hospital Anuar Auad (HDT/GO), Goiânia-GO, entre 2009 e 2012, 27 apresentavam leishmaniose cutânea localizada (LCL) e 1 leishmaniose mucosa (LM). Destes pacientes, 27 eram do sexo masculino e 13, feminino. A idade dos pacientes com LCL variou entre 18 a 65 anos e com LM, de 25 a 61 anos. Os pacientes com LCL apresentavam de uma a 20 lesões, os tamanhos das lesões variaram entre 0,2 a 7 cm e o tempo das lesões ao diagnóstico variou de 0,5 a 4 meses e com LM de 0,5 a 120 meses. A positividade dos testes laboratoriais AH, IDRM, IF e Elisa para IgG foi de 80%, 67,5%, 42,5% e 87,5% respectivamente. Foi realizada análise de reatividade cruzada com amostras de pacientes com blastomicose, malária, leishmaniose visceral (LV), doença de chagas e toxoplasmose e apenas uma amostra de paciente com LV (10%) foi reagente, a especificidade do teste foi de 98,3%. A detecção de IgE específica ocorreu em 40% dos pacientes com LTA. Não houve correlação entre as quantidades de IgE e os resultados da IDRM. Não foi observada correlação entre os índices de IgE e o número de lesões em pacientes com LCL. Não foi detectada uma associação entre cura ou não cura clínica e os índices de reatividade de IgE. Um aumento do número de amostras de pacientes é necessário para melhorar a acurácia dos resultados. Leishmaniases comprise a disease group with a great epidemiological and clinical diversity. The detection of IgE can be an important tool for prognosis of American Tegumentary Leishmaniasis (LTA). The objective of this present work was to assess if the immunoglobulin E (IgE), anti - Leishmania can be used as biomarkers for prognosis in LTA. The standardization of an enzyme-linked immunosorbent assay (ELISA) was carried out applying antigenic extract of L. (V.) braziliensis to detect IgE specific for leishmania in LTA patients’ plasmas. It was determined that the dilution which differed positive from negative samples was 1/20 for plasma and 1/100 for peroxidase labeled anti-IgE antibody. Forty LTA patients were assisted in the laboratory of endemic diseases at AnuarAuad Hospital (HDT/GO), Goiânia - GO, between 2009 and 2012. Twenty-seven of those patients showed localized cutaneous leishmaniasis (LCL) and 1 showed mucosal leishmaniasis (LM). Twenty-seven of those patients were male and thirteen were female. The LCL patients’ ages varied between 18 to 65 years and LM patients’ ages varied from 25 to 61 years. The LCL patients showed 1 to 20 lesions whose sizes varied from 0,2 to 7 cm and the duration of the lesion diagnostic varied from 0,5 to 4 months, and in LM patients it varied from 0,5 to 120 months. The positive result of the AH, IDRM, IF and Elisa lab tests for IgG was 80%, 67,5%, 42,5% and 87,5% respectively. A cross - reactivity analysis was performed using samples of patients with blastomycosis, malaria, visceral leishmaniasis (LV), Chagas disease and toxoplasmosis and only one sample from an LV patient (10%) was positive, the test was 98,3% precise. The specific IgE detection occurred in 40% of the LTA patients. No correlation between the quantity of IgE and IDRM results was found. No correlation between the index of IgE and the number of lesions in LCL patients was observed. No association between clinical cure or no cure and the index of IgE reactivity was detected. A higher number of patients’ samples are necessary to obtain more accurate results. Outro
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- 2015
46. Evaluation of innate immune response cell recruitment by BCG Moreau in the absence of cytokine IL-17 and IL-22
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Resende, Danilo Pires de, Junqueira-Kipnis, Ana Paula, kipnis, André, Nagib, Patrícia Resende Alo Loyola, and Dorta, Miriam Leandro
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Innate immune responce ,IMUNOLOGIA [CIENCIAS BIOLOGICAS] ,Tuberculose ,BCG ,Tuberculsis ,Resposta imune inata - Abstract
