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Item Acarbose presents in vitro and in vivo antileishmanial activity against Leishmania infantum and is a promising therapeutic candidate against visceral leishmaniasis.(2020) Costa, Rafaella Rodrigues; Silva, João Augusto Oliveira da; Reis, Thiago Alves Rosa dos; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Freitas, Camila Simões de; Lage, Daniela Pagliara; Martins, Vívian Tamietti; Antinarelli, Luciana Maria Ribeiro; Machado, Amanda Sanchez; Bandeira, Raquel Soares; Ribeiro, Fernanda Ludolf; Santos, Thaís Teodoro de Oliveira; Brito, Rory Cristiane Fortes de; Humbert, Maria Victoria; Souza, Daniel Menezes; Duarte, Mariana Costa; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Coimbra, Elaine Soares; Coelho, Eduardo Antônio FerrazTreatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identifcation of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specifc inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specifc antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/ Mic proved efective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented signifcant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals sufered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.Item 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 Digitoxigenin presents an effective and selective antileishmanial action against Leishmania infantum and is a potential therapeutic agent for visceral leishmaniasis.(2020) Freitas, Camila Simões de; Silva, João Augusto Oliveira da; Lage, Daniela Pagliara; Costa, Rafaella Rodrigues; Mendonça, Débora Vasconcelos Costa; Martins, Vívian Tamietti; Reis, Thiago Alves Rosa dos; Antinarelli, Luciana Maria Ribeiro; Machado, Amanda Sanchez; Tavares, Grasiele de Sousa Vieira; Ramos, Fernanda Fonseca; Coelho, Vinicio Tadeu da Silva; Brito, Rory Cristiane Fortes de; Ribeiro, Fernanda Ludolf; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Ramos, Gabriela S.; Munkert, Jennifer; Ottoni, Flaviano Melo; Campana, Priscilla Rodrigues Valadares; Humbert, Maria Victoria; Coimbra, Elaine Soares; Braga, Fernão Castro; Pádua, Rodrigo Maia de; Coelho, Eduardo Antônio FerrazTreatment for visceral leishmaniasis (VL) is hampered mainly by drug toxicity, their high cost, and parasite resistance. Drug development is a long and pricey process, and therefore, drug repositioning may be an alternative worth pursuing. Cardenolides are used to treat cardiac diseases, especially those obtained from Digitalis species. In the present study, cardenolide digitoxigenin (DIGI) obtained from a methanolic extract of Digitalis lanata leaves was tested for its antileishmanial activity against Leishmania infantum species. Results showed that 50% Leishmania and murine macrophage inhibitory concentrations (IC50 and CC50, respectively) were of 6.9 ± 1.5 and 295.3 ± 14.5 μg/mL, respectively. With amphotericin B (AmpB) deoxycholate, used as a control drug, values of 0.13 ± 0.02 and 0.79 ± 0.12 μg/mL, respectively, were observed. Selectivity index (SI) values were of 42.8 and 6.1 for DIGI and AmpB, respectively. Preliminary studies suggested that the mechanism of action for DIGI is to cause alterations in the mitochondrial membrane potential, to increase the levels of reactive oxygen species and induce accumulation of lipid bodies in the parasites. DIGI was incorporated into Pluronic® F127-based polymeric micelles, and the formula (DIGI/Mic) was used to treat L. infantum–infected mice. Miltefosine was used as a control drug. Results showed that animals treated with either miltefosine, DIGI, or DIGI/Mic presented significant reductions in the parasite load in their spleens, livers, bone marrows, and draining lymph nodes, as well as the development of a specific Th1-type response, when compared with the controls. Results obtained 1 day after treatment were corroborated with data corresponding to 15 days after therapy. Importantly, treatment with DIGI/Mic induced better parasitological and immunological responses when compared with miltefosine- and DIGI-treated mice. In conclusion, DIGI/Mic has the potential to be used as a therapeutic agent to protect against L. infantum infection, and it is therefore worth of consideration in future studies addressing VL treatment.Item Exploring drug repositioning for leishmaniasis treatment : ivermectin plus polymeric micelles induce immunological response and protection against tegumentary leishmaniasis.