Navegando por Autor "Melo, Maria Norma"
Agora exibindo 1 - 13 de 13
Resultados por página
Opções de Ordenação
Item American tegumentary leishmaniasis : effectiveness of an immunohistochemical protocol for the detection of leishmania in skin.(2013) Alves, Cibele Fontes; Alves, Cíntia Fontes; Figueiredo, Maria Marta; Souza, Carolina Carvalho de; Coelho, George Luiz Lins Machado; Melo, Maria Norma; Tafuri, Washington Luiz; Raso, Pedro; Soares, Rodrigo Pedro Pinto; Tafuri, Wagner LuizBackground: American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody. Methodology: Skin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples. Results: The IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a nonspecific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system). Conclusions: The data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.Item An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein.(2013) Rocha, Marcele Neves; Corrêa, Célia Maria; Melo, Maria Norma; Beverley, Stephen M.; Martins Filho, Olindo Assis; Madureira, Ana Paula; Soares, Rodrigo Pedro PintoFluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy islabor intensive and subject to observer variation. In this work, we propose afluorimetric method for drugscreening using redfluorescent parasites. FluorescentLeishmania amazonensiswere developed aftertransfection with integration plasmids containing either red (RFP) or greenfluorescent protein (GFP)genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected toflow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents andpropranolol derivatives previously shown to be active againstTrypanosoma cruzi. Flow cytometry analysisdiscriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. Withmicroscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity andsusceptibility to amphotericin B and propranolol derivatives. Retention offluorescence remained in theintracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFPparasites was only achieved in thefluorimeter. Murine BALB/c macrophages were infected with LaRFPparasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) andtested molecules. Although it was possible to determine IC50values for 4 propranolol derivatives (1, 2b, 3, and4b), all compounds were considered inactive. This study is thefirst to develop afluorimetric drug screeningtest forL. amazonensisRFP. Thefluorimetric test was comparable to microscopy with the advantage of beingfaster and not requiring manual counting.Item Cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants : saponin, incomplete Freund's adjuvant, and monophosphoryl Lipid A.(2012) Souza, Juliana Vitoriano de; Moreira, Nádia das Dores; Carvalho, Andréa Teixeira de; Carneiro, Cláudia Martins; Mathias, Fernando Augusto Siqueira; Vieira, Paula Melo de Abreu; Giunchetti, Rodolfo Cordeiro; Moura, Sandra Aparecida Lima de; Fujiwara, Ricardo Toshio; Melo, Maria Norma; Reis, Alexandre BarbosaVaccine adjuvants are substances associated with antigens that are fundamental to the formation of an intense, durable, and fast immune response. In this context, the use of vaccine adjuvants to generate an effective cellular immune response is crucial for the design and development of vaccines against visceral leishmaniasis. The objective of this study was to evaluate innate inflammatory response induced by the vaccine adjuvants saponin (SAP), incomplete Freund’s adjuvant (IFA), and monophosphoryl lipid A (MPL). After a single dose of adjuvant was injected into the skin of mice, we analyzed inflammatory reaction, selective cell migration, and cytokine production at the injection site, and inflammatory cell influx in the peripheral blood. We found that all vaccine adjuvants were able to promote cell recruitment to the site without tissue damage. In addition, they induced selective migration of neutrophils, macrophages, and lymphocytes. The influx of neutrophils was notable at 12 h in all groups, but at other time points it was most evident after inoculation with SAP. With regard to cytokines, the SAP led to production of interleukin (IL)-2, IL-6, and IL-4. IFA promoted production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, IL-17, IL-4, and IL-10. We also observed that MPL induced high production of IL-2, TNF-α, and IFN-γ, in addition to IL-6, IL-17, and IL-10. In peripheral blood, values of certain cell populations in the local response changed after stimulation. Our data demonstrate that the three vaccine adjuvants stimulate the early events of innate immune response at the injection site, suggesting their ability to increase the immunogenicity of co-administered antigens. Moreover, this work provides relevant information about elements of innate and acquired immune response induced by vaccine adjuvants administered alone.Item Despite Leishvaccine and Leishmune ® trigger distinct immune profiles, their ability to activate phagocytes and CD8 + T-cells support their high-quality immunogenic potential against canine visceral leishmaniasis.