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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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 Barbosa
Dogs 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.
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 Barbosa
Visceral 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 interleukin
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 Barbosa
Polymerase 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.
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 Cordeiro
In 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.
Kinetics of cell migration to the dermis and hypodermis in dogs vaccinated with antigenic compounds of Leishmania braziliensis plus saponin.
(2008) Souza, Juliana Vitoriano de; Reis, Alexandre Barbosa; Moreira, Nádia das Dores; Giunchetti, Rodolfo Cordeiro; Oliveira, Rodrigo Corrêa de; Carneiro, Cláudia Martins
The search for new immunobiologicals against canine visceral leishmaniasis (CVL) has intensified in the last decade. However, it still remains to be elucidated that mechanisms of the innate immune response in situ after immunization (a.i.). The aim of this study was to investigate the kinetics of cell migration in the skin dogs with distinct antigenic compounds of the LBSap vaccine. Our major findings indicated that saponin adjuvant alone or combined with Leishmania braziliensis antigen induced strong local acute inflammatory reaction. However, these reactions not progressed to ulcerated lesions. Overall, the cell profile found in Sap and LBSap was composed of neutrophils, lymphocytes and eosinophils. There was also increased production of iNOS in Sap and LBSap groups. Thus, we can conclude that dogs immunized by LBSap and the saponin adjuvant elicited a potential innate-immune activations status compatible with effective control of the resistance to infection by Leishmania and contributing to a better understanding of the innate-immunity events induced by the LBSap vaccine.
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 Barbosa
The 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.
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 Barbosa
Hematological 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.
Systemic and compartmentalized immune response in canine visceral leishmaniasis.
(2009) Reis, Alexandre Barbosa; Martins Filho, Olindo Assis; Carvalho, Andréa Teixeira de; Giunchetti, Rodolfo Cordeiro; Carneiro, Cláudia Martins; Mayrink, Wilson; Tafuri, Washington Luiz; Oliveira, Rodrigo Corrêa de
Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most important emerging diseases with high prevalence in Latin American countries and are mainly caused by Leishmania (L.) chagasi (Syn = L. infantum). CVL has a great impact on Brazilian public health because domestic dogs are the most important VL peri-domicile reservoirs in both urban and peri-urban areas. Our findings highlight the complexity of cellular immunological events related to the natural infection from dogs by L. chagasi, additionally correlating major peripheral blood phenotypic markers with clinical status and tissues parasite density. Our main results demonstrated that lower frequency ofcirculating B cells and monocytes are important markers of severe CVL, whereas increased levels of CD8+ lymphocytes appear to be the major phenotypic feature of asymptomatic disease. Determination of the isotypes patterns during CVL demonstrated thatasymptomatic dogs and those with low parasitism are associated with an increase of IgG1, while the symptomatic dogs and those with high parasitism are associated with an increase of IgG, IgG2, IgM, IgA and IgE immunoglobulins. Pioneer findings obtained by our group showed a correlation between clinical status of CVL with degree of tissue parasite density. This data demonstrated that asymptomatic dogs presented low parasitism while symptomatic dogs are associated with high parasite load in various tissues such as skin, bone marrow and spleen. We have also investigated the association between tissue parasitism and CVL clinical forms. Regardless of clinical status, skin and spleen are the major sites of high parasite density during ongoing CVL. Furthermore, we demonstrated that bone marrow and spleen parasite density are the most reliable parasitological markers to decode the clinical status of CVL. In this article, we have reviewed some aspectsof the histopathological and immunological events occurring in natural and experimentalL. chagasi/L. infantum infection, pointing out the main L. chagasi-parasitized tissue. Wehave discussed the importance of the association between parasite density, immunological/ histopathological aspects and clinical status of the CVL, their current applications, challenges for the future and potential opportunities in CVL research.
Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis.
(2008) Giunchetti, Rodolfo Cordeiro; Martins Filho, Olindo Assis; Carneiro, Cláudia Martins; Mayrink, Wilson; Marques, Marcos José; Tafuri, Wagner Luiz; Oliveira, Rodrigo Corrêa de; Reis, Alexandre Barbosa
While enlargement of popliteal lymph nodes (LN) is frequently described in canine visceral leishmaniasis (CVL), there are few histopathologic studies of lymph nodes during this chronic immunopathological condition. Besides a detailed histopathologic analysis, we have characterized the parasite load and major immunophenotypic features of the LN inLeishmania (Leishmania ) chagasi-infected dogs. Our major histopathological findings highlight that hypertrophy/hyperplasia of LN cortical and medullary zones was the principal characteristic observed in asymptomatic dogs (AD), whereas atrophy of LN cortical zone was predominant in symptomatic animals (SD). The LN parasite density detected by anti- Leishmania immunohistochemical assay or expressed as Leishman Donovan Units was also highly correlated with the skin parasitism, the most reliable parameter to decode the clinical status of CVL. The major LN immunophenotypic changes during ongoing CVL were an increased frequency of T-lymphocytes, particularly CD8 + T-cells, up-regulation of MHC-II expression by lymphocytes and decreased levels of CD21 + B-cells. Our findings further demonstrated that changes in the LN B-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitism and LN T-cell-mediated immunity, suggesting that T-cells, especially CD8 + lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL.
Relationship of Leishmania-specific IgG levels and IgG avidity with parasite density and clinical signs in canine leishmaniasis.
(2010) Teixeira Neto, Rafael Gonçalves; Giunchetti, Rodolfo Cordeiro; Carneiro, Cláudia Martins; Vitor, Ricardo Wagner de Almeida; Vital, Wendel Coura; Quaresma, Patrícia Flávia; Ker, Henrique Gama; Melo, Lutiana Amaral de; Gontijo, Célia Maria Ferreira; Reis, Alexandre Barbosa
The clinical status and tissue parasite burden of the skin and spleen of 40 dogs naturally infected with Leishmania chagasi (syn. Leishmania infantum), together with 5 uninfected control dogs, were assessed. On the basis of the clinical evaluation, infected dogs were classified as asymptomatic (AD) or symptomatic (SD). Infected animals were also grouped according to their parasite load as exhibiting low (LP), medium (MP) and high (HP) parasitism. The results indicated a high parasite load in the skin samples of SD animals in relation to the AD group. The serum immunoglobin isotype profiles of the studied animals revealed increased levels of IgG 1 in the AD and LP dogs, whereas high levels of IgG 2 were correlated with SD and HP dogs. The avidity index (AI) of IgG total in the SD group was high in comparison of that of the AD group. Moreover, animals with a larger parasite burden either in the spleen or skin showed higher AI values than animals with lower parasitism. Based on these findings, it is suggested that CVL commences with an asymptomatic clinical form with low parasitism, high production of IgG 1 and low affinity of IgG total molecules, and evolves into a symptomatic clinical form with higher parasitism intensity, higher IgG 2 levels, and high affinity of IgG total

EFAR - Escola de Farmácia

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