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 Establishment of monoclonal antibodies to evaluate the cellular immunity in a hamster model of L. infantum infection.(2021) Carvalho, Lívia Mendes; Brito, Rory Cristiane Fortes de; Gusmão, Miriã Rodrigues; Soares, Rodrigo Dian de Oliveira Aguiar; Reis, Alexandre Barbosa; Roatt, Bruno MendesSyrian hamsters (Mesocricetus auratus) are largely used as a model for infectious diseases because it is very susceptible to several pathogens, including Leishmania spp. parasites. However, the research community faces limitations in its use due to the lack of immunological reagents and tools to study the immune system in this model. In this context, we proposed the validation of some important commercially antimouse mAbs (CD4, TNF-α, IFN-γ and IL-10) and how this could be useful to evaluate a specific cellular immune response in Leishmania-infected hamster using flow cytometry experiments. Our data demonstrated a cross-reactivity between these anti-mouse mAbs and hamster molecules that were herein studied. Beyond that, it was able to characterize the development of a specific cellular immune response through cytokine production in L infantum-infected hamsters when compared to uninfected ones. These data not only aid the usage of hamsters as experimental model to investigate various infectious diseases, but they contribute to the design of novel approaches to further investigate the immunological mechanisms associated to pathogen infections.Item Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NK T and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas'disease morbidity?.(2006) Avelar, Danielle Marchetti Vitelli; Avelar, Renato Sathler; Massara, Rodrigo Lima; Borges, Jaila Dias; Lage, Paula Souza; Lana, Marta de; Carvalho, Andréa Teixeira de; Dias, João Carlos Pinto; Santos, Silvana Maria Elói; Martins Filho, Olindo AssisThe immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi - infected children, characterizing the early stages of the indeterminate clinical form of Chagas’ disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre-natural killer (NK)-cells (CD3 – CD16 + CD56 – ), and higher values of proinflammatory monocytes (CD14 + CD16 + HLA-DR ++ ). The higher values of activated B lymphocytes (CD19 + CD23 + ) contrasted with impaired T cell activation, indicated by lower values of CD4 + CD38 + and CD4 + HLA-DR + lymphocytes, a lower frequency of CD8 + CD38 + and CD8 + HLA-DR + cells; a decreased frequency of CD4 + CD25 HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas’ disease. Comparative cross-sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage-like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell-mediated inflammatory immunoprofile characterized by high levels of activated CD8 + cells and basal levels of mature NK cells, NKT and CD4 + CD25 HIGH cells might lead to late chronic pathologies associated with chagasic heart disease.Item Histopathological features, parasite density and cell phenotype 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, Washington Luiz; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaWhile 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 andmajor immunophenotypic features of theLNin Leishmania (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, upregulation of MHC-II expression by lymphocytes and decreased levels of CD21+ B-cells. Our findings further demonstrated that changes in the LNB-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitismandLNT-cell-mediated immunity, suggesting thatT-cells, especiallyCD8+ lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL.Item Development of a fluorescent based immunosensor for the serodiagnosis of canine Leishmaniasis combining immunomagnetic separation and flow cytometry.(2013) Sousa, Susana; Cardoso, Luís; Reed, Steven G.; Reis, Alexandre Barbosa; Martins Filho, Olindo Assis; Silvestre, Ricardo; Silva, Anabela Cordeiro daAn accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce. Methodology: Ni-NTA magnetic microspheres with 1,7 mm and 8,07 mm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry. Principal Findings: A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%). Conclusions: Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis.Item Innovations in diagnosis and post-therapeutic monitoring of Chagas disease : simultaneous flow cytometric detection of IgG1 antibodies anti-live amastigote, anti-live trypomastigote, and anti-fixed epimastigote forms of Trypanosoma cruzi.(2014) Alessio, Glaucia Diniz; Côrtes, Denise Fonseca; Assis, Girley Francisco Machado de; Sales Júnior, Policarpo Ademar; Ferro, Eloisa Amália Vieira; Antonelli, Lis Ribeiro do Valle; Carvalho, Andréa Teixeira de; Martins Filho, Olindo Assis; Lana, Marta deThis study developed a remarkable methodological innovation (FC-ATE) which enables simultaneous detection of antibodies specific to the three evolutive forms of Trypanosoma cruzi: live amastigote (AMA), live trypomastigote (TRYPO), and fixed epimastigote (EPI) using a differential fluorescence staining as low (AMA), intermediate (TRYPO), and high (EPI). An outstanding performance (100%) was observed in the discrimination of the chagasic (CH) and non-chagasic (NCH) patients. In the applicability of FC-ATE in the diagnosis of Chagas disease, 100% of the CH samples presented positivity in the percentage of positive fluorescent parasites (PPFP) for all the three forms of T. cruzi. Moreover, 94% of the samples of NCH presented negative values of PPFP with AMA and TRYPO, and 88% with EPI. Samples from the NCH group with falsepositive results were those belonging to the leishmaniasis patients. Considering the applicability of this technique in post-therapeuticmonitoring of Chagas disease, 100% of non-treated (NT) and treated non-cured (TNC) samples were positive with the three T. cruzi evolutive forms, while a percentage of 100% fromsamples of the treated cured (TC) patientswere negativewith AMA, 93% with TRYPO and 96% with EPI. The comparison between FC-ATE and two other flow cytometric tests using the same samples of patients NT, TNC and TC showed that the three techniques presented different reactivities, although categorical correlation between the methodologies was observed. Taken together, the results obtained with the novel FC-ATE method have shown an outstanding performance in the diagnosis and post-therapeutic monitoring of Chagas disease.Item Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes.(2005) Reis, Alexandre Barbosa; Carneiro, Cláudia Martins; Carvalho, Maria das Graças; Carvalho, Andréa Teixeira de; Giunchetti, Rodolfo Cordeiro; Mayrink, Wilson; Genaro, Odair; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo AssisThe effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1 + /CD5 + /CD4 + /CD8 + /CD21 + and CD14 + ) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5 + -T-cells within the lymphocyte-gate and increased percentage of CD21 + -B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1 + , CD4 + , CD8 + and CD21 + lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/ CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process.Item Differential impact of metacyclic and blood trypomastigotes on parasitological, serological and phenotypic features triggered during acute Trypanosoma cruzi infection in dogs.(2007) Carneiro, Cláudia Martins; Martins Filho, Olindo Assis; Reis, Alexandre Barbosa; Veloso, Vanja Maria; Araújo, Flávio Marcos Gomes de; Bahia, Maria Terezinha; Lana, Marta de; Coelho, George Luiz Lins Machado; Gazzinelli, Giovanni; Oliveira, Rodrigo Corrêa de; Tafuri, Wagner LuizDifferential impact of metacyclic and blood trypomastigotes on parasitological, serological and phenotypic features triggered during acute Trypanosoma cruzi infection in dogs A detailed follow-up investigation of the major parasitological, serological and phenotypic features in dogs experimentally infected with metacyclic (MT) and blood (BT) trypomastigotes of Trypanosoma cruzi strain Berenice-78, typifying vectorial and transfusional transmission of human Chagas disease, has been conducted. Although there were no changes with respect to the window of patent-parasitaemia, significant differences between MT- and BT-infected dogs in both the prepatent period (days 23 and 19, respectively) and the day of maximum parasitaemia (days 26 and 22, respectively) were recorded. A progressive enhancement in the level of T. cruzi -specific antibodies accompanied infection by both MT and BT forms, although higher IgG titres developed on days 14 and 21 following infection with MT forms. Higher Thy-1 + /CD21 + and lower CD4 + /CD8 + cell ratios, occasioned by increased levels of Thy-1 + and CD8 + T-cells and reduced frequencies of CD4 + T-cells and CD21 + B-lymphocytes, were observed in both MT- and BT-infected animals. The reduced frequency of CD14 + leukocytes was revealed as the most relevant phenotypic feature intrinsic to T. cruzi infection independent of inoculum source. BT-specific phenotypic features included an early reductionItem Clinical value of anti-Leishmania (Leishmania) chagasi IgG titers detected by flow cytometry to distinguish infected from vaccinated dogs.(2007) Andrade, Renata Aline de; Reis, Alexandre Barbosa; Gontijo, Célia Maria Ferreira; Braga, Lidiane Bento; Rocha, Roberta Dias Rodrigues; Araújo, Márcio Sobreira Silva; Vianna, Leonardo Rocha; Martins Filho, Olindo AssisLeishmune® vaccination covers a broader number of endemic areas of canine visceral leishmaniasis (CVL) and therefore the development of new serological devices able to discriminate CVL from Leishmune® vaccinees becomes an urgent need considering the post-vaccine seroconversion detected throughout conventional methodologies. Herein, we have described the establishment of a flow cytometry based methodology to detect anti-fixedL. ( L. ) chagasipromastigotes antibodies (FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2) in sera samples from Leishmania ( Leishmania ) chagasiinfected dogs and Leishmune® vaccinees. The results of FC-AFPA were reported along the sera titration curve (1:128–1:524,288), as percentage-of-positive-fluorescent-parasite (PPFP). The use of PPFP = 20% as a cut-off edge to segregate negative and positive results at sera dilution 1:2048 revealed outstanding performance indexes that elect FC-AFPA-IgG and IgG2 (both detected by polyclonal FITC-labeled second step reagent) applicable to the serological diagnosis of CVL, with 100% of specificity for both IgG and IgG2 and 97 and 93% of sensitivity, respectively. Moreover, FC-AFPA-IgG, applied at sera dilution 1:2048, also appeared as a useful tool to discriminate L. chagasiinfected dogs from Leishmune® vaccinees, with 76% of specificity. Outstanding likelihood indexes further support the performance of FC-AFPA-IgG for exclusion diagnosis of CVL in Leishmune® vaccinees. Analysis of FC-AFPA-IgG at sera dilution 1:8192 revealed the most outstanding indexes, demonstrating that besides the ability of PPFP 20% to exclude the diagnosis of CVL, a PPFP values higher 80%, mostly observed for infected dogs (INF) have a minimal change to come from a non-infected animal (NI) or Leishmune®.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 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 BarbosaWhile 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.