Navegando por Autor "Carvalho, Ana Maria Ravena Severino"
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Item An 8-hydroxyquinoline-containing polymeric micelle system is effective for the treatment of murine tegumentary leishmaniasis.(2016) Lage, Letícia Martins dos Reis; Barichello, José Mario; Lage, Daniela Pagliara; Mendonça, Débora Vasconcelos Costa; Carvalho, Ana Maria Ravena Severino; Rodrigues, Marcella Rezende; Souza, Daniel Menezes; Roatt, Bruno Mendes; Alves, Ricardo José; Tavares, Carlos Alberto Pereira; Coelho, Eduardo Antônio Ferraz; Duarte, Mariana CostaItem A clioquinol-containing Pluronic ® F127 polymeric micelle system is effective in the treatment of visceral leishmaniasis in a murine model.(2020) Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Pereira, Isabela Amorim Gonçalves; Silva, João Augusto Oliveira da; Ramos, Fernanda Fonseca; Lage, Daniela Pagliara; Machado, Amanda Sanchez; Carvalho, Lívia Mendes; Reis, Thiago Alves Rosa dos; Melo, Luísa Helena Perin de; Carvalho, Ana Maria Ravena Severino; Ottoni, Flaviano Melo; Ribeiro, Fernanda Ludolf; Freitas, Camila Simões de; Bandeira, Raquel Soares; Silva, Alessandra M.; Chávez Fumagalli, Miguel Angel; Duarte, Mariana Costa; Souza, Daniel Menezes; Alves, Ricardo José; Roatt, Bruno Mendes; Coelho, Eduardo Antônio FerrazA clioquinol (ICHQ)-containing Pluronic F127 polymeric micelle system (ICHQ/Mic) was recently shown to be effective against Leishmania amazonensis infection in a murine model. In the present study, ICHQ/ Mic was tested against L. infantum infection. BALB/c mice (n = 12 per group) were infected with L. infantum stationary promastigotes through subcutaneous injection and, 45 days after challenge, received saline or were treated via the subcutaneous route with empty micelles, ICHQ or ICHQ/Mic. In addition, animals were treated with miltefosine by the oral route, as a drug control. Half of the animals were euthanized 1 and 15 days after treatment, aiming to evaluate two endpoints after therapy, when parasitological and immunological parameters were investigated. Results showed that the treatment using miltefosine, ICHQ or ICHQ/Mic induced significantly higher anti-parasite IFN-c, IL-12, GM-CSF, nitrite and IgG2a isotype antibody levels, which were associated with low IL-4 and IL-10 production. In addition, a higher frequency of IFN-c and TNF-a-producing CD4+ and CD8+ T-cells was found in these animals. The parasite load was evaluated in distinct organs, and results showed that the treatment using miltefosine, ICHQ or ICHQ/Mic induced significant reductions in organic parasitism in the treated and infected mice. A comparison between the treatments suggested that ICHQ/Mic was the most effective in inducing a highly polarized Th1-type response, as well as reducing the parasite load in significant levels in the treated and infected animals. Data obtained 15 days after treatment suggested maintenance of the immunological and parasitological responses. In conclusion, ICHQ/Mic could be considered in future studies for the treatment of visceral leishmaniasis.Item Comparing the therapeutic efficacy of different amphotericin Bcarrying delivery systems against visceral leishmaniasis.(2018) Mendonça, Débora Vasconcelos Costa; Martins, Vivian Tamietti; Lage, Daniela Pagliara; Ribeiro, Patrícia Aparecida Fernandes; Carvalho, Ana Maria Ravena Severino; Dias, Anna Leticia Teotonio; Miyazaki, Carolina Kei; Souza, Daniel Menezes; Roatt, Bruno Mendes; Tavares, Carlos Alberto Pereira; Duarte, Mariana Costa; Coelho, Eduardo Antônio FerrazAmphotericin B (Amp) has been well-successfully used to treat against Leishmania infection, although high toxicity has been found in patients. In the present study, Amp was administered in Leishmania infantum-infected BALB/c mice by three distinct delivery systems aiming to compare their efficacy against challenge infection, as well as their side effects in a murine visceral leishmaniasis (VL) model. This product was administered in a Poloxamer P407 (Pluronic® F127)-based polymeric micelle system (Amp/M), in the Ambisome® formulation (Lip-Amp) or in a free format (free Amp). Glucantime® (Gluc) was used as a comparative drug. Aiming to evaluate different endpoints of the treatments, the efficacy of the compounds was investigated one and 15-days after the therapeutic