Navegando por Autor "Souza, Daniel Menezes"
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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 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 Avaliação da influência do inóculo em açaí (Euterpe oleracea) na infecção oral pelo Trypanosoma cruzi em camundongos BALB/c.(2024) Marques, Flávia de Souza; Vieira, Paula Melo de Abreu; Borges, William de Castro; Vieira, Paula Melo de Abreu; Borges, William de Castro; Lima, Wanderson Geraldo; Amaral, Joana Ferreira do; Souza, Daniel Menezes; Toledo, Max Jean de OrnelasPor muito tempo, o principal mecanismo de transmissão da doença de Chagas foi o vetorial. Entretanto, após uma série de medidas de controle ao principal vetor, Triatoma infestans, ocorreu um decréscimo na transmissão vetorial no Brasil. Nesse cenário, a infecção por via oral se tornou o principal mecanismo de transmissão da doença de Chagas no Brasil. Essa via de infecção é caracterizada pela ingestão de alimentos ou bebidas contaminados com asformas metacíclicas (TM) do Trypanosoma cruzi, sendo o açaí (Euterpe oleracea) o principal alimento envolvido. A alta capacidade antioxidante da pasta de açaí, com alto teor de polifenóis, especialmente antocianinas, já foi confirmada em vários estudos, entretanto existem poucos trabalhos na literatura que abordam a via oral, e complementarmente, a participação do açaí na interação parasito/hospedeiro. Nesse sentido, um estudo sobre a influência do açaí, frente a infecção por via oral possibilitará a compreensão de como o alimento no qual o parasito é inserido no organismo interfere na interação entre T. cruzi/hospedeiro. Baseado nisso, o objetivo desse trabalho foi avaliar a influência do inóculo em açaí na infecção experimental pelo T. cruzi. Para cumprir com esse objetivo, camundongos BALB/c foram divididos em três grupos experimentais, o grupo Controle (animais não infectados) e os grupos de animais infectados por via oral com formas TM da cepa Y (DTU II) do T. cruzi adicionadas ao meio RPMI ou ao açaí. Os animais inoculados com os parasitos em meio RPMI apresentaram 83% de sobrevida, enquanto essa porcentagem no grupo inoculado com parasitos em açaí foi de 98%. Apesar do grupo Açaí apresentar período pré- patente de parasitemia mais longo, as curvas de parasitemia dos animais infectados foram semelhantes. A quantificação da carga parasitária tecidual mostrou um aumento do parasitismo na bochecha no grupo RPMI em relação ao grupo Açaí no 14o DAI (dias após a infecção). Já no estômago esse aumento foi observado precocemente no 5o DAI. Entretanto, no coração observou-se um perfil inverso, onde houve um aumento da carga no grupo Açaí no 7o DAI em relação ao grupo RPMI. Já no 14o DAI ocorre uma inversão. A avaliação do processo inflamatório mostrou que tanto no músculo próximo a bochecha, quanto no estômago há presença de processo inflamatório nos dias 2 e 5 após a infecção para ambos os grupos infectados. No 14o DAI esse aumento foi observado apenas na bochecha para animais infectados em meio RPMI. No coração apesar de não ter sido observado processo inflamatório, foram encontrados ninhos de amastigota no 14o DAI no grupo RPMI. A quantificação de citocinas no músculo próximo a bochecha mostrou que ocorre uma maior produção da citocina pró-inflamatória TNF aos 14 dias após a infecção no grupo RPMI em relação ao grupo Açaí. Já no estômago, a avaliação das citocinas mostrou que nos animais infectados com Açaí há uma menor produção de citocinas pró-inflamatórias e imunoreguladoras. A avaliação proteômica do estômago revelou 110 proteínas diferencialmente abundantes nos três grupos experimentais. Destas, 24 proteínas encontraram- se aumentadas no grupo Açaí e diminuídas no grupo RPMI em relação ao grupo Controle. Estas proteínas estão envolvidas no controle do processo inflamatório e na manutenção da integridade da barreira epitelial. Já os grupos infectados partilharam 17 proteínas com aumento da abundância em relação ao grupo Controle, entre as quais se destacam proteínas relacionadas à invasão celular e integridade da mucosa gástrica. Esses resultados sugerem que a infecção oral por ingestão de formas metacíclicas em meio RPMI, além de promover uma parasitemia mais precoce, foi responsável pelo estabelecimento da infecção no estômago no 5o DAI. Já no grupo Açaí, a infecção ocorre de forma maissilenciosa, com parasitemia mais tardia e estabelecimento da infecção apenas no 14o DAI. Este fato pode estar relacionado à uma menor produção de citocinas pró-inflamatórias nos animais infectados com o Açaí, além do aumento de proteínas que dificultam a invasão celular pelo T. cruzi.Item Avaliação de quimeras como candidatos vacinais em modelo hamster (Mesocricetus auratus) desafiados por Leishmania infantum.