EFAR - Escola de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/451
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O curso de Farmácia em Ouro Preto foi criado em 1839, sendo a mais antiga Escola de Farmácia da América Latina.
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Item LBMPL vaccine therapy induces progressive organization of the spleen microarchitecture, improved Th1 adaptative immune response and control of parasitism in Leishmania infantum naturally infected dogs.(2022) Roatt, Bruno Mendes; Cardoso, Jamille Mirelle de Oliveira; Reis, Levi Eduardo Soares; Moreira, Gabriel José Lucas; Gonçalves, Letícia Captein; Marques, Flávia de Souza; Moreira, Nádia das Dores; Vieira, Paula Melo de Abreu; Soares, Rodrigo Dian de Oliveira Aguiar; Giunchetti, Rodolfo Cordeiro; Reis, Alexandre BarbosaThe spleen plays a central role in human and canine visceral leishmaniasis, where the activation of the immune response occurs in one of the tissues where Leishmania infantum reproduces. Therefore, this organ is both a target to understand the mechanisms involved in the parasite control and a parameter for assessing the therapeutic response. In this sense, this study aimed to evaluate the main histological, immunological and parasitological aspects in the spleen of symptomatic dogs naturally infected by L. infantum treated with the therapeutic vaccine LBMPL. For this, dogs were divided into four groups: dogs uninfected and untreated (NI group); L. infantum-infected dogs that were not treated (INT group); L. infantum-infected dogs that received treatment only with monophosphoryl lipid A adjuvant (MPL group); and L. infantum-infected dogs that received treatment with the vaccine composed by L. braziliensis promastigote proteins associated with MPL adjuvant (LBMPL group). Ninety days after the therapeutics protocol, the dogs were euthanized and the spleen was collected for the proposed evaluations. Our results demonstrated a reduction of hyperplasia of red pulp and follicular area of white pulp, increased mRNA expression of IFN-γ, TNF-α, IL-12 and iNOS, and decreased IL-10 and TGF-β1, and intense reduction of splenic parasitism in dogs treated with the LBMPL vaccine. These results possibly suggest that the pro-inflammatory environment promoted the progressive organization of the splenic architecture favoring the cellular activation, with consequent parasite control. Along with previously obtained data, our results propose the LBMPL vaccine as a possible treatment strategy for canine visceral leishmaniasis (CVL).Item CD4+ T-lymphocytes from asymptomatic dogs infected with Leishmania infantum are able to activate macrophages for higher leishmanicidal ability in an in vitro co-culture experiment.(2022) Vieira, João Filipe Pereira; Cardoso, Jamille Mirelle de Oliveira; Brito, Rory Cristiane Fortes de; Roatt, Bruno Mendes; Carneiro, Cláudia Martins; Valadares, Diogo Garcia; Soares, Rodrigo Dian de Oliveira Aguiar; Reis, Alexandre BarbosaDogs are the most common domestic reservoir of Leishmania infantum, making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4+, CD8+ T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP+ promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4+ and CD8+ T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL.Item Down regulation of IL-10 and TGF-β1 mRNA expression associated with reduced inflammatory process correlates with control of parasitism in the liver after treating L. infantum infected dogs with the LBMPL vaccine therapy.(2022) Roatt, Bruno Mendes; Cardoso, Jamille Mirelle de Oliveira; Brito, Rory Cristiane Fortes de; Reis, Levi Eduardo Soares; Moreira, Gabriel José Lucas; Vieira, Paula Melo de Abreu; Souza, Flávia Marques de; Lima, Wanderson Geraldo de; Soares, Rodrigo Dian de Oliveira Aguiar; Giunchetti, Rodolfo Cordeiro; Reis, Alexandre BarbosaThe liver plays an important role in human and canine visceral leishmaniasis, then it is considered as target to understand the mechanisms involved in the parasite control and a parameter to assess therapeutic responses. In this sense, our study focuses on evaluating the major alterations in the liver by histological (morphometric parenchyma inflammation/semi-quantitative portal inflammation), immunohistochemical assays (parasitism), and qPCR (parasitism and cytokine gene expression) in Leishmania infantum naturally infected dogs and treated with LBMPL vaccine. Animals were divided in four groups: NI group (n = 5): uninfected and untreated dogs; INT group (n = 7): L. infantum-infected dogs and not treated; MPL group (n = 6): L. infantum-infected dogs