(2023) Freitas, Camila Simões de; Lage, Daniela Pagliara; Machado, Amanda Sanchez; Vale, Danniele Luciana; Martins, Vívian Tamietti; Cardoso, Jamille Mirelle de Oliveira; Silva, João Augusto Oliveira da; Reis, Thiago Alves Rosa dos; Tavares, Grasiele de Sousa Vieira; Ramos, Fernanda Fonseca; Ribeiro, Fernanda Ludolf; Pereira, Isabela Amorim Gonçalves; Bandeira, Raquel Soares; Fujiwara, Ricardo Toshio; Bueno, Lilian Lacerda; Roatt, Bruno Mendes; Chávez Fumagalli, Miguel Angel; Coelho, Eduardo Antônio FerrazLeishmania amazonensis can cause a wide spectrum of the clinical manifestations of leishmaniasis in humans. The development of new therapeutics is a long and expensive task; in this context, drug repositioning could be considered a strategy to identify new biological actions of known products. In the present study, ivermectin (IVE) was tested against distinct Leishmania species able to cause disease in humans. In vitro experiments showed that IVE was effective to reduce the infection degree and parasite load in Leishmania donovani- and L. amazonensisinfected macrophages that were treated with it. In addition, using the culture supernatant of treated macrophages, higher production of IFN-γ and IL-12 and lower levels of IL-4 and IL-10 were found. Then, IVE was used in a pure form or incorporated into Poloxamer 407-based polymeric micelles (IVE/M) for the treatment of L. amazonensis-infected BALB/c mice. Animals (n = 16 per group) were infected and later received saline, empty micelles, amphotericin B (AmpB), IVE, or IVE/M. They were euthanized at one (n = 8 per group) and 30 (n = 8 per group) days after treatment and, in both endpoints, immunological, parasitological, and biochemical evaluations were performed. Results showed that both IVE and IVE/M induced higher levels of IFN-γ, IL-12, GM-CSF, nitrite, and IgG2a antibodies, as well as higher IFN-γ expression evaluated by RT-qPCR in spleen cell cultures. Such animals showed low organic toxicity, as well as significant reductions in the lesion’s average diameter and parasite load in their infected tissue, spleen, liver, and draining lymph node. The efficacy was maintained 30 days post-therapy, while control mice developed a polarized Th2-type response and high parasite load. In this context, IVE could be considered as a new candidate to be applied in future studies for the treatment against distinct Leishmania species.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 In vitro and in vivo antileishmanial activity of b-acetyl-digitoxin, a cardenolide of Digitalis lanata potentially useful to treat visceral leishmaniasis.(2021) Freitas, Camila Simões de; Lage, Daniela Pagliara; Silva, João Augusto Oliveira da; Costa, Rafaella Rodrigues; Mendonça, Débora Vasconcelos Costa; Martins, Vívian Tamietti; Reis, Thiago Alves Rosa dos; Antinarelli, Luciana Maria Ribeiro; Machado, Amanda Sanchez; Tavares, Grasiele de Sousa Vieira; Ramos, Fernanda Fonseca; Brito, Rory Cristiane Fortes de; Ribeiro, Fernanda Ludolf; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Ramos, Gabriela S.; Munkert, Jennifer; Ottoni, Flaviano Melo; Campana, Priscilla Rodrigues Valadares; Duarte, Mariana Costa; Gonçalves, Denise Utsch; Coimbra, Elaine Soares; Braga, Fernão Castro; Pádua, Rodrigo Maia de; Coelho, Eduardo Antônio FerrazCurrent treatments of visceral leishmaniasis face limitations due to drug side effects and/or high cost, along with the emergence of parasite resistance. Novel and low-cost antileishmanial agents are therefore required. We