(2020) Gusmão, Miriã Rodrigues; Roatt, Bruno Mendes; Brito, Rory Cristiane Fortes de; Roatt, Bruno Mendes; Souza, Daniel Menezes; Béla, Samantha RibeiroA leishmaniose visceral é considerada a mais grave dentre as formas clínicas das leishmanioses e até o presente momento não há uma vacina que possa ser empregada em campanhas de controle e profilaxia dessa doença. No intuito de aprimorar estratégias para o desenvolvimento de vacinas, a vacinologia reversa, por meio da imunoinformática é uma moderna tecnologia que aborda metodologias computacionais para a busca de novos alvos e candidatos vacinais. Em um estudo prévio do nosso grupo de pesquisa, duas quimeras foram desenhadas empregando a estratégia da imunoinformática, Quimera A e Quimera B, e quando associadas ao adjuvante saponina, apresentaram resultados promissores em modelo murino. Entretanto, este modelo não reflete a evolução clínica observada em cães e humanos infectados pelo parasito. Neste sentido, é fundamental avaliar estes potenciais imunobiológicos em um modelo experimental que desenvolva de forma semelhante aspectos, clínicos-patológicos, imunológicos e parasitológicos observados em cães e humanos durante a progressão da LV. Desse modo, o objetivo deste trabalho foi avaliar a imunogenicidade e eficácia das quimeras isoladas e/ou associadas ao sistema de adjuvantes saponina e monofosforil lipídeo A (MPL) em modelo hamster (M. auratus) desafiados com L. infantum. Os animais foram divididos em seis grupos experimentais compreendendo: grupo salina (SAL), sitema de adjuvantes saponina e MPL (SM), quimera A (QA), quimera A associada a saponina+MPL (QASM), quimera B (QB) e quimera B associada a saponina+MPL (QBSM). Esses animais foram imunizados com três doses de cada uma das composições vacinais em intervalos de 15 dias entre as doses. Após 21 dias da última dose, os hamsters foram desafiados com promastigotas de L. infantum em fase estacionária de crescimento, pela via intraperitoneal e após 60 dias os animais foram necropsiados. Amostras de sangue, soro, fragmentos do baço foram obtidos para a realização de diferentes análises laboratoriais através de análises hematológicas, bioquímicas, imunológicas e parasitológicas. Em relação às análises hemato-bioquímicas, observamos que os grupos imunizados apresentaram parâmetros de leucograma, eritrograma e das funções renais, hepáticas e proteinograma normais. Entretanto, foram observadas nos grupos controle (SAL) e sistema de adjuvantes (SM) queda nos valores de plaquetas e alterações nas dosagens hepáticas, principalmente de ALT. A análise da resposta humoral pelo perfil de IgG-total anti-Leishmania, evidenciou uma redução na reatividade desse anticorpo nos grupos imunizados em relação aos grupos controles. Além disso, foi evidenciado nos grupos imunizados uma elevada resposta próinflamatória de esplenócitos com aumento da produção de citocinas como IFN- e TNF-, tanto por linfócitos totais e pela subpopulação CD4+ , bem como uma redução na produção da citocina IL-10 por estas células. As vacinas demonstraram eficácia constatada pela redução do parasitismo esplênico nos grupos imunizados chegando a mais de 90% de redução da carga parasitária o que pode estar relacionado também com a alta produção de óxido nítrico (NO) encontrada nesses grupos. Dessa forma, os resultados obtidos no presente trabalho sugerem o potencial de imunogenicidade e proteção das quimeras avaliadas, a relevância da utilização do hamster como modelo experimental para LV, bem como a importância de novas estratégias como a imunoinformática para o desenvolvimento de vacinas para a LV.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 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 Cytokine and transcription factor profiles in the skin of dogs naturally infected by Leishmania (Leishmania) chagasi presenting distinct cutaneous parasite density and clinical status.