A tuberculose é uma doença infecto-contagiosa responsável por milhões de mortes todos os anos. BCG (Bacille Calmette-Guérin) é a única vacina autorizada para a prevenção da tuberculose (TB) humana. A produção precoce de IL-17 e IL-22, como a induzida pela BCG, é importante para formação de células de memória protetoras na TB. Neste contexto, avaliamos o papel destas citocinas no recrutamento e ativação das células da imunidade inata induzida pela vacina BCG. Foram utilizados camundongos C57BL/6, IL22_/_ e IL17_/_. Células do lavado peritoneal foram avaliadas por citospin e citometria 24 horas, 2º, 3º, 5º e 7º dias após infecção com BCG, via intraperitoneal (1x106/mL). O recrutamento de macrófagos (F4/80+CD11b+) para o peritônio foi semelhante em todos os animais avaliados, porém nos camundongos IL17_/_ estas células expressavam mais moléculas de MHC de classe II. Os neutrófilos (Gr1+) foram inicialmente mais recrutados nos camundongos IL17_/_ e IL22_/_ em relação ao C57BL/6 (50,9±5,4; 31,7±1,7). Após 5 dias da inoculação, estas células apresentavam-se mais ativadas (Gr1+CD11b+) no camundongo IL17_/_ que nos outros animais. Quanto as células NK (NK1.1+), dois dias após a inoculação, houve recrutamento nos C57BL/6 enquanto que poucas células NK migraram para o peritônio nos camundongos IL22_/_ e IL17_/_. Novamente essas células encontravam-se mais ativadas nos IL17_/_ que nos outros animais (NK1.1+CD11b+). A vacinação intraperitoneal com BCG recruta macrófagos, neutrófilos e células NK para o peritônio. Na ausência de IL17 estas células são mais ativadas do que nos outros camundongos, indicando um perfil de ativação superior. Tuberculosis is an infectious disease responsible for millions of deaths every year. BCG (Bacille Calmette-Guérin) is the only licensed vaccine for the prevention of human tuberculosis (TB). Early production of IL-17 and IL-22 as induced by BCG is important for protective memory cells formation in TB. In this context, we evaluated the role of these cytokines in the recruitment and activation of cells of the innate immunity induced by BCG vaccine. C57BL/6, IL22 _ /_ and IL17_ /_ mice were used. Cells obtained after peritoneal lavage were evaluated by flow cytometry and citospin 24 hours, 2, 3, 5 and 7 days after infection with BCG, intraperitoneally (1x106/ml). The macrophages recruitment (F4/80 + CD11+) to the peritoneum was similar for all animals, but in IL17_ /_ mice, these cells expressed more MHCII molecules. Neutrophils (Gr1+), initially presented higher percentage in IL17_/_ and IL22_/_ mice compared with C57BL/6 (50.9 ± 5.4, 31.7 ± 1.7). These cells where more activated (Gr1+CD11b+) in the IL17_/_ mice at 5 days post inoculation. NK cells (NK1.1+) was recruited in C57BL/6, mice whereas few NK cells migrated to the peritoneum in IL22_ /_ and IL17_/_ mice, two days after inoculation. Again, these cells were also more activated in IL17_/_ than in other animals (NK1.1+ CD11b+). In summary, intraperitoneal immunization with BCG recruit macrophages, neutrophils and NK cells into the peritoneum. In the absence of IL17 these cells are more activated than in the other mice, indicating a higher activated profile, although these cells were reduced. Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
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- 2015
47. Evaluation of accuracy of tests immunochromatographic rK39 in diagnosis of visceral leishmaniasis in patients coinfected with HIV
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Silva , Mauro Roberto Biá da, Oliveira, Milton Adriano Pelli de, Costa, Carlos Henrique Nery, Pereira, Ledice Inácia de Araújo, Lino Junior, Ruy de Souza, and Dorta, Miriam Leandro
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Visceral leishmaniasis ,IMUNOLOGIA [CIENCIAS BIOLOGICAS] ,Saliva humana ,Soro humano ,Leishmaniose visceral ,HIV / AIDS ,HIV/AIDS ,Teste imunocromatográfico rK39 ,Saliva ,rK39 - Abstract
Introdução: A Leishmaniose Visceral (LV) é uma antropozoonose causada por protozoários do gênero Leishmania, especialmente Leishmania (Leishmania) infantum ou Leishmania (Leishmania) donovani. Está presente em regiões tropicais e subtropicais e é considerada uma doença negligenciada. O diagnóstico preciso da LV é geralmente difícil, principalmente em pacientes coinfectados pelo HIV, porque a LV apresenta formas clínicas atípicas e porque o diagnóstico sorológico se torna pouco confiável. Objetivo: Avaliar a acurácia de Testes Imunocromatográficos rK39 no diagnóstico da LV em soro e saliva de pacientes coinfectados com HIV. Métodos: Pacientes suspeitos de LV foram atendidos no Instituto de Doenças Tropicais Natan Portela - IDTNP, Teresina, Piauí, Brasil, no período de março de 2011 a outubro de 2012. Além do exame clínico, foi realizado o teste IC rK39 em saliva e sangue, aspiração de medula óssea para exames parasitológicos e PCR- RFLP. Como rotina na