report herein the antileishmanial activity of b-acetyl-digitoxin (b-AD), a cardenolide isolated from Digitalis lanata leaves, assayed in vitro and in vivo against Leishmania infantum. Results showed direct action of b-AD against parasites, as well as efficacy for the treatment of Leishmania-infected macrophages. In vivo experiments using b-AD-containing Pluronic F127 polymeric micelles (b-AD/Mic) to treat L. infantum-infected mice showed that this composition reduced the parasite load in distinct organs in more significant levels. It also induced the development of anti-parasite Th1-type immunity, attested by high levels of IFN-c, IL-12, TNF-a, GM-CSF, nitrite and specific IgG2a antibodies, in addition to low IL-4 and IL-10 contents, along with higher IFN-c-producing CD4+ and CD8+ T-cell frequency. Furthermore, low toxicity was found in the organs of the treated animals. Comparing the therapeutic effect between the treatments, b-AD/Mic was the most effective in protecting animals against infection, when compared to the other groups including miltefosine used as a drug control. Data found 15 days after treatment were similar to those obtained one day post-therapy. In conclusion, the results obtained suggest that b-AD/Mic is a promising antileishmanial agent and deserves further studies to investigate its potential to treat visceral leishmaniasis.Item Ivermectin presents effective and selective antileishmanial activity in vitro and in vivo against Leishmania infantum and is therapeutic against visceral leishmaniasis.(2021) Reis, Thiago Alves Rosa dos; Silva, João Augusto Oliveira da; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Freitas, Camila Simões de; Costa, Rafaella Rodrigues; Lage, Daniela Pagliara; Martins, Vívian Tamietti; Machado, Amanda Sanchez; Ramos, Fernanda Fonseca; Silva, Alessandra M.; Ribeiro, Fernanda Ludolf; Antinarelli, Luciana Maria Ribeiro; Brito, Rory Cristiane Fortes de; Chávez Fumagalli, Miguel Angel; Humbert, Maria Victoria; Roatt, Bruno Mendes; Coimbra, Elaine Soares; Coelho, Eduardo Antônio FerrazTreatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species. Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 μM and 427.50 ± 17.60 μM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 μM and 1.06 ± 0.23 μM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite’s mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.Item A Leishmania amastigote-specific hypothetical protein evaluated as recombinant protein plus Th1 adjuvant or DNA plasmid-based vaccine to protect against visceral leishmaniasis.(2020) Silva, João Augusto Oliveira da; Machado, Amanda Sanchez; Ramos, Fernanda Fonseca; Tavares, Grasiele de Sousa Vieira; Lage, Daniela Pagliara; Mendonça, Débora Vasconcelos Costa; Pereira, Isabela Amorim Gonçalves; Santos, Thaís Teodoro de Oliveira; Martins, Vivian Tamietti; Carvalho, Lívia Mendes; Freitas, Camila Simões de; Ribeiro, Fernanda Ludolf; Reis, Thiago Alves Rosa dos; Bandeira, Raquel Soares; Silva, Alessandra M.; Costa, Lourena Emanuele; Oliveira, Jamil Silvano de; Duarte, Mariana Costa; Roatt, Bruno Mendes; Teixeira, Antônio Lúcio; Coelho, Eduardo Antônio FerrazMost studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigoteexpressed antigens; however, Leishmania proteins expressed in the amastigote forms should be considered, since few hours after infection this stage comes into contact with the host immune system and is responsible for the development of the disease. In this context, in the present study, a Leishmania amastigote-specific hypothetical protein, called LiHyJ, was evaluated as a recombinant protein plus saponin as an adjuvant or DNA vaccine to protect against VL. The vaccine effect was evaluated by means of the evaluation of immunological and parasitological analyses performed in BALB/c mice against Leishmania infantum infection. Results showed that rLiHyJ/saponin and DNA LiHyJ induced significantly higher levels of anti-protein and anti-parasite IFN-γ, IL-12, GM-CSF, and IgG2a isotype antibodies, which were associated with a low presence of IL-4 and IL-10. DNA vaccination induced higher IFN-γ production, mainly by CD8+ T cells, while rLiHyJ/saponin stimulated the production of this cytokine, mainly by CD4+ T cells. The parasite load evaluated in distinct organs showed that both immunization schedules significantly reduced organic parasitism, when compared to the controls. Similar results were found in the immunological and parasitological assays when using the recombinant protein or DNA, although the vaccination with rLiHyJ plus saponin induced a slightly higher Th1 response and lower parasite load, when compared to the use of DNA plasmid. The protein also proved to be immunogenic when peripheral blood mononuclear cells of treated VL patients and healthy subjects were in vitro stimulated, since higher IFN-γ and lower IL-4 and IL-10 levels were found in the culture supernatants. In conclusion, LiHyJ should be considered in future studies as a vaccine candidate to protect against VL.Item Leishmania eukaryotic elongation Factor-1 beta protein is immunogenic and induces parasitological protection in mice against Leishmania infantum infection.(2021) Santos, Thaís Teodoro de Oliveira; Machado, Amanda Sanchez; Ramos, Fernanda Fonseca; Silva, João Augusto Oliveira da; Lage, Daniela Pagliara; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Cardoso, Mariana Santos; Siqueira, Williane Fernanda; Martins, Vívian Tamietti; Ribeiro, Fernanda Ludolf; Reis, Thiago Alves Rosa dos; Carvalho, Lívia Mendes; Freitas, Camila Simões de; Bandeira, Raquel Soares; Silva, Alessandra M.; Oliveira, Jamil Silvano de; Moreira, Ricardo Luiz Fontes; Fujiwara, Ricardo Toshio; Roatt, Bruno Mendes; Chávez Fumagalli, Miguel Angel; Humbert, Maria Victoria; Teixeira, Antônio Lúcio; Coelho, Eduardo Antônio FerrazTreatment for visceral leishmaniasis (VL) is hampered mainly by the toxicity and/or high cost of antileishmanial drugs. What is more, variability on sensitivity and/or specificity of diagnostic tests hinders effective disease management. In this context, prophylactic vaccination should be considered as a strategy to prevent disease. In the present study, immunogenicity of the Leishmania eukaryotic Elongation Factor-1 beta (EF1b) protein, classified as a Leishmania virulence factor, was evaluated in vitro and in vivo and tested, for the first time, as a vaccine candidate against Leishmania infantum infection. The antigen was administered as DNA vaccine or as recombinant protein (rEF1b) delivered in saponin. BALB/c mice immunization with a DNA plasmid and recombinant protein plus saponin induced development of specific Th1-type immunity, characterized by high levels of IFN-γ, IL-12, GM-CSF, both T cell subtypes and antileishmanial IgG2a isotype antibodies, before and after infection. This immunological response to the vaccines was corroborated further by parasitological analysis of the vaccinated and then challenged mice, which showed significant reductions in the parasite load in their liver, spleen, bone marrow and draining lymph nodes, when compared to the controls. Vaccination using rEF1b/saponin induced a more robust Th1 response and parasitological protection when compared to the DNA vaccine. Furthermore, in vitro analysis of lymphoproliferation, IFN-γ and IL-10 levels in human PBMC cultures showed as well development of a specific Th1-type response. In conclusion, data suggest that EF1b could be a promising vaccine candidate to protect against L. infantum infection.Item Leishmania infantum pyridoxal kinase evaluated in a recombinant protein and DNA vaccine to protects against visceral leishmaniasis.