Navegando por Autor "Gontijo, Nelder de Figueiredo"
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Item Antibodies from dogs with canine visceral leishmaniasis (CVL) recognise two proteins from the saliva of lutzomyia longipalpis.(2007) Bahia, Diana; Gontijo, Nelder de Figueiredo; León, Ileana Rodríguez; Perales, Jonas; Pereira, Marcos Horácio; Oliveira, Guilherme Corrêa de; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaThe saliva of the sand fly Lutzomyia longipalpis, a major vector of Leishmania, exhibits pharmacological and immunomodulatory activities that may facilitate entry and establishment of parasites into the vertebrate host. Salivary gland components of the sand fly are, therefore, potential candidates in the development of a vaccine against human leishmaniasis. With the objective of identifying sand fly saliva proteins that could be used to immunise animals against canine visceral leishmaniasis, we have evaluated anti-saliva antibody reactivity using serum samples collected from dogs naturally infected with Leishmania chagasi. Two proteins with molecular weights of 28.6 and 47.3 kDa were recognised by dog antibodies in Western blot assays. Protein bands were excised from an SDS-PAGE gel and the sequences determined by mass spectrometry. The proteins were identified as LuLo-D7 and Lulo YELLOW, respectively. The significance of these findings in the context of the development of multicomponent vaccination experiments is discussed.Item Infectividade e patogenicidade de diferentes isolados de Leishmania (Leishmania) chagasi obtidos de cães naturalmente infectados.(2016) Oliveira, Lucilene Aparecida Resende; Giunchetti, Rodolfo Cordeiro; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro; Machado, Evandro Marques de Menezes; Lana, Marta de; Gontijo, Célia Maria Ferreira; Gontijo, Nelder de FigueiredoNo Brasil, a Leishmaniose Visceral (LV), que tem como agente etiológico a Leishmania chagasi (sinonímia Leishmania infantum), vem sendo amplamente distribuída. O cão se destaca como reservatório doméstico deste parasito, favorecendo sua transmissão no ambiente urbano. Considerando que a transmissão da L. chagasi ocorre necessariamente com a participação dos cães no ambiente urbano, e que muitos deles podem ter sido tratados ou estar em tratamento contra esta infecção, sem induzir cura parasitológica, é possível especular que em ambientes domésticos possam estar circulando cepas com características de resistência ao tratamento, bem como alta infectividade e patogenicidade. Neste contexto, torna-se fundamental a caracterização de isolados de L. chagasi, oriundos de cães naturalmente infectados, bem como o estudo do comportamento in vitro e in vivo destes isolados para identificação de padrões de infectividade e patogenicidade. Dentre os resultados obtidos neste estudo, foi possível confirmar, pela PCR-RFLP, que todos os isolados são da espécie L. chagasi. De forma interessante, o cultivo dos diferentes isolados em linhagem de macrófago canino (DH82) evidenciou perfis distintos de infectividade e carga parasitária. Esta abordagem permitiu identificar os isolados com comportamentos polares in vitro, representados por infectividade e carga parasitária elevada (isolado 616) ou reduzida (isolado 614). Após este estudo inicial in vitro, foi realizado uma análise do perfil de infecção em hamsters, utilizando estes isolados, bem como a cepa padrão PP75 e a cepa selvagem OP46. Em um segundo experimento com estes isolados e com as cepas padrão (PP75 e OP46), os animais de cada um dos grupos foram submetidos a uma intervenção com a administração de Glucantime® em dose subterapêutica. De forma interessante, a taxa de sobrevida nos animais infectados com o isolado 616 foi de 100%, enquanto que para os animais infectados com o isolado 614 (com ou sem intervenção medicamentosa), bem como com o isolado 616 (nos animais que receberam Glucantime®) foi em torno de 50%. De um modo em geral, o isolado 614 induziu: (i) maiores alterações clinicopatológicas, (ii) indicativo de alteração hepática pelas análises bioquímicas, (iii) intenso infiltrado inflamatório no fígado, (iv) elevação da carga parasitária avaliada por qPCR no baço e no fígado. Estes resultados mostraram que o isolado que foi menos infectivo (614) nos ensaios in vitro foi o mais infectivo e patogênico em hamsters. Por outro lado, na presença de infecção com o isolado 616 e com administração de Glucantime®, o perfil da infecção mostrou maior gravidade, alterando, portanto, a evolução da infecção. Em conjunto, os dados obtidos pela análise in vitro em macrófagos caninos da linhagem DH82, a partir de isolados de L. chagasi apresentando perfis polares de infectividade, não refletiram o mesmo padrão nos ensaios in vivo utilizando hamsters. Além disto, foi observado que o tratamento não efetivo da leishmaniose visceral experimental pode induzir alteração no curso da infecção, tornando-a mais agressiva.Item A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs.(2008) Giunchetti, Rodolfo Cordeiro; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis; Carvalho, Andréa Teixeira de; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Vital, Wendel Coura; Abreu, Raquel Trópia de; Malaquias, Luiz Cosme Cotta; Gontijo, Nelder de Figueiredo; Brodskyn, Cláudia; Oliveira, Camila Indiani de; Costa, Dirceu Joaquim; Lana, Marta de; Reis, Alexandre BarbosaA vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensispromastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was eval-uated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE andLeishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmaniainfection. Immunophenotypic analysis revealed increased circulating CD21 + B-cells and CD5 + T-cells, reflected by higher counts of CD4 + and CD8 + T-cells. The observed interac-tion between potential antigen-presenting cells (evaluated as CD14 + monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8 + T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy.Item LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4+ and CD8+) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis.(2014) Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Ker, Henrique Gama; Moreira, Nádia das Dores; Mathias, Fernando Augusto Siqueira; Cardoso, Jamille Mirelle de Oliveira; Gontijo, Nelder de Figueiredo; Romero, Oscar Bruna; Carvalho, Andréa Teixeira de; Martins Filho, Olindo Assis; Oliveira, Rodrigo Corrêa de; Giunchetti, Rodolfo Cordeiro; Reis, Alexandre BarbosaBackground: The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8+ T cells, and high NO production. Methods: We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 × 107 late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge. Results: The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5+, CD4+, and CD8+ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load. Conclusions: These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector.Item Leishmania metacyclogenesis is promoted in the absence of purines.(2012) Serafim, Tiago Donatelli; Figueiredo, Amanda Braga de; Costa, Pedro Augusto Carvalho; Silva, Eduardo de Almeida Marques da; Gonçalves, Ricardo; Moura, Sandra Aparecida Lima de; Gontijo, Nelder de Figueiredo; Silva, Sydnei Magno da; Michalick, Marilene Susan Marques; Fernandes, José Roberto Meyer; Carvalho, Roberto Paes de; Uliana, Silvia Reni Bortolin; Fietto, Juliana Lopes Rangel; Afonso, Luís Carlos CroccoLeishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that ‘‘stressful conditions’’ will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.Item Performance of LBSap vaccine after intradermal challenge with L. infantum and saliva of Lu. longipalpis : immunogenicity and parasitological evaluation.(2012) Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Souza, Juliana Vitoriano de; Vital, Wendel Coura; Braga, Samuel Leôncio; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis; Carvalho, Andréa Teixeira de; Lana, Marta de; Gontijo, Nelder de Figueiredo; Marques, Marcos José; Giunchetti, Rodolfo Cordeiro; Reis, Alexandre BarbosaIn the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21+) and both CD4+ and CD8+ T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4+ T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-c and low IL-10 and TGF-b1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.Item A vaccine therapy for canine visceral leishmaniasis promoted significant improvement of clinical and immune status with reduction in parasite burden.(2017) Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Reis, Levi Eduardo Soares; Cardoso, Jamille Mirelle de Oliveira; Mathias, Fernando Augusto Siqueira; Brito, Rory Cristiane Fortes de; Silva, Sydnei Magno da; Gontijo, Nelder de Figueiredo; Ferreira, Sidney de Almeida; Valenzuela, Jesus G.; Oliveira, Rodrigo Corrêa de; Giunchetti, Rodolfo Cordeiro; Reis, Alexandre BarbosaHerein, we evaluated the treatment strategy employing a therapeutic heterologous vaccine composed of antigens of Leishmania braziliensis associated with MPL adjuvant (LBMPL vaccine) for visceral leishmaniasis (VL) in symptomatic dogs naturally infected by Leishmania infantum. Sixteen dogs received immunotherapy with MPL adjuvant (n = 6) or with a vaccine composed of antigens of L. braziliensis associated with MPL (LBMPL vaccine therapy, n = 10). Dogs were submitted to an immunotherapeutic scheme consisting of 3 series composed of 10 subcutaneous doses with 10-day interval between each series. The animals were evaluated before (T0) and 90 days after treatment (T90) for their biochemical/hematological, immunological, clinical, and parasitological variables. Our major results showed that the vaccine therapy with LBMPL was able to restore and normalize main biochemical (urea, AST, ALP, and bilirubin) and hematological (erythrocytes, hemoglobin, hematocrit, and platelets) parameters. In addition, in an ex vivo analysis using flow cytometry, dogs treated with LBMPL vaccine showed increased CD3+ T lymphocytes and their subpopulations (TCD4+ and TCD8+), reduction of CD21+ B lymphocytes, increased NK cells (CD5−CD16+) and CD14+ monocytes. Under in vitro conditions, the animals developed a strong antigen- specific lymphoproliferation mainly by TCD4+ and TCD8+ cells; increasing in both TCD4+IFN-γ+ and TCD8+IFN-γ+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-α and reduced levels of IL-10. Concerning the clinical signs of canine visceral leishmaniasis, the animals showed an important reduction in the number and intensity of the disease signs; increase body weight as well as reduction of splenomegaly. In addition, the LBMPL immunotherapy also promoted a reduction in parasite burden assessed by real-time PCR. In the bone marrow, we observed seven times less parasites in LBMPL animals compared with MPL group. The skin tissue showed a reduction in parasite burden in LBMPL dogs 127.5 times higher than MPL. As expected, with skin parasite reduction promoted by immunotherapy, we observed a blocking transmission to sand flies in LBMPL dogs with only three positive dogs after xenodiagnosis. The results obtained in this study highlighted the strong potential for the use of this heterologous vaccine therapy as an important strategy for VL treatment.