that received only monophosphoryl lipid A adjuvant, and LBMPL group (n = 10): L. infantum-infected dogs that received treatment with the vaccine composed by L. braziliensis disrupted promastigotes associated with MPL adjuvant. Ninety days after the end of treatments, the dogs were euthanized, and the liver was collected for the proposed evaluations. Significantly lower portal inflammatory reactions, and lower parenchyma inflammation were observed in the LBMPL group compared to INT and MPL groups. iNOS mRNA expression was higher in LBMPL group and in contrast, IL-10 and TGF-β1 mRNA expression was lower in this group when compared to INT group. Immunohistochemical and qPCR analysis showed significant parasite load reduction in LBMPL group compared to INT and MPL animals. Our data suggest that in naturally Leishmania-infected dogs, LBMPL vaccine reduces the damage in the hepatic tissue, being able to attenuate the type 2 immune response. It could be associated with a marked reduction in the parasitism decreasing liver inflammation in treated dogs. Along with previously obtained data, our results suggest that LBMPL vaccine can significantly contribute to the therapy strategy for L. infantum infected dogs.Item IL-10 receptor blockade controls the in vitro infectivity of Leishmania infantum and promotes a Th1 activation in PBMC of dogs with visceral leishmaniasis.(2021) Cardoso, Jamille Mirelle de Oliveira; Brito, Rory Cristiane Fortes de; Costa, Ana Flávia Pereira; Mathias, Fernando Augusto Siqueira; Reis, Levi Eduardo Soares; Vieira, João Filipe Pereira; Soares, Rodrigo Dian de Oliveira Aguiar; Reis, Alexandre Barbosa; Roatt, Bruno MendesAn important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anticanine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+ Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFNγ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment.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 Immunological profile of resistance and susceptibility innaturally infected dogs by Leishmania infantum.(2014) Leal, Gleisiane Gomes de Almeida; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Carneiro, Cláudia Martins; Giunchetti, Rodolfo Cordeiro; Carvalho, Andréa Teixeira de; Martins Filho, Olindo Assis; Francisco, Amanda Fortes; Cardoso, Jamille Mirelle de Oliveira; Mathias, Fernando Augusto Siqueira; Oliveira, Rodrigo Corrêa de; Carneiro, Mariângela; Vital, Wendel Coura; Reis, Alexandre BarbosaVisceral leishmaniasis has a great impact on public health, and dogs are considered the maindomestic reservoir of Leishmania infantum, the causal parasite. In this study, 159 animalsnaturally infected by L. infantum from an endemic area of Brazil were evaluated through ananalysis of cellular responses, using flow cytometry, and of the hematological parameters.The results confirmed that disease progression is associated with anemia and reductionsin eosinophils, monocytes and lymphocytes. The investigation of the immune response,based on the immunophenotypic profile of peripheral blood, showed declines in the abso-lute numbers of T lymphocytes CD5+and their subsets (CD4+and CD8+) and a drop of Blymphocytes in asymptomatic seropositive (AD-II) and symptomatic seropositive (SD) dogs.Neutrophils, when stimulated with soluble antigen of L. infantum, showed higher synthesisof interferon (IFN)- _+in AD-II and SD groups, with decreased production of interleukinItem Molecular diagnosis of canine visceral leishmaniasis : a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection.(2013) Reis, Levi Eduardo Soares; Vital, Wendel Coura; Roatt, Bruno Mendes; Bouillet, Leoneide Érica Maduro; Ker, Henrique Gama; Brito, Rory Cristiane Fortes de; Resende, Daniela de Melo; Carneiro, Cláudia Martins; Reis, Alexandre BarbosaPolymerase chain reaction (PCR) and its variations represent highly sensitive and specificmethods for Leishmania DNA detection and subsequent canine visceral leishmaniasis (CVL)diagnosis. The aim of this work was to compare three different molecular diagnosis tech-niques (conventional PCR [cPCR], seminested PCR [snPCR], and quantitative PCR [qPCR])in samples of skin and spleen from 60 seropositive dogs by immunofluorescence antibodytest and enzyme-linked immunosorbent assay. Parasitological analysis was conducted byculture of bone marrow aspirate and optical microscopic assessment of ear skin and spleensamples stained with Giemsa, the standard tests for CVL diagnosis. The primers L150/L152and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNAminicircle in the cPCR, and snPCR and qPCR were performed using the DNA polymerasegene (DNA pol _) primers from Leishmania infantum. The parasitological analysis revealedparasites in 61.7% of the samples. Sensitivities were 89.2%, 86.5%, and 97.3% in the skin and81.1%, 94.6%, and 100.0% in spleen samples used for cPCR, snPCR, and qPCR, respectively.We demonstrated that the qPCR method was the best technique to detect L. infantum inboth skin and spleen samples. However, we recommend the use of skin due to the highsensitivity and sampling being less invasive.Item Cytokine and nitric oxide patterns in dogs immunized with LBSap vaccine, before and after experimental challenge with Leishmania chagasi plus saliva of Lutzomyia longipalpis.