EFAR - Escola de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/451
Notícias
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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93 resultados
Resultados da Pesquisa
Item The use of an adjuvant system improves innate and adaptive immune response when associated with a Leishmania (Viannia) braziliensis antigen in a vaccine candidate against L. (Leishmania) infantum infection.(2023) Mathias, Fernando Augusto Siqueira; Ostolin, Thais Lopes Valentim Di Paschoale; Reis, Levi Eduardo Soares; Cardoso, Jamille Mirelle de Oliveira; Brito, Rory Cristiane Fortes de; Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Vieira, Paula Melo de Abreu; Reis, Alexandre BarbosaBackground: The adjuvants’ optimal dose and the administration route can directly influence the epitope recognition patterns and profiles of innate response. We aimed to establish the effect and the optimal dose of adjuvant systems for proposing a vaccine candidate to be employed with Leishmania (Viannia) braziliensis. Methods: We evaluated the adjuvants saponin (SAP), monophosphoryl lipid A (MPL) and resiquimod (R-848) isolated and combined as adjuvant systems in a lower dose corresponding to 25%, 33%, and 50% of each adjuvant total dose. Male outbred BALB/c mice were divided into 13 groups, SAP, MPL, and R-848 isolated, and the adjuvant systems SAP plus MPL (SM), SAP plus R-848 (SR), and MPL plus R-848 (MR). Results: SM50 increased levels of all chemokines analyzed and TNF production, while it presented an increased inflammatory cell infiltrate in the skin with macrophage recruitment. Thus, we proposed a vaccine candidate employing L. (V.) braziliensis antigen associated with the SM adjuvant system against experimental L. (Leishmania) infantum challenge. We observed a significant increase in the frequency of cells expressing the central and effector memory CD4+ T cells phenotype in immunized mice with the LBSM50. In the liver, there was a decreased parasite load when mice received LBSM50. Conclusions: When combined with L. (V.) braziliensis antigen, SM50 increases TNF and IFN-γ, which generates central and effector memory CD4+ T cells. Therefore, using an adjuvant system can promote an effective innate immune response with the potential to compose future vaccines.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 ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of chagas’ disease.(2023) Castro, Júlia Teixeira de; Brito, Rory Cristiane Fortes de; Souza, Natália Satchiko Hojo de; Azevedo, Bárbara Ribeiro Batista Vaz de; Castro, Natália Salazar de; Ferreira, Camila Pontes; Giusta, Caroline Junqueira; Fernandes, Ana Paula Salles Moura; Vasconcellos, José Ronnie Carvalho de; Cardoso, Jamille Mirelle de Oliveira; Soares, Rodrigo Dian de Oliveira Aguiar; Vieira, Paula Melo de Abreu; Carneiro, Cláudia Martins; Valiate, Bruno Vinícius Santos; Toledo, Cristiane; Salazar, Andres M.; Caballero, Otávia; Vieira, Joseli Lannes; Teixeira, Santuza Maria Ribeiro; Reis, Alexandre Barbosa; Gazzinelli, Ricardo TostesImmunization with the Amastigote Surface Protein-2 (ASP-2) and Trans-sialidase (TS) antigens either in the form of recombinant protein, encoded in plasmids or human adenovirus 5 (hAd5) confers robust protection against various lineages of Trypanosoma cruzi. Herein we generated a chimeric protein containing the most immunogenic regions for T and B cells from TS and ASP-2 (TRASP) and evaluated its immunogenicity in comparison with our standard protocol of heterologous prime-boost using plasmids and hAd5. Mice immunized with TRASP protein associated to Poly-ICLC (Hiltonol) were highly resistant to challenge with T. cruzi, showing a large decrease in tissue parasitism, parasitemia and no lethality. This protection lasted for at least 3 months after the last boost of immunization, being equivalent to the protection induced by DNA/hAd5 protocol. TRASP induced high levels of T. cruzispecific antibodies and IFNγ-producing T cells and protection was primarily mediated by CD8+ T cells and IFN-γ. We also evaluated the toxicity, immunogenicity, and efficacy of TRASP and DNA/hAd5 formulations in dogs. Mild collateral effects were detected at the site of vaccine inoculation. While the chimeric protein associated with Poly-ICLC induced high levels of antibodies and CD4+ T cell responses, the DNA/hAd5 induced no antibodies, but a strong CD8+ T cell response. Immunization with either vaccine protected dogs against challenge with T. cruzi. Despite the similar efficacy, we conclude that moving ahead with TRASP together with Hiltonol is advantageous over the DNA/hAd5 vaccine due to pre-existing immunity to the adenovirus vector, as well as the cost-benefit for development and large-scale production.Item Influence of climatic variables on the number of cases of visceral leishmaniasis in an endemic urban area.