(2011) Souza, Daniel Menezes; Oliveira, Rodrigo Corrêa de; Cota, Renata Guerra de Sá; Giunchetti, Rodolfo Cordeiro; Carvalho, Andréa Teixeira de; Martins Filho, Olindo Assis; Oliveira, Guilherme Corrêa de; Reis, Alexandre BarbosaThe immune response in the skin of dogs infected withLeishmania chagasi and its asso-ciation with distinct levels of tissue parasitism and clinical progression of canine visceral leishmaniasis (CVL) are poorly understood and limited studies are available. A detailed analysis of the profiles of cytokines (IFN- , IL-4, IL-5, IL-10, IL-12, IL-13, TGF- 1 and TNF- ) and transcription factors (T-bet, GATA-3 and FOXP3) in the skin of 35 naturally infected dogs was carried out using real-time PCR alongside determinations of skin parasite density and the clinical status of CVL. A mixed cytokine profile with high levels of expression of IFN- , TNF- and IL-13 was determined in asymptomatic dogs. Additionally, the levels of transcription factors GATA-3 and FOXP3 were correlated with the asymptomatic disease. A mixed cytokine profile was also observed during active CVL. Moreover, high levels of IL-10 and TGF- 1, concomitant with the low expression of IL-12, may represent a key condition that allows persistence of parasite replication in the skin. The results obtained indicate that in asymptomatic disease or lower levels of skin parasite density, a mixed inflammatory, regulatory immune response profile may be of major relevance for both the maintenance of the clinical status of the dogs as well as for parasite persistence and replication at low levels.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 Dogs immunized with LBSap vaccine displayed high levels of IL-12and IL-10 cytokines and CCL4, CCL5 and CXCL8 chemokinesin the dermis.(2013) Souza, Juliana Vitoriano de; Moreira, Nádia das Dores; Souza, Daniel Menezes; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Mathias, Fernando Augusto Siqueira; Cardoso, Jamille Mirelle de Oliveira; Giunchetti, Rodolfo Cordeiro; Cota, Renata Guerra de Sá; Oliveira, Rodrigo Corrêa de; Carneiro, Cláudia Martins; Reis, Alexandre BarbosaThe complex interplay between cytokines and chemokines regulates innate and adaptive immuneresponses against pathogens; specifically, cytokine and chemokine expression drives activation ofimmune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs withLeishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components,and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12,IFN- _, TNF- _, IL-4, IL-13, TGF- _ and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24and 48 h after inoculation. We also evaluated the correlation between cytokine and chemokine expres-sion and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and24 h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression,IFN- _ and IL-13 expression, and IL-13 and TGF- _ expression, suggesting that a mixed cytokine microen-vironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant aloneinduced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initiallyhighest in the LBSap group, but at 48 h, expression was highest in the LB group. Information about thekinetics of the immune response to this vaccine gained using this dog model will help to elucidate themechanisms of and factors involved in a protective response against Leishmania infection and will aid inestablishing rational approaches for the development of vaccines against canine visceral leishmaniasis.Item Epitope mapping of the HSP83.1 protein of Leishmania braziliensis discloses novel targets for immunodiagnosis of tegumentary and visceral clinical forms of leishmaniasis.(2014) Souza, Daniel Menezes; Mendes, Tiago Antônio de Oliveira; Gomes, Matheus de Souza; Cunha, João Luís Reis; Nagem, Ronaldo Alves Pinto; Carneiro, Cláudia Martins; Coelho, Eduardo Antônio Ferraz; Galvão, Lúcia Maria da Cunha; Fujiwara, Ricardo Toshio; Bartholomeu, Daniella CastanheiraGold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution.Item Evaluation of a Leishmania hypothetical protein administered as DNA vaccine or recombinant protein against Leishmania infantum infection and its immunogenicity in humans.