(2013) Resende, Lucilene Aparecida; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Viana, Kelvinson Fernandes; Mendonça, Ludmila Zanandreis de; Lanna, Mariana Ferreira; Lemos, Denise da Silveira; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis; Fujiwara, Ricardo Toshio; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa; Giunchetti, Rodolfo CordeiroIn the studies presented here, dogs were vaccinated against Leishmania (Leishmania) cha-gasi challenge infection using a preparation of Leishmania braziliensis promastigote proteinsand saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1immune response that was characterized by increased levels of interleukin (IL-) 12 andinterferon gamma (IFN- _) production by peripheral blood mononuclear cells (PBMC) uponstimulation with soluble vaccine antigen. Importantly, results showed that this type ofresponsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasichallenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN- _ andconcomitant nitric oxide (NO) when stimulated with Leishmania antigens as comparedto PBMCs from respective control groups (saponin, LB- treated, or non-treated controldogs). Moreover, transforming growth factor (TGF)- _ decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysisrevealed decreased frequency of parasitism in the presence of vaccine antigen. It is con-cluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immuneresponse against L. chagasi challenge infection.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 Clinical forms of canine visceral Leishmaniasis in naturally Leishmania infantum – infected dogs and related myelogram and hemogram changes.(2013) Nicolato, Roney Luiz de Carvalho; Abreu, Raquel Trópia de; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Reis, Levi Eduardo Soares; Carvalho, Maria das Graças; Carneiro, Cláudia Martins; Giunchetti, Rodolfo Cordeiro; Bouillet, Leoneide Érica Maduro; Lemos, Denise da Silveira; Vital, Wendel Coura; Reis, Alexandre BarbosaHematological analysis has limited applications for disease diagnosis in Leishmania infantum–infected dogs, but it can be very important in evaluating the clinical forms of the disease and in understanding the evolution of canine visceral leishmaniasis (CVL) pathogenesis. Recently, we demonstrated that alterations in leucopoiesis and erythropoiesis are related to clinical status and bone marrow parasite density in dogs naturally infected by L. infantum. To further characterize these alterations, we evaluated the association between the hematological parameters in bone marrow and peripheral blood alterations in groups of L. infantum–infected dogs: asymptomatic I (AD-I: serum negative/PCR+), asymptomatic II (AD-II: serum positive), oligosymptomatic (OD), and symptomatic (SD). Results were compared with those from noninfected dogs (NID). The SD group was found to present a decrease in erythropoietic lineage with concomitant reductions in erythrocytes, hemoglobin, and hematocrit parameters, resulting in anemia. The SD group also had increased neutrophils and precursors and decreased band eosinophils and eosinophils, leading to peripheral blood leucopenia. In the AD-II group, lymphocytosis occurred in both the peripheral blood and the bone marrow compartments. The SD group exhibited lymphocytosis in the bone marrow, with lymphopenia in the peripheral blood. In contrast, the AD-I group, showed no significant changes suggestive of CVL, presenting normal counts in bone marrow and peripheral blood. Our results showed for the first time that important changes in hematopoiesis and hematological parameters occur during ongoing CVL in naturally infected dogs, mainly in symptomatic disease. Taken together, our results based on myelogram and hemogram parameters enable better understanding of the pathogenesis of the anemia, lymphocytosis, and lymphopenia, as well as the leucopenia (eosinopenia and monocytopenia), that contribute to CVL prognosis.