(2022) Duarte, Rafael Vieira; Monteiro, Josefa Clara Lafuente; Cruz, Tamara Coelho; Ribeiro, Lucas Moreira; Morais, Maria Helena Franco; Carneiro, Mariângela; Reis, Alexandre Barbosa; Ribeiro, Sérvio Pontes; Vital, Wendel CouraBackground Visceral leishmaniasis (VL) is a neglected tropical disease endemic in several countries, with as much as 97% of cases in the Americas attributable to Brazil. Despite considerable investment in disease control, Belo Horizonte is one of the Brazilian municipalities with the highest mortality rates. The present study aimed to investigate the association between climatic variables and the increase in the number of VL cases in Belo Horizonte. Methods The study analyzed 1,897 laboratory-confirmed cases of VL registered by the municipality’s Epidemiological Surveillance from 1994 to 2019. The climatic variables were obtained from the National Institute of Meteorology. Results The first reported VL case was detected in 1994, and since then, the disease has spread throughout the municipality. The disease was most common in the very young age groups, 1 to 4 years, and older groups, 40 to 49 years old. Approximately 63% of detected cases were men. A peak number of cases were observed in 2004, 2006, 2008 and 2017. Total precipitation was significantly associated with the number of VL cases, with more cases depending on rainfall (P=0.006), thus confirming that climate contributes to the disease spread. Conclusions These results demonstrate the need for the actions of the Visceral Leishmaniasis Surveillance and Control Program to be reinforced in years with above-average rainfall, a situation that is increasingly more frequent in Southeastern Brazil due to global climate change.Item Replacement of Leishmania (Leishmania) infantum populations in an endemic focus of visceral leishmaniasis in Brazil.(2022) Valdivia Rodríguez, Hugo Oswaldo; Roatt, Bruno Mendes; Baptista, Rodrigo de Paula; Ottino, Jennifer; Santos, Anderson Coqueiro dos; Sanders, Mandy J.; Reis, Alexandre Barbosa; Cotton, James A.; Bartholomeu, Daniella CastanheiraVisceral leishmaniasis is an important global health problem with an estimated of 50,000 to 90,000 new cases per year. VL is the most serious form of leishmaniasis as it can be fatal in 95% of the cases if it remains untreated. VL is a particularly acute problem in Brazil which contributed with 97% of all cases reported in 2020 in the Americas. In this country, VL affects mainly the poorest people in both urban and rural areas and continues to have a high mortality rate estimated around 8.15%. Here, we performed a temporal parasite population study using whole genome sequence data from a set of 34 canine isolates sampled in 2008, 2012 and 2015 from a re-emergent focus in Southeastern Brazil. Our study found the presence of two distinct sexual subpopulations that corresponded to two isolation periods. These subpopulations diverged hundreds of years ago with no apparent gene flow between them suggesting a process of rapid replacement during a two-year period. Sequence comparisons and analysis of nucleotide diversity also showed evidence of balancing selection acting on transport-related genes and antigenic families. To our knowledge this is the first population genomic study showing a turn-over of parasite populations in an endemic region for leishmaniasis. The complexity and rapid adaptability of these parasites pose new challenges to control activities and demand more integrated approaches to understand this disease in New World foci.Item The entrance route : oral, mucous, cutaneous, or systemic has a marked influence on the outcome of Trypanosoma cruzi experimental infection.