(2018) Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Lage, Daniela Pagliara; Costa, Lourena Emanuele; Martins, Vivian Tamietti; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Lima, Mariana Pedrosa; Oliveira, Jamil Silvano de; Steiner, Bethina Trevisol; Ávila, Ricardo Andrez Machado de; Roatt, Bruno Mendes; Chávez Fumagalli, Miguel Angel; Souza, Daniel Menezes; Duarte, Mariana Costa; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazVisceral leishmaniasis (VL) is a fatal disease when acute and untreated. The treatment against this disease is long and presents toxicity and/or high costs. Moreover, parasite resistance has been increasing. Therefore, alternative control measures to avoid the spread of disease should be considered. It is accepted that the development of the T helper (Th)1 immune response, based on the production of pro-inflammatory cytokines, is required for the control of parasites. Although recombinant protein-based vaccines have been tested against VL, they require supplementation with immune adjuvants. In addition, there is a scarcity of studies that comparatively evaluate the efficacy of the immunogens when administered by different delivery systems in mammalian hosts. In the present study, a Leishmania hypothetical protein, LiHyR, was cloned and evaluated by immunization as a plasmid deoxyribonucleic acid (DNA) vaccine or in a recombinant format plus saponin against Leishmania infantum infection. Results showed that both vaccination regimens induced a Th1 cell-based immunity, since high levels of interferon-gamma (IFN-γ), interleukin (IL)-2, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α) were found, and were associated with the low production of IL-4, IL-10, and anti-parasite immunoglobulin (IgG)1 isotype. In addition, significant reductions in the parasite load were found in the evaluated organs of the DNA LiHyR or rLiHyR/saponin-vaccinated animals. No significant difference was achieved between groups vaccinated with DNA or the recombinant protein. The antigen proved to be also immunogenic in human peripheral blood mononuclear cells (PBMCs) collected from healthy subjects and from untreated and treated VL patients. A higher IgG2 isotype was also found in sera samples of these subjects, thus demonstrating its possible use as a human vaccine. This study demonstrates the protective efficacy of a new Leishmania protein against VL, when it is administered as a DNA vaccine or a recombinant protein plus saponin, and points out its use as a human vaccine against disease.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 High-through identification of T cell-specific phage-exposed mimotopes using PBMCs from tegumentary leishmaniasis patients and their use as vaccine candidates against Leishmania amazonensis infection.(2019) Carvalho, Gerusa Brandão de; Costa, Lourena Emanuele; Lage, Daniela Pagliara; Ramos, Fernanda Fonseca; Santos, Thaís Teodoro de Oliveira; Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Salles, Beatriz Cristina Silveira; Lima, Mariana Pedrosa; Carvalho, Lívia Mendes; Dias, Ana Carolina Silva; Alves, Patrícia Terra; Franklin, Michelle Lucrécio; Silva, Renata A. M.; Duarte, Mariana Costa; Souza, Daniel Menezes; Roatt, Bruno Mendes; Chávez Fumagalli, Miguel Angel; Goulart Filho, Luiz Ricardo; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazIn the current study, phage-exposed mimotopes as targets against tegumentary leishmaniasis (TL) were selected by means of bio-panning cycles employing sera of TL patients and healthy subjects, besides the immune stimulation of peripheral blood mononuclear cells (PBMCs) collected from untreated and treated TL patients and healthy subjects. The clones were evaluated regarding their specific interferon-γ (IFN-γ) and interleukin-4 (IL-4) production in the in vitro cultures, and selectivity and specificity values were calculated, and those presenting the best results were selected for the in vivo experiments. Two clones, namely A4 and A8, were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of IFN-γ, IL-2, IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. In conclusion, we presented a strategy to identify new mimotopes able to induce Th1 response in PBMCs from TL patients and healthy subjects, and that were successfully used to protect against L. amazonensis infectionItem Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine Visceral Leishmaniasis.(2012) Souza, Daniel Menezes; Cota, Renata Guerra de Sá; Carneiro, Cláudia Martins; Souza, Juliana Vitoriano de; Giunchetti, Rodolfo Cordeiro; Carvalho, Andréa Teixeira de; Lemos, Denise da Silveira; Oliveira, Guilherme Corrêa de; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaBackground: The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL). Methodology/Principal Findings: A detailed analysis of inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes, and macrophages) as well as the expression of chemokines (CCL2, CCL4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8) was carried out in dermis skin samples from 35 dogs that were naturally infected with L. infantum. The analysis was based on real-time polymerase chain reaction (PCR) in the context of skin parasitism and the clinical status of CVL. We demonstrated increased inflammatory infiltrate composed mainly of mononuclear cells in the skin of animals with severe forms of CVL and high parasite density. Analysis of the inflammatory cell profile of the skin revealed an increase in the number of macrophages and reductions in lymphocytes, eosinophils, and mast cells that correlated with clinical progression of the disease. Additionally, enhanced parasite density was correlated with an increase in macrophages and decreases in eosinophils and mast cells. The chemokine mRNA expression demonstrated that enhanced parasite density was positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression. Conclusions/Significance: These findings represent an advance in the knowledge about skin inflammatory infiltrates in CVL and the systemic consequences. Additionally, the findings may contribute to the design of new and more efficient prophylactic tools and immunological therapies against CVL.Item Immunodiagnosis of canine visceral leishmaniasis using mimotope peptides selected from phage is played combinatorial libraries.(2015) Machado, Christina Monerat Toledo; Ávila, Ricardo Andrez Machado de; NGuyen, Christophe; Granier, Claude; Bueno, Lilian Lacerda; Carneiro, Cláudia Martins; Souza, Daniel Menezes; Carneiro, Rubens Antônio; Chávez Orlórtegui, Carlos; Fujiwara, ToshioELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur.We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptideswere synthesized, polymerizedwith glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVLItem Immunodiagnosis of human and canine visceral leishmaniasis using recombinant Leishmania infantum Prohibitin protein and a synthetic peptide containing its conformational B-cell epitope.(2019) Rodrigues, Marcella Rezende; Santos, Lucas Magno Oliveira; Miyazaki, Carolina Kei; Martins, Vivian Tamietti; Ribeiro, Fernanda Ludolf; Kursancew, Amanda Christine da Silva; Ramos, Fernanda Fonseca; Dias, Daniel Silva; Oliveira, Jamil Silvano de; Vieira, Paula Melo de Abreu; Roatt, Bruno Mendes; Ávila, Ricardo Andrez Machado de; Gonçalves, Denise Utsch; Souza, Daniel Menezes; Coelho, Eduardo Antônio Ferraz; Duarte, Mariana CostaIn the present study, Leishmania infantum's Prohibitin was cloned and, alongside a synthetic peptide, evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis (CVL and TL, respectively) in dogs and humans. For TL diagnosis, this study analyzed serum samples from cutaneous (n=20) or mucosal (n=39) leishmaniasis patients, and from Chagas disease (CD) patients (n=8) and non-infected patients (n=45). For CVL diagnosis, serum samples from asymptomatic (n=14), symptomatic (n=71), non-infected (n=116), and Leish-Tec®- vaccinated (n=79) dogs were examined, as well as T. cruzi (n=11) and Ehrlichia canis (n=10) infected animals. An indirect ELISA method using rProhibitin showed diagnostic sensitivity and specificity values of 91.76% and 89.91%, respectively. L. infantum SLA showed 86.11% and 48.24% of specificity and sensitivity, respectively, for CVL serodiagnosis, and 98.31% and 84.91% sensitivity and specificity, respectively for TL diagnosis. L. braziliensis SLA showed 75.47% and 83.05% of specificity and sensitivity, respectively, for TL diagnosis. The synthetic peptide showed a better result in TL than in CVL diagnosis. In conclusion, preliminar results suggest that the detection of antibodies against the rProhibitin protein and the synthetic peptide improves the serodiagnosis of TL and CVL.Item Immunogenicity and protective efficacy of a new Leishmania hypothetical protein applied as a DNA vaccine or in a recombinant form against Leishmania infantum infection.