(2020) Silva, João Augusto Oliveira da; Lage, Daniela Pagliara; Ramos, Fernanda Fonseca; Machado, Amanda Sanchez; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Pereira, Isabela Amorim Gonçalves; Martins, Vívian Tamietti; Carvalho, Lívia Mendes; Ribeiro, Fernanda Ludolf; Santos, Thaís Teodoro de Oliveira; Reis, Thiago Alves Rosa dos; Oliveira, Camila S.; Bandeira, Raquel Soares; Silva, Alessandra M.; Costa, Lourena Emanuele; Oliveira, Jamil Silvano de; Duarte, Mariana Costa; Souza, Daniel Menezes; Roatt, Bruno Mendes; Teixeira, Antonio Lucio; Coelho, Eduardo Antônio FerrazLeishmania infantum pyridoxal kinase (PK) protein was characterized after an immunoproteomics screening performed with the sera from patients suffering visceral leishmaniasis (VL). Since it was recognized by sera of mammalian hosts infected by a viscerotropic Leishmania species, PK could emerge as a new vaccine candidate against disease, due to its antigenicity and immunogenicity. In this context, in the present study, the effects of the immunization using PK were evaluated when administered as a DNA plasmid (pDNAA3/PK) or recombinant protein (rPK) plus saponin. The immune response elicited by both vaccination regimens reduced in significant levels the parasite load in spleen, liver, draining lymph nodes and bone marrow, being associated with the development of Th1-type immune response, which was characterized by high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD8+ T cells were more important in the IFN-γ production in the pDNAA3/PK group, while CD4+ T cells contributed more significantly to production of this cytokine in the rPK/Saponin group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from VL patients after treatment and healthy individuals were stimulated with the protein. In conclusion, when administered either as a DNA plasmid or recombinant protein plus adjuvant, PK can direct the immune response towards a Th1-type immune profile, protecting mice against L. infantum challenge; therefore, it can be seen as a promising immunogen against human VL.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 Recombinant guanosine-5--triphosphate (GTP)-binding protein associated with Poloxamer 407-based polymeric micelles protects against Leishmania infantum infection.(2022) Lage, Daniela Pagliara; Machado, Amanda Sanchez; Vale, Danniele Luciana; Freitas, Camila Simões de; Linhares, Flávia Prata; Cardoso, Jamille Mirelle de Oliveira; Pereira, Isabela Amorim Gonçalves; Ramos, Fernanda Fonseca; Tavares, Grasiele de Sousa Vieira; Ribeiro, Fernanda Ludolf; Silva, João Augusto Oliveira da; Bandeira, Raquel Soares; Silva, Alessandra M.; Simões, Luciana C.; Reis, Thiago Alves Rosa dos; Oliveira, Jamil Silvano de; Christodoulides, Myron; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Martins, Vivian Tamietti; Coelho, Eduardo Antônio FerrazLeishmania virulence proteins should be considered as vaccine candidates against disease, since they are involved in developing infection in mammalian hosts. In a previous study, a Leishmania guanosine-5′ -triphosphate (GTP)- binding protein was identified as a potential parasite virulence factor. In the present work, the gene encoding GTP was cloned and the recombinant protein (rGTP) was evaluated as a vaccine candidate against Leishmania infantum infection. The protein was associated with saponin (rGTP/Sap) or Poloxamer 407-based micelles (rGTP/ Mic) as adjuvants, and protective efficacy was investigated in BALB/c mice after parasite challenge. Both rGTP/ Sap and rGTP/Mic compositions induced a Th1-type immune response in vaccinated animals, with significantly higher levels of IFN-γ, IL-12, IL-2, TNF-α, GM-CSF, nitrite, specific IgG2a isotype antibody and positive lym- phoproliferation, when compared to the control groups. This response was accompanied by significantly lower parasite load in the spleens, livers, bone marrows and draining lymph nodes of the animals. Immunological and parasitological evaluations indicated that rGTP/Mic induced a more polarized Th1-type response and higher reduction in the organ parasitism, and with lower hepatotoxicity, when compared to the use of rGTP/Sap. In conclusion, our preliminary data suggest that rGTP could be considered for further development as a vaccine candidate to protect against VL.