instituição, pacientes sugestivos de LV também foram investigados para HIV. Foram coletadas amostras de medula óssea para pesquisa de Leishmania em esfregaço, cultura e PCR. Para pesquisa de Leishmania spp. em aspirado de medula óssea, utilizou-se a coloração pelo Panótico (RANYLAB Química Farmacêutica). A leitura das lâminas coradas foi realizada em microscópio óptico com objetiva de imersão, aumento de 1000x. Para realização da cultura de aspirado de medula óssea, o aspirado foi cultivado em 3 mL de meio NNN (McNeal, Novy & Nicolle) e 500 μL de meio Schneider a 26ºC. A pesquisa de formas promastigotas de Leishmania spp. foi realizada a cada sete dias em lâmina – lamínula em microscópio óptico. Foram coletados 5 mL de sangue periférico em tubos Vaccutainer® sem anticoagulante para obtenção do soro. Em seguida, os soros foram aliquotados em tubos de crioensaio e mantidos em freezer a -70ºC. A saliva foi recolhida em tubos de polipropileno de 50 mL e para aumentar a quantidade de saliva, os pacientes receberam um pedaço de Parafilm® para mastigar. A saliva foi aliquotada em tubos de crioensaio e mantidas em freezer a -70ºC. Resultados: Pacientes com LV que possuiam o teste IC rK39 positivo no soro, apresentaram uma positividade de 58,6% (n = 17) na saliva (SalPos), enquanto todos os doadores saudáveis (CT) (n = 20) foram negativos no soro e na saliva. A quantidade de IgG total e específica no soro do paciente com LV foi significativamente mais elevada do que no grupo CT. A IgG xiii específica do grupo SalPos foi maior do que no grupo de pacientes com LV com saliva negativa (SalNeg), mas não foi estatisticamente significativa. A quantidade de IgA total ou específica foi semelhante em todos os grupos. A quantidade de IgG específica para Leishmania observada na saliva de ambos os grupos SalPos e SalNeg foi maior do que no grupo controle, mas o grupo com saliva SalPos apresentou mais IgG específico para Leishmania do que o grupo SalNeg. Dos 86 pacientes com LV, 33 possuíam sorologia positiva para HIV, cinco apresentaram sorologia negativa, porém, eram portadores do vírus e estavam em tratamento, totalizando 37 indivíduos infectados. A sensibilidade para LV na PCR foi de 100%, já que este foi o teste considerado ouro neste estudo. Os demais testes tiveram sensibilidade de 57,14% (n= 49) (cultura de aspirado medular), 48,71% (n = 78) (análise de esfregaço de aspirado medular), 55,81% (n = 86) e 56,25% (n = 85) para os testes IC rK39 no soro Orangelife ou Kalazar Detect respectivamente. No caso do uso do teste parasitológico como padrão ouro, a sensibilidade dos testes sorológicos foi superior a 82%. Ainda não está bem esclarecido se a infecção pelo HIV é capaz de interferir nos testes sorológicos. Conclusões: Os testes IC rK39, com utilização de soro, tem uma baixa sensibilidade quando aplicados a pacientes coinfectados com LV-HIV, e o uso da saliva para o diagnóstico da LV é limitado. Background: Visceral Leishmaniasis (VL) is an anthropozoonosis caused by protozoa of the genus Leishmania, especially Leishmania (Leishmania) infantum or Leishmania (Leishmania) donovani. It is present in tropical and subtropical regions and it is considered a neglected disease. The LV diagnosis is dificult, mainly for patients co infected with HIV, because LV paients present atipical clinic forms and the sorology is not trustfull Objectives: The aim of this thesis is to evaluate the accuracy of Immunochromatographic rK39 tests in the diagnosis of Visceral Leishmaniasis in serum and saliva of HIV-co-infected patients Methods: VL suspected patients were treated at the Institute of Tropical Diseases Natan Portela - IDTNP, Teresina, Piauí, Brazil, from March 2011 to October 2012. In addition to the clinical examination, it was performed the IC rK39 test in saliva and blood and also the bone marrow aspiration for parasitological and the PCR-RFLP tests. As routine in the institution, VL suggestive patients were also investigated for HIV. Bone marrow samples were collected for Leishmania research in smear, culture and PCR. For the research of Leishmania spp. in bone marrow aspirate, the Panoptic stain was used (RANYLAB Pharmaceutical Chemistry). The analyses of the stained slides were done in an optical microscope with immersion objective, magnification of 1000x. To perform the bone marrow aspirate culture, the aspirate was cultivated in 3 mL of NNN medium (McNeal, Novy & Nicolle) and 500 μL Schneider medium at 26°C. The search for promastigotes. was performed every seven days in blade - cover slip in an optical microscope. 