(2019) Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Lage, Daniela Pagliara; Martins, Vivian Tamietti; Costa, Lourena Emanuele; Santos, Thaís Teodoro de Oliveira; Ramos, Fernanda Fonseca; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Ribeiro, Fernanda Ludolf; Gomes, Dawidson Assis; Rodrigues, Michele Angela; Chávez Fumagalli, Miguel Angel; Silva, Eduardo Sergio da; Galdino, Alexsandro Sobreira; Duarte, Mariana Costa; Roatt, Bruno Mendes; Souza, Daniel Menezes; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazVaccination is one the most important strategies for the prevention of visceral leishmaniasis (VL). In the current study, a new Leishmania hypothetical protein, LiHyP, which was previously showed as antigenic in an immunoproteomic search in canine VL, was evaluated regarding its immunogenicity and protective efficacy against Leishmania infantum infection. The effects of the immunization using LiHyP were evaluated when administered as a DNA plasmid (DNA LiHyP) or recombinant protein (rLiHyP) associated with saponin. The immunity elicited by both vaccination regimens reduced the parasitism in liver, spleen, bone marrow and draining lymph nodes, being associated with 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. CD4+ T cells contributed more significantly to IFN-γ production in the rLiHyP/saponin group, while CD8+ T cells were more important in the production of this cytokine in the DNA LiHyP group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from treated VL subject and healthy individuals were stimulated with the recombinant protein. In conclusion, when administered either as a DNA plasmid or recombinant protein, LiHyP can direct the immune response towards a Th1 immune profile, protecting animals against L. infantum infection; therefore, it can be seen as a promising immunogen against human 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 Immunotherapy using immunogenic mimotopes selected by phage display plus amphotericin B inducing a therapeutic response in mice infected with Leishmania amazonenses.(2023) Soyer, Tauane Gonçalves; Ramos, Fernanda Fonseca; Pereira, Isabela Amorim Gonçalves; Lage, Daniela Pagliara; Bandeira, Raquel Soares; Jesus, Marcelo Moreira de; Costa, Guilherme de Paula; Machado, Amanda Sanchez; Freitas, Camila Simões de; Vale, Danniele Luciana; Martins, Vivian Tamietti; Galdino, Alexsandro Sobreira; Chávez Fumagalli, Miguel Angel; Souza, Daniel Menezes; Duarte, Mariana Costa; Roatt, Bruno Mendes; Coelho, Eduardo Antônio Ferraz; Tavares, Grasiele de Sousa VieiraLeishmania amazonensis can cause cutaneous and visceral clinical manifestations of leish- maniasis in infected hosts. Once the treatment against disease is toxic, presents high cost, and/or there is the emergence of parasite-resistant strains, alternative means through which to control the disease must be developed. In this context, immunotherapeutics combining known drugs with immunogens could be applied to control infections and allow hosts to recover from the disease. In this study, immunotherapeutics protocols associating mimotopes selected by phage display and amphotericin B (AmpB) were evaluated in L. amazonensis-infected mice. Immunogens, A4 and A8 phages, were administered alone or associated with AmpB. Other animals received saline, AmpB, a wild-type phage (WTP), or WTP/AmpB as controls. Evaluations performed one and thirty days after the application of immunotherapeutics showed that the A4/AmpB and A8/AmpB combinations induced the most polarized Th1-type immune responses, which reflected in significant reductions in the lesion’s average diameter and in the parasite load in the infected tissue and distinct organs of the animals. In addition, the combination also reduced the drug toxicity, as compared to values found using it alone. In this context, preliminary data presented here suggest the potential to associate A4 and A8 phages with AmpB to be applied in future studies for treatment against leishmaniasis.