regimens, determining the parasite load by a limiting dilution assay and a quantitative PCR (qPCR) technique, as well as evaluating the immune response generated in the infected and treated animals. In the results, Amp/M or Lip-Amp-treated mice presented the best outcomes, since significant parasite load reductions were found in the evaluated organs, as well as a parasite-specific Th1 immune response was observed in the animals. In addition, no hepatic or renal damage was found in these mice. On the other hand, free Amp or Gluc induced toxicity in the animals, which was associated with a low Th1 immune response. Comparatively, Amp/M was the most effective drug in our experimental model, and results showed that the Amp-carrying system could be considered as a future alternative in studies against VL.Item Development of an immunogen containing CD4+/CD8+ T‐cell epitopes for the prophylaxis of tegumentary leishmaniasis.(2022) Ferraz, Isabela de Andrade; Carvalho, Ana Maria Ravena Severino; Brito, Rory Cristiane Fortes de; Roatt, Bruno Mendes; Martins, Vivian Tamietti; Lage, Daniela Pagliara; Cruz, Luiza dos Reis; Medeiros, Fernanda Alvarenga Cardoso; Gonçalves, Denise Utsch; Rocha, Manoel Otávio da Costa; Coelho, Eduardo Antônio Ferraz; Mendes, Tiago Antônio de Oliveira; Duarte, Mariana Costa; Souza, Daniel MenezesTegumentary leishmaniasis (TL) is a disease of high severity and incidence in Brazil, and Leishmania braziliensis is its main etiological agent. The inefciency of control measures, such as high toxicity and costs of current treatments and the lack of efective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present work developed a gene encoding multiple T-cell (CD4+/CD8+) epitope, derived from conserved proteins found in Leishmania species and associated with TL, to generate a chimeric protein (rMEP/TL) and compose a vaccine formulation. For this, six T-cell epitopes were selected by immunoinformatics approaches from proteins present in the amastigote stage and associated with host-parasite interactions. The following formulations were then tested in an L. braziliensis murine infection model: rMEP/TL in saline or associated with MPLA-PHAD®. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging, rMEP/TL and rMEP/TL+MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG2a, IgG2a/IgG1, NO, CD4+, and CD8+ T-cells with IFN-γ and TNF-α production, associated with a reduction in CD4+IL-10+ and CD8+IL-10+ T-cells. Vaccines also induced the development of central (CD44highCD62Lhigh) and efector (CD44highCD62Llow) memory of CD4+ and CD8+ T-cells. These fndings, associated with the observation of lower rates of parasite burdens in the vaccinated groups, when compared to the control groups, suggest that immunization with rMEP/TL and, preferably, associated with an adjuvant, may be considered an efective tool to prevent TL.Item Evaluation of the protective efficacy of a Leishmania protein associated with distinct adjuvants against visceral leishmaniasis and in vitro immunogenicity in human cells.(2020) Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Lage, Daniela Pagliara; Mendonça, Débora Vasconcelos Costa; Vale, Danniele Luciana; Ramos, Fernanda Fonseca; Carvalho, Lívia Mendes; Carvalho, Ana Maria Ravena Severino; Steiner, Bethina Trevisol; Roque, Marjorie Coimbra; Silva, João Augusto Oliveira da; Oliveira, Jamil Silvano de; Tavares, Grasiele de Sousa Vieira; Martins, Vivian Tamietti; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Moreira, Ricardo Luiz Fontes; Souza, Daniel Menezes; Duarte, Mariana Costa; Oliveira, Mônica Cristina de; Ávila, Ricardo Andrez Machado de; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazThe treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients’ and healthy subjects’ cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL..Item Flau-A, a naphthoquinone derivative, is a promising therapeutic candidate against visceral leishmaniasis : a preliminary study.(2022) Mendonça, Débora Vasconcelos Costa; Tavares, Grasiele de Sousa Vieira; Pereira, Isabela Amorim Gonçalves; Silva, João Augusto Oliveira da; Ramos, Fernanda Fonseca; Lage, Daniela Pagliara; Machado, Amanda Sanchez; Carvalho, Lívia Mendes; Reis, Thiago Alves Rosa dos; Carvalho, Ana Maria Ravena Severino; Ottoni, Flaviano Melo; Ribeiro, Fernanda Ludolf; Freitas, Camila Simões de; Martins, Vivian Tamietti; Chávez Fumagalli, Miguel Angel; Duarte, Mariana Costa; Humbertf, Maria V.; Roatt, Bruno Mendes; Souza, Daniel Menezes; Alves, Ricardo José; Coelho, Eduardo Antônio FerrazVisceral leishmaniasis (VL) is a neglected tropical disease found in tropical and subtropical regions in the world. The therapeutics used for the treatment against disease presents problems, mainly related to drug toxicity, route of administration, high cost and/or by emergence of resistant strains. In this context, the search for alternative antileishmanial candidates is desirable. Recently, a naphthoquinone derivative namely 2-(2,3,4-tri-O-acetyl-6- deoxy-β-L-galactopyranosyloxy)-1,4-naphthoquinone or Flau-A showed an effective in vitro biological action against Leishmania infantum. In the present study, the efficacy of this naphthoquinone derivative was evaluated in an in vivo infection model. BALB/c mice (n = 12 per group) were infected and later received saline or were treated with empty micelles (B/Mic), free Flau-A or it incorporated in Poloxamer 407-based micelles (Flau-A/ Mic). The products were administered subcutaneously in the infected animals, which were then euthanized one (n = 6 per group) and 15 (n = 6 per group) days post-therapy, when immunological and parasitological eval- uations were performed. Results showed that animals treated with Flau-A or Flau-A/Mic produced significantly higher levels of antileishmanial IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibody, when compared to data found in the control (saline and B/Mic) groups; which showed significantly higher levels of parasite- specific IL-4, IL-10 and IgG1 antibody. In addition, animals receiving free Flau-A or Flau-A/Mic presented also significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, when compared to the controls. A low hepatic and renal toxicity was also found. Overall, Flau-A/Mic showed better immunological and parasitological results, when compared to the use of free molecule. In conclusion, pre- liminary data suggest that this composition could be considered in future studies as promising therapeutic candidate against VL.Item Immunoproteomics approach for the discovery of antigens applied to the diagnosis of canine visceral leishmaniasis.(2023) Costa, Scarleth Silva; Santos, Lucas Magno Oliveira; Freire, Larissa Chaves; Tedeschi, Ana Luiza Filizzola; Ribeiro, Naianda Rezende; Queiroz, Mariana Helena Rodrigues; Beraldo Neto, Emídio; Pimenta, Daniel Carvalho; Galvani, Nathália Coral; Luiz, Gabriel Paulino; Oliveira, Maria Eduarda de; Ávila, Ricardo Andrez Machado de; Carvalho, Ana Maria Ravena Severino; Brigido, Bryan Victor Serafim; Reis, Alexandre Barbosa; Fernandes, Ana Paula Salles Moura; Coelho, Eduardo Antônio Ferraz; Roatt, Bruno Mendes; Souza, Daniel Menezes; Duarte, Mariana CostaIn the present study, an immunoproteomic approach using Leishmania infantum parasites isolated from naturally infected dogs from an endemic region of the disease, was carried out to identify new antigens to be used in the diagnosis of canine visceral leishmaniasis (CVL). Protein extracts, obtained from parasites isolated from asymptomatic (CanLA) and symptomatic (CanLS) dogs, were used to perform the two-dimensional gels. Western Blotting assays were carried out by employing a pool of sera from dogs with visceral leishmaniasis (CanLA or CanLS), healthy dogs from an endemic area, or dogs with similar diseases associated with cross-reactions (babesiosis and ehrlichiosis). With these results, it was possible to exclude the spots that showed a crossreactivity of the sera from groups of healthy dogs, and those with babesiosis or ehrlichiosis. Taken together, 20 proteins were identified, 15 of which have already been described in the literature and 5 of which are hypothetical. An immunogenomic screen strategy was applied to identify conserved linear B-cell epitopes in the identified hypothetical proteins. Two peptides were synthesized and tested in ELISA experiments as a proof of concept for the validation of our immunoproteomics findings. The results demonstrated that the antigens presented sensitivity and specificity values ranging from 81.93% to 97.59% and 78.14 to 85.12%, respectively. As a comparative antigen, a preparation of a Leishmania extract showed sensitivity and specificity values of 75.90% and 74.88%, respectively. The present study was able to identify proteins capable of being used for the serodiagnosis of canine visceral leishmaniasis.Item Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.