(2022) Gonçalves, Karolina Ribeiro; Mazzeti, Ana Lia; Nascimento, Alvaro Fernando da Silva do; Lacerda, Jéssica Mara Castro; Paiva, Nívia Carolina Nogueira de; Mathias, Fernando Augusto Siqueira; Reis, Alexandre Barbosa; Caldas, Sérgio; Bahia, Maria TerezinhaIn recent decades, the oral infection of Trypanosoma cruzi has gathered increased attention due to frequent outbreaks that can lead to more severe clinical signs than those usually found in the areas of vector transmission. This study addresses the main routes of infection using metacyclic trypomastigotes (MT) and blood trypomastigotes (BT). Herein, BALB/c mice were infected with the Colombian (TcI) strain via intraperitoneal (IP), oral, intragastric (IG), ocular (OC) and cutaneous (CT) routes with 106 culture-derived MT or BT. Parasitemia was intermittent and low in animals inoculated with MT, in contrast, high parasitemia levels were found in BT-mice. A tropism for the muscles was observed in oral or IG infection with BT. Differently, the parasite was widely distributed in the tissues of mice infected with MT. However, the intensity of the inflammation infiltrating the tissues was higher in oral or IG infection with BT. Animals inoculated with BT via the IG route had similar serum levels of IFN-γ and smaller IL-10 compared to those infected with MT via the IG route. TNF-α levels were higher in the serum from BT-animals, which could explain the higher intensity of heart inflammation in these animals. Our results suggest that the infective form and the route of infection differentially modulated the outcome of Trypanosoma cruzi mice infection.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 A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis.(2022) Ostolin, Thais Lopes Valentim Di Paschoale; Gusmão, Miriã Rodrigues; Mathias, Fernando Augusto Siqueira; Cardoso, Jamille Mirelle de Oliveira; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Ruiz, Jeronimo Conceição; Resende, Daniela de Melo; Brito, Rory Cristiane Fortes de; Reis, Alexandre BarbosaThe development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 107 stationary-phase Leishmania infantum promastigotes via tail vein. Immunogenicity and parasitism in spleen and liver of immunized mice were evaluated 45 days postchallenge. Our results revealed that the immunization with Poly-T Leish and Poly-T Leish/SM increases the percentage of specific T (CD4+ and CD8+) lymphocytes proliferation in vitro after antigen-specific stimulation. Also, Poly-T Leish and Poly-T Leish/SM groups showed a high percentage of IFN-γ and TNF-α-producing T cells, meanwhile, the Poly-T Leish/SM group also showed an increased percentage of multifunctional T cells producing double and triple-positive (IFN-γ+TNF-α+IL-2+) cytokines. The immunization with Poly-T Leish or Poly-T Leish/ SM stimulated a decreased IL-4 and IL-10 compared to the Saline and adjuvant group. Poly-T Leish/SM immunized mice exhibit a noteworthy reduction in the parasite burden (spleen and liver) through real-time PCR (96%). Moreover, we observed higher nitrite secretion in 120-hour stimulated-culture supernatant using Griess method. We demonstrated that the Poly-T Leish/SM candidate was potentially immunogenic, providing enhancement of protective immune mechanisms, and conferred protection reducing parasitism. Our candidate was considered potential against visceral leishmaniasis, and eventually, could be tested in phase I and II clinical trials in dogs.Item Immunoprophylaxis using polypeptide chimera vaccines plus adjuvant system promote Th1 response controlling the spleen parasitism in hamster model of visceral leishmaniasis.(2022) Gusmão, Miriã Rodrigues; Ostolin, Thais Lopes Valentim Di Paschoale; Carvalho, Lívia Mendes; Costa, Ana Flávia Pereira; Moreira, Gabriel José Lucas; Cardoso, Jamille Mirelle de Oliveira; Soares, Rodrigo Dian de Oliveira Aguiar; Reis, Alexandre Barbosa; Brito, Rory Cristiane Fortes de; Roatt, Bruno MendesIn recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras in Mesocricetus auratus hamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-c and TNF-a cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease.