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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Resultados da Pesquisa
Item Immunogenicity in dogs of three recombinant antigens (TSA, LeiF and LmSTI1) potential vaccine for canine visceral leishmaniasis.(2005) Fujiwara, Ricardo Toshio; Vale, André Macedo; Silva, João Carlos França da; Costa, Roberto Teodoro da; Quetz, Josiane da Silva; Martins Filho, Olindo Assis; Reis, Alexandre Barbosa; Oliveira, Rodrigo Corrêa de; Coelho, George Luiz Lins Machado; Bueno, Lilian Lacerda; Bethony, Jeffrey Michael; Frank, Glen; Nascimento, Evaldo do; Genaro, Odair; Mayrink, Wilson; Reed, Steven G.; Campos Neto, AntonioControl of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE® and AdjuPrime®. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE® or AdjuPrime® resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE®. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime® as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE® in efficacy field trials against canine VL.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 Citometria de fluxo no diagnóstico da leishmaniose visceral canina.(2006) Carvalho Neta, Alcina Vieira de; Rocha, Roberta Dias Rodrigues; Gontijo, Célia Maria Ferreira; Reis, Alexandre Barbosa; Martins Filho, Olindo AssisDescreve-se a padronização de nova metodologia para detecção de anticorpos antiformas promastigotas fixadas de L. (L.) chagasi, por citometria de fluxo (AAPF-IgG), sua aplicabilidade e desempenho na identificação de casos de leishmaniose visceral canina (LVC). Foram avaliados dois grupos de cães classificados pela reação de imunofluorescência indireta (RIFI), como: não reatores (NR, n=10) e reatores (R, n=50) dos quais foram coletadas amostras de sangue (soro) para realização dos testes laboratoriais. Os resultados relacionados ao estabelecimento, aplicabilidade e desempenho da metodologia AAPF-IgG demonstraram que essa metodologia possibilita a identificação de uma região de reatividade diferencial entre cães NR e R, no soro diluído a 1:2048 e o valor de 20% de parasitos fluorescentes positivos (PPFP) como ponto de corte entre resultados positivos e negativos, mostrando que a AAPF-IgG aplica-se na identificação de casos de LVC, possibilitando distinguir 96% de cães R como positivos e 100% de cães NR como negativos. Esses resultados em conjunto sugerem que a utilização da AAPF-IgG pode ser um novo instrumento para ensaios clínicos de diagnóstico sorológico da LVC.Item Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections.(2007) Zanin, Francisca Helena Calheiros; Coelho, Eduardo Antônio Ferraz; Tavares, Carlos Alberto Pereira; Silva, Eduardo de Almeida Marques da; Costa, Miriam Maria Silva; Rezende, Simone Aparecida; Gazzinelli, Ricardo Tostes; Fernandes, Ana Paula Salles MouraSeveral antigens have been tested as vaccine candidates against Leishmania infections but controversial results have been reported when different antigens are co-administered in combined vaccination protocols. Immunization with A2 or nucleoside hydrolase (NH) antigens was previously shown to induce Th1 immune responses and protection in BALB/c mice against Leishmania donovani and L. amazonensis (A2) or L. donovani and L. mexicana (NH) infections. In this work, we investigated the protective efficacy of A2 and NH DNA vaccines, in BALB/c mice, against L. amazonensis or L. chagasi challenge infection. Immunization with either A2 (A2-pCDNA3) or NH (NH-VR1012) DNA induced an elevated IFN-g production before infection; however, only A2 DNA immunized mice were protected against both Leishmania species and displayed a sustained IFN-g production and very low IL-4 and IL-10 levels, after challenge. Mice immunized with NH/A2 DNA produced higher levels of IFN-g in response to both specific recombinant proteins (rNH or rA2), but displayed higher IL-4 and IL-10 levels and increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals. These data extend the characterization of the immune responses induced by NH and A2 antigens as potential candidates to compose a defined vaccine and indicate that a highly polarized type 1 immune response is required for improvement of protective levels of combined vaccines against both L. amazonensis and L. chagasi infections.Item Successful vaccination against Leishmania chagasi infection in BALB/c mice with freeze-thawed Leishmania antigen and Corynebacterium parvum.(2007) Vilela, Márcia de Carvalho; Gomes, Daniel Cláudio de Oliveira; Silva, Eduardo de Almeida Marques da; Serafim, Tiago Donatelli; Afonso, Luís Carlos Crocco; Rezende, Simone AparecidaThis study evaluated the potential of a Leishmania antigen vaccine in protecting BALB/c mice against Leishmania chagasi. Mice received two subcutaneous doses of L. amazonensis vaccine with Corynebacterium parvum and subsequent boost was done without adjuvant. One week later, mice were challenged with L. chagasi. We observed that this vaccine caused a significant reduction in parasite load in liver and spleen and induced a high production of IFN-_ and IL-4 by spleen cells from vaccinated mice in response to Leishmania antigen. Together, our data show that this vaccine is capable of inducing a Th1/Th2 response that is important to control parasite replication.Item Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis.(2005) Rosário, Eliza Yoshie do; Genaro, Odair; Silva, João Carlos França da; Costa, Roberto Teodoro da; Mayrink, Wilson; Reis, Alexandre Barbosa; Carneiro, MariângelaThe performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.Item Isotype patterns of immunoglobulins : hallmarks for clinical status and tissue parasite density in brazilian dogs naturally infected by Leishmania (Leishmania) chagasi.(2006) Reis, Alexandre Barbosa; Carvalho, Andréa Teixeira de; Vale, André Macedo; Marques, Marcos José; Giunchetti, Rodolfo Cordeiro; Mayrink, Wilson; Guerra, Luanda Liboreiro; Andrade, Renata Aline de; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo AssisThe role of anti-leishmanial immune response underlying the susceptibility/resistance during canine visceral leishmaniasis (CVL) has been recognized throughout ex vivo and in vitro investigations. Recently, we demonstrated that immunoglobulin levels (Igs), as well as the parasite load are relevant hallmarks of distinct clinical status of CVL. To further characterize and upgrade the background on this issue, herein, we have evaluated, inLeishmania ( Leishmania ) chagasinaturally infected dogs, the relationship between tissue parasitism (skin, bone marrow, spleen, liver and lymph node), the CVL clinical status (asymptomatic (AD), with no suggestive signs of the disease; oligosymptomatic (OD), with maximum three clinical signs—opaque bristles; localized alopecia and moderate loss of weight; symptomatic (SD), serologically positive with severe clinical signs of visceral leishmaniasis), and the humoral immunological profile of anti-Leishmania immunoglobulins (IgG, IgG1, IgG2, IgM, IgA and IgE). Our major statistically significant findings revealed distinct patterns of tissue parasite density within L. chagasi-infected dogs despite their clinical status, pointing out the spleen and skin as the most relevant sites of high parasitism during ongoing CVL. Parasite density of bone marrow and spleen were the most reliable parasitological markers to decode the clinical status of CVL. Moreover, the parasite density of bone marrow better correlates with most anti- Leishmania Igs reactivity. Additionally, a prognostic hallmark for canine visceral leishmaniasis was found, highlighting strong correlation between IgG1 and asymptomatic disease, but with IgA, IgE and IgG2 displaying better association with symptomatic disease. The new aspects of this study highlighted pioneer findings that correlated the degree of tissue parasite density (low (LP), medium (MP) and high (HP) parasitism) with distinct patterns of anti- Leishmania Igs reactivity. In this scope, our data re-enforce the anti- Leishmania IgG but with IgA reactivity as the better marker for overall tissue parasitism. The association between clinical status, Ig profile and the tissue parasitism support a novel investigation on the impact of humoral immune response and susceptibility/resistance mechanism during ongoing CVLItem Relationship between Canine Visceral Leishmaniosis and the Leishmania (Leishmania) chagasi Burden in Dermal Inflammatory Foci.(2006) Giunchetti, Rodolfo Cordeiro; Mayrink, Wilson; Genaro, Odair; Carneiro, Cláudia Martins; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis; Marques, Marcos José; Tafuri, Wagner Luiz; Reis, Alexandre BarbosaThe skin is the first point of contact with organisms of the genus Leishmania from sand fly vectors, and apparently normal skin of sick dogs harbours amastigote forms of Leishmania chagasi. In relation to canine visceral leishmaniosis ( CVL), the ear skin was examined in 10 uninfecte d dog s ( UD s ) and in 31 dog s dog s naturally infected with L. chagasi . The infected animals consisted of 10 symptomless dogs ( SLDs ), 12 mildly affected dog s ( M A D s ) and nine affected dog s ( A D s ). A higher parasite burden was demonstrated in A D s than in SLD s by anti-Leishmania immunohistochemistry (P o0.01), and by Leishman Donivan Unit ( LDU ) indices ( P ¼ 0.0024) obtained from Giemsa- stained impression smears. Sect ions stained with haematoxylin and eosin demonstrated a higher intensity of inflammatory changes in ADs than in SLD s ( P o0.05), and in t he latter group low cytometry demonstrated a correlation (P ¼ 0. 0 5/ r ¼ 0.7454) between the percentage of CD14 + monocytes in peripheral blood and chronic der mal inflammation. Extracellular matrix assessment for reticular fibres by staining of sect ions with Masson trichrome and Gomori ammoniacal silver demonstrated a decrease in collagen typeI and an increase in collagen type III as the clinical signs increase d. The data on correlation between cellular phenotypes and histological changes seemed to reflect cellular activation and migration from peripheral blood to the skin, mediated by antigenic stimulation. The results suggested that chronic dermal inflammation and cutaneous parasitism were directly related to t he sever it y of clinical disease.Item 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 Analysis of the cytokine profile in spleen cells from dogs naturally infected by Leishmania chagasi.(2007) Lage, Ramon Silva; Oliveira, Guilherme Corrêa de; Busek, Solange Cristina Uber; Guerra, Luanda Liboreiro; Giunchetti, Rodolfo Cordeiro; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaRecent studies suggest that asymptomatic dogs infected with canine visceral leishmaniasis (CVL) develop a Th1 immunological profile whilst oligosymptomatic and symptomatic CVL-infected animals present a Th2 profile. In the present study, an RT-PCR method has been standardised and employed to evaluate the frequency and the semi-quantitative level of expression of the cytokines IL-4, IL-10, IL-12, INF- g and TNF- a in splenocytes of 30 dogs naturally infected with Leishmania chagasiand of 7 non-infected dogs (NID). An increase in the level of expression of IL-12 ( p = 0.059) was detected in all CVL-infected dogs compared with NID. In dogs exhibiting high parasitism, the frequency of expression of IL-10 was higher ( p = 0.011) than in animals presenting low parasitism or medium parasitism (MP) and in NID animals, whilst the level of expression of IL-10 was higher (p = 0.0094) than in animals exhibiting MP and in the NID group. Positive correlations between the levels of expression of IL-10 with respect to the progression of the disease (IL-10: r = 0.3510; p = 0.0337) and the levels of expression of IL-10 and INF- g increase in parasitism (IL-10: r = 0.3428; p = 0.0438 and INF- g: r = 0.4690; p = 0.0045) were observed. Such data suggest that CVL is marked by a balanced production of Th1 and Th2 cytokines, with a predominant accumulation of IL-10 as a consequence of an increase in parasitic load and progression of the disease, and INF- g was related with the increase in parasitic load.