5 mL were collected from peripheral blood in Vaccutainer® tubes without any anticoagulant in order to obtain the serum. Sera were then aliquoted in cryoassay tubes and kept in a freezer at -70°C. Saliva was collected in 50 mL polypropylene tubes and, in order to increase the amount of saliva, the patients received a piece of Parafilm® for chewing. Saliva was aliquoted in cryoessay tubes and kept in a freezer at -70°C. Results: VL patients who possessed the IC rK39 test with positive serum showed 58.6% of positivity (n = 17) in saliva (SalPos), while all of the healthy donors (CT) (n = 20) were negative in serum and saliva. The amount of total and specific IgG in the serum of patients with VL was significantly higher than that in the CT group. The specific IgG of the SalPos group was greater than in VL patients with negative saliva (SalNeg), but it was not statistically xv significant. The amount of total or specific IgA was similar in all groups. The amount of specific IgG for Leishmania found in saliva of both the SalPos and SalNeg groups was higher than in the control group, but the group with SalPos saliva presented more specific IgG to Leishmania than the SalNeg group. Among the 86 VL patients, 33 had positive serology for HIV and five tested negative serology, however, they were carriers of the virus and were being treated, totalizing 37 infected individuals. The sensitivity of VL in PCR was of 100%, since this test was considered gold standard in this study. The other tests had sensitivity of 57.14% (n = 49) (bone marrow aspiration culture), 48.71% (n = 78) (marrow aspirate smear analysis), 55.81% (n = 86) and 56 25% (n = 85) for the IC rK39 tests in Orangelife or Kalazar Detect serum, respectively. In the case of using the parasitological test as the gold standard, the sensitivity of serological tests was greater than 82%. It is not clear if HIV infection is able to interfere in the serological tests yet. Conclusions: The rK39 IC tests, with the utilization of serum, have a low sensitivity when applied to patients co-infected with HIV and VL, and the use of saliva for VL diagnosis is limited. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
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- 2014
48. Cutaneous Leishmaniasis in the Midwest region: review of clinical, epidemiological, laboratory and molecular data
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Balian, Rosana Pereira Morais, Dorta, Miriam Leandro, Moraes, Sandra do Lago, and Loyola, Patrícia Resende Alo Nagib
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LTA ,Cutaneous Leishmaniasis ,Centro-Oeste ,DOENCAS INFECCIOSAS E PARASITARIAS [CLINICA MEDICA] ,Dados epidemiológicos, laboratoriais e moleculares ,Midwest ,Clinical, epidemiological, laboratory and molecular - Abstract
A leishmaniose tegumentar americana (LTA) é causada por protozoários do gênero Leishmania, que acomete pele e/ou mucosas. É uma zoonose endêmica, cujos números de casos na região Centro-Oeste são crescentes. No Estado de Goiás, foram notificados 2798 casos de LTA, entre os anos de 2007 e 2013. No presente trabalho a nossa proposta foi Investigar as características epidemiológicas e moleculares da LTA de pacientes atendidos no ambulatório de endemias do hospital Anuar Auad no período de 2000 a 2006 e identificar as espécies de Leishmania spp. Participaram do estudo 152 pacientes com LTA, 124 do Estado de Goiás e 28 do Mato Grosso, com faixa etária de 6 a 79 anos. Destes, 113 eram do sexo masculino. Para o diagnóstico foi realizada a avaliação clínicoepidemiológica e laboratorial, tais como exame direto (ED), análise histopatológica (AH), intradermorreação de Montenegro (IRM), imunofluorescência indireta (IFI) e ELISA utilizando extrato total de L. (Viannia) braziliensis. Foi feita a caracterização das espécies de leishmânias por reação em cadeia da polimerase (PCR). A positividade do ED, AH, IRM, IFI e ELISA foi de 70,6%, 80,9%, 68,9%, 44,2% e 73,0%, respectivamente. Foram detectadas IgG específica para L. (V.) braziliensis em 84,7% dos pacientes com leishmaniose mucosa (LM), sendo significativamente superiores às encontradas em pacientes com leishmaniose cutânea (LC), que foi de 69,0% (p
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- 2014
49. Evaluation of antigenicity of recombinant proteins of L. (V.) braziliensis
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Alves, José Vitor Ferreira, Dorta, Miriam Leandro, Moraes, Sandra do Lago, and Dias, Fátima Ribeiro
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Leishmania ,Recombinant proteins ,IMUNOLOGIA [CIENCIAS BIOLOGICAS] ,Imunodiagnóstico ,LTA ,ATL ,ELISA ,Immunodiagnostic ,Proteínas recombinantes - Abstract
As leishmanioses compreendem um grupo de doenças que apresentam características clínicas, histopatológicas e imunológicas distintas, é causada por parasitos intracelulares que pertencem ao gênero Leishmania. Os testes sorológicos utilizados até o presente, para o diagnóstico, apresentam várias limitações e por isto é de grande importância identificar proteínas imunogênicas da Leishmania, para que sejam testadas como potenciais antígenos para o desenvolvimento de técnicas para o diagnóstico da leishmaniose tegumentar americana (LTA). O objetivo deste estudo foi produzir e purificar as proteínas recombinantes “Leishmania activated C kinase” (rLACK); “Thiol Specific Antioxidant” (TSA), “Leishmania elongation initiation factor” (LeIF) e “Leishmania braziliensis stress inducible protein 1” (LbSTI) e avaliar a sua antigenicidade. As proteínas recombinantes foram produzidas pela técnica de DNA recombinante segundo descrito por Salay et al. (2007). Para a realização do ELISA foram cultivadas as espécies L. (V.) braziliensis (MHOM/BR/1975/M2903) e L. (L.) amazonensis (IFLA/BR/67/PH8) e seus extratos antigênicos e as proteínas recombinantes produzidas foram utilizados como antígenos. Foram utilizadas 60 amostras de pacientes com LTA, atendidos no hospital Anuar Auad, Goiânia, Goiás, sendo que destas, 45 eram de pacientes com leishmaniose cutânea localizada (LCL) e 15 eram de leishmaniose mucosa (LM). Para a análise da especificidade foram utilizados o soro de pacientes com outras patologias. O extrato total de L. (L.) amazonensis, rLACK, rTSA, rLbSTI, rLeIF apresentarem sensibilidade de 85%, 75%, 70%, 76,7%, e 56,1% respectivamente. O extrato total de L. (L.) amazonensis, rLACK, rTSA, rLbSTI, rLeIF obtiveram especificidade de 72,5%, 80%, 60%, 30% e 62,5%, respectivamente. Os resultados do presente trabalho demonstraram que as proteínas recombinantes e o extrato total de L. (L.) amazonensis foram antigênicas e o extrato total de L. (L.) amazonensis e a rLACK foram os antígenos que tiveram as melhores sensibilidades e especificidades. Leishmaniasis is a group of diseases with distinct clinical, histopathological and immunological characteristics, caused by parasites belonging to the Leishmania genus. The serological tests used until the moment have several limitations. There are a great interest to identify immunogenic proteins of Leishmania to be tested as potential antigens for the development of techniques for the diagnosis of American cutaneous leishmaniasis (ACL). The aim of this study was to produce and purify the recombinant proteins "Leishmania activated C kinase" (rLACK); "Thiol Specific Antioxidant" (TSA), "Leishmania elongation initiation factor" (LeIF) and "Leishmania braziliensis stress inducible protein 1" (LbSTI) to evaluate the antigenicity. The recombinant proteins were produced by recombinant DNA techniques as described by Salay et al. (2007). To perform the ELISA, L.(V.) braziliensis (MHOM/BR/1975/M2903) and L.(L.) amazonensis (IFLA/BR/67/PH8) species were cultivated and the antigenic extracts and recombinant proteins were used as antigens. Sixty serum samples from patients with ATL assisted at Anuar Auad hospital, Goiânia, Goiás, were assayed, and from them, 45 were from patients with localized cutaneous leishmaniasis (LCL) and 15 were from mucosal leishmaniasis (ML). To analyze the specificity of the response of total extract and the recombinant proteins, sera from patients with other pathologies were tested. The total extract of L. (L.) amazonensis, rLACK, rTSA, rLbSTI and rLeIF showed a sensitivity of 85%, 75%, 70%, 76.7% and 56.1% respectively. The specificity of total extract of L. (L.) amazonensis, rLACK, rTSA, rLbSTI, rLeIF were 72.5%, 80%, 60%, 30% and 62.5% respectively. Thus, these results showed that recombinants proteins and total extract of L. (L.) amazonensis were antigenic and the total extract of L. (L.) amazonensis and rLACK were the antigen that had the best sensibility and specificity.
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- 2014
50. In vitro evaluation of metacyclogenesis of L. (V.) braziliensis e L. (L.) amazonensis
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Silva Junior, Ildefonso Alves da, Dias, Fátima Ribeiro, Dorta, Miriam Leandro, and Horta, Maria de Fátima
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Macrófago humano ,IMUNOLOGIA [CIENCIAS BIOLOGICAS] ,Leishmania amazonensis ,Human macrophage ,Leishmania braziliensis ,Metaciclogênese ,Metacyclogenesis - Abstract
A Leishmaniose Tegumentar Americana (LTA), causada por protozoários Leishmania, acomete a pele e/ou as mucosas nasal e orofaríngea. Durante o ciclo de transmissão, as formas promastigotas procíclicas crescem no intestino do inseto vetor e diferenciam-se em formas promastigotas metacíclicas infectantes, por um processo chamado de metaciclogênese, que também ocorre em culturas axênicas in vitro. O objetivo deste estudo foi avaliar a metaciclogênese in vitro de isolados L. (Viannia) braziliensis e L. (L.) amazonensis, provenientes de pacientes com diferentes formas clínicas de LTA, usando diferentes metodologias, e a capacidade destes isolados em infectar macrófagos humanos. As curvas de crescimento (L. (V.) braziliensis: IMG3, PPS6m, M2903 e L. (L.) amazonensis: MAB6 e PH8) foram realizadas em meio Grace e após 2, 6 e 10 dias de cultivo, a quantidade de formas metacíclicas foi avaliada por meio de morfometria, ensaio de seleção com a lectina de Bauhinia purpurea (BPL, para L. (V.) braziliensis) ou anticorpo 3A1-La (para L. (L.) amazonensis), citometria de fluxo e ensaio de resistência ao Complemento. Macrófagos humanos derivados de monócitos de doadores sadios, ativados ou não com interferon gama (IFN e lipopolissacarídeo (LPS), foram infectados com os parasitos de diferentes dias de cultivo ou formas metacíclicas selecionadas com BPL/3A1-La (1:1, parasito:macrófago) por 24 ou 72 h. Os perfis das curvas de crescimento in vitro foram similares para todos os isolados. Parasitos da fase estacionária do crescimento (6, 10 dias) apresentaram alterações morfológicas, como a diminuição do tamanho do corpo celular e um aumento do comprimento do flagelo, em relação aos parasitos do 2º dia. As formas promastigotas L. (L.) amazonensis do 6º e do 10º dia de cultura apresentaram maiores comprimentos do corpo celular e do flagelo do que os parasitos L. (V.) braziliensis. Na fase estacionária houve um aumento significante da porcentagem de formas metacíclicas detectadas pelas quatro técnicas (M2903: 71-86%; IMG3: 41-85%; PPS6m: 39-76%; PH8: 38-69% e MAB6: 47-84%; 6º dia, valores min e max). Apesar das quatro técnicas mostrarem um similar perfil de metaciclogênese para todos os isolados avaliados, as densidades de formas metacíclicas variaram conforme a técnica utilizada, dependendo do dia de cultura. A técnica de seleção com BPL/3A1-La foi a que detectou maiores frequências de formas metacíclicas para todos os isolados no 6o dia. A metaciclogênese dos isolados foi confirmada nos ensaios de infecção in vitro dos macrófagos, mostrando um aumento significante no índice de infecção com parasitos do 6º e 10º dia em relação àqueles de 2o dia (24 ou 72 h). Não foram atestadas diferenças entre os índices de infecção de macrófagos incubados com parasitos totais ou formas metacíclicas selecionadas do 6º dia. Os isolados mostraram diferentes perfis de suscetibilidade aos mecanismos microbicidas dos macrófagos. As cepas M2903 e PH8 foram mais suscetíveis, enquanto o isolado MAB6, mais resistente. Como as quantidades de formas metacíclicas nas culturas, considerando a técnica de seleção BPL/3A1-La, foram elevadas para todos os isolados, os resultados sugerem que a resistência/suscetibilidade aos mecanismos de controle dos macrófagos seja uma característica intrínseca do isolado. Avaliar a metaciclogênese de isolados clínicos, simultaneamente à capacidade dos parasitos em infectar macrófagos humanos, define as condições necessárias para a obtenção de parasitos eficientes em estabelecer uma infecção, para uma adequada avaliação da patogenia e da resposta imune natural na leishmaniose. The American Tegumentary Leishmaniasis (ATL), caused by Leishmania protozoa, affects the skin and oropharyngeal/nasal mucosa. During transmission cycle, the procyclic promastigote forms grow in the vector gut and differentiate into infective metacyclic promastigotes by a process called metacyclogenesis, which also occurs in axenic cultures. The aim of this study was to evaluate the in vitro metacyclogenesis of L. (Viannia) braziliensis and L. (L.) amazonensis isolates obtained from patients with different clinical forms of ATL, using different methodologies, and the ability of these isolates to infect human macrophages. Parasites (L. (Viannia) braziliensis: IMG3, PPS6m, M2903 and L. (L.) amazonensis: MAB6 and PH8) were cultured in Grace´s insect medium and the amount of metacyclic forms was evaluated by using morphometry, Bauhinia purpurea lectin (BPL, to L. (Viannia) braziliensis) or 3A1-La antibody (to L. (L.) amazonensis) negative selection, flow cytometry and Complement-resistance test after 2, 6 and 10 days of culture. Human monocyte-derived macrophages from healthy donors, activated or not with interferon gamma (IFN) and lipopolysaccharide (LPS), were infected with parasites from different days of culture or with selected metacyclic forms with BPL/3A1-La (1:1 parasite: macrophage) for 24 or 72 h. Growth patterns were similar among the isolates. Stationary-phase promastigotes (6, 10 days) showed morphological changes such as a reduction of the cell body and an increase in the flagellum length, in comparison to the parasites of the 2nd day. Leishmania. (L.) amazonensis promastigotes of the 6th and 10th day of culture presented higher length of the cell body and flagellum than L. (Viannia) braziliensis isolates. In stationary phase there was a significant increase in the percentage of metacyclic forms detected by the four techniques used (M2903: 71-86%; IMG3: 41-85%; PPS6m: 39-76%; PH8: 38-69% e MAB6: 47-84%; 6th day, medians of the techniques used). Despite the four techniques demonstrated similar metacyclogenesis profiles for all isolates evaluated, metacyclic forms densities varied with the technique and the day of culture. The BLP/3A1-La selection consistently identified the highest frequencies of metacyclic forms for all isolates (6th day) among the four techniques. Metacyclogenesis of the isolates was confirmed in in vitro macrophage infection assays which showed a significant increase of the index of infection with parasites of the 6th and 10th day compared to those of the 2nd day (24 or 72 h). No significant differences in capability to infect macrophages were detected between unfractionated or 6th day-selected metacyclic forms. The isolates showed different profiles of susceptibility to macrophage microbicidal mechanisms. The M2903 and PH8 strains were the most susceptible whereas MAB6 isolate was the most resistant among the isolates. As high amounts of metacyclic forms were present in all isolate cultures, considering BPL/3A1-La selection, the results suggest that resistance/susceptibility to macrophage control mechanisms is an intrinsic characteristic of the isolates. To evaluate the metacyclogenesis of clinical isolates together with the ability of parasites to infect human macrophages sets the conditions for obtaining efficient parasites to establish a successful infection what is critical for the evaluation of the pathogenesis and natural immune response in leishmaniasis.
- Published
- 2013
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