(2020) Ribeiro, Patrícia Aparecida Fernandes; Vale, Danniele Luciana; Dias, Daniel Silva; Lage, Daniela Pagliara; Mendonça, Débora Vasconcelos Costa; Ramos, Fernanda Fonseca; Carvalho, Lívia Mendes; Carvalho, Ana Maria Ravena Severino; Steiner, Bethina Trevisol; Roque, Marjorie Coimbra; Silva, João Augusto Oliveira da; Oliveira, Jamil Silvano de; Tavares, Grasiele de Sousa Vieira; Galvani, Nathália Coral; Martins, Vivian Tamietti; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Moreira, Ricardo Luiz Fontes; Souza, Daniel Menezes; Oliveira, Mônica Cristina de; Ávila, Ricardo Andrez Machado de; Teixeira, Antonio Lucio; Coelho, Eduardo Antônio FerrazThe control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.Item Parasitological and immunological evaluation of a novel chemotherapeutic agent against visceral leishmaniasis.(2020) Pereira, Isabela Amorim Gonçalves; Mendonça, Débora Vasconcelos Costa; Tavares, Grasiele de Sousa Vieira; Lage, Daniela Pagliara; Ramos, Fernanda Fonseca; Silva, João Augusto Oliveira da; Antinarelli, Luciana Maria Ribeiro; Machado, Amanda Sanchez; Carvalho, Lívia Mendes; Carvalho, Ana Maria Ravena Severino; Salustiano, Iorrana Vieira; Reis, Thiago Alves Rosa dos; Bandeira, Raquel Soares; Silva, Alessandra M.; Martins, Vivian Tamietti; Chávez Fumagalli, Miguel Angel; Humbert, Maria Victoria; Roatt, Bruno Mendes; Duarte, Mariana Costa; Souza, Daniel Menezes; Coimbra, Elaine Soares; Leite, João Paulo Viana; Coelho, Eduardo Antônio Ferraz; Gonçalves, Denise UtschAims: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. Methods and Results: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4’-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. Conclusion: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent against VL.Item Treatment of murine visceral leishmaniasis using an 8-hydroxyquinoline-containing polymeric micelle system.(2016) Duarte, Mariana Costa; Lage, Letícia Martins dos Reis; Lage, Daniela Pagliara; Martins, Vivian Tamietti; Carvalho, Ana Maria Ravena Severino; Roatt, Bruno Mendes; Souza, Daniel Menezes; Tavares, Carlos Alberto Pereira; Alves, Ricardo José; Barichello, José Mario; Coelho, Eduardo Antônio FerrazNewtherapeutics are urgently needed to treat visceral leishmaniasis (VL). Due to the fact that drug discovery is a long and expensive process, the development of delivery systems to carry old and toxic drugs could be considered, as well as the evaluation of new molecules that have already shown to present biological activity. In this context, the present study evaluated the in vitro and in vivo antileishmanial activity of an 8-hydroxyquinoline (8-HQN)-containing polymeric micelle (8-HQN/M) system against Leishmania infantum, the main causative agent of VL in the Americas. The experimental strategy used was based on the evaluation of the parasite load by a limiting-dilution technique in the spleen, liver, bone marrow and draining lymph nodes of the infected and treated animals, as well as by a quantitative PCR (qPCR) technique to also assess the splenic parasite load. The immune response developed was evaluated by the production of IFN-γ, IL-4, IL-10, IL-12 and GM-CSF cytokines, as well as by antileishmanial nitrite dosage and antibodies production. Hepatic and renal enzymes were also investigated to verify cellular injury as a result of treatments toxicity. In the results, 8-HQN/M-treated mice, when compared to the other groups: saline, free amphotericin B (AmpB, as a drug control), 8-HQN and B-8-HQN/M (as a micelle control) showed more significant reductions in their parasite burden in all evaluated organs. These animals also showed an antileishmanial Th1 immunity, which was represented by high levels of IFN-γ, IL-12, GM-CSF and nitrite, associated with a low production of IL-4 and IL-10 and anti-Leishmania IgG1 isotype antibodies. In addition, any hepatic or renal damage was found in these treated animals. In conclusion, 8-HQN/M was effective in treating L. infantum-infected BALB/c mice, and can be considered alone, or combined with other drugs, as an alternative treatment for VL.