(2008) Araújo, Márcio Sobreira Silva; Andrade, Renata Aline de; Vianna, Leonardo Rocha; Mayrink, Wilson; Reis, Alexandre Barbosa; Avelar, Renato Sathler; Carvalho, Andréa Teixeira de; Andrade, Mariléia Chaves; Melo, Maria Norma; Martins Filho, Olindo AssisPhenotypic features of peripheral blood leukocytes have been investigated as a prerequisite to characterize the protective immunity attributed to both Leishvaccine and Leishmune ® . Our results showed that either those vaccine were accompanied by distinct profiles on innate immune compartment. While Leishvaccine promoted early changes in phenotypic fea-tures of neutrophils and eosinophils with late involvement of monocytes, Leishmune ® induced early and persistent activation of neutrophils and monocytes, without changes on eosinophil activation status. Regarding the adaptive immunity, Leishvaccine sponsored a mixed profile, associated with phenotypic changes of T and B-lymphocytes. Major phenotypic changes in CD4 + T-cells with transient activation of CD8 + T-cell, besides decreased frequency of B-cell expressingItem Ecto-nucleotidase activities of promastigotes from leishmania (Viannia) braziliensis relates to parasite infectivity and disease clinical outcome.(2012) Leite, Pauline Martins; Gomes, Rodrigo Saar; Figueiredo, Amanda Braga de; Serafim, Tiago Donatelli; Tafuri, Wagner Luiz; Gomes, Carolina Cavaliéri; Moura, Sandra Aparecida Lima de; Fietto, Juliana Lopes Rangel; Melo, Maria Norma; Dias, Fátima Ribeiro; Oliveira, Milton Adriano Pelli de; Rabello, Ana Lúcia Teles; Afonso, Luís Carlos CroccoBackground: Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. Methodology/Principal Findings: Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. Conclusions/Significance: Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.Item Effect on cellular recruitment and the innate immune response by combining saponin, monophosphoryl lipid-A and incomplete freund’s adjuvant with Leishmania (Viannia) braziliensis antigens for a vaccine formulation.(2019) Souza, Juliana Vitoriano de; Mathias, Fernando Augusto Siqueira; Moreira, Nádia das Dores; Soares, Rodrigo Dian de Oliveira Aguiar; Vieira, Paula Melo de Abreu; Carvalho, Andréa Teixeira de; Carneiro, Cláudia Martins; Giunchetti, Rodolfo Cordeiro; Brito, Rory Cristiane Fortes de; Fujiwara, Ricardo Toshio; Roatt, Bruno Mendes; Melo, Maria Norma; Reis, Alexandre BarbosaThe poor immunogenicity displayed by some antigens has encouraged the development of strategies to improve the immune response and safety of vaccine candidates, resulting in an intense search for substances that potentiate vaccine response. Adjuvants have these properties helping vaccine candidates to induce a strong, durable, and fast immune response. In this study, we evaluated the specific immune response of adjuvants alone, Saponin (SAP), Incomplete Freund’s Adjuvant (IFA) and Monophosphoryl lipid-A SE (MPL-SE ) and in combination with total antigen of L. braziliensis (LB): LBSAP, LBIFA and LBMPL. The specific immune response induced by these compositions demonstrated that they were powerfully immunogenic, increasing cellular infiltration in the skin. Draining lymph nodes cultures showed that LBIFA and LBMPL have higher ability to increase the capacity of APCs to present antigens, with increased frequency of CD11c+ CD86+ cells. SAP, MPL, LBSAP, LBIFA and LBMPL could activate lymphocytes increasing expression of CD69 and CD25. LBSAP group was an excellent inducer of pro-inflammatory cytokines at 24 h. At 48 h, higher cytokines production was observed in IFA, LBIFA, MPL and LBMPL groups. Our data demonstrate that LBSAP and LBMPL are potential formulations to be tested in other experimental models. Also, the data obtained could expand the knowledge about immune response after sensitization and also contribute to the development of safe, immunogenic and effective vaccines.Item Germ-free mice produce high levels of interferon-gamma in response to infection with Leishmania major but fail to heal lesions.(2005) Oliveira, Marcia Rosa de; Tafuri, Wagner Luiz; Afonso, Luís Carlos Crocco; Oliveira, Milton Adriano Pelli de; Nicoli, Jacques Robert; Vieira, Etel Rocha; Scott, Phillip; Melo, Maria Norma; Vieira, Leda QuerciaIn order to investigate the importance of the host microbiota on differentiation of T cell subsets in response to infection, Swiss/NIH germ-free mice and conventional (microbiota-bearing) mice were infected with Leishmania major, and lesion development, parasite loads, and cytokine production were assessed. Germ-free mice failed to heal lesions and presented a higher number of parasites at the site of infection than their conventional counterparts. In addition, histopathological analysis indicated a higher density of parasitized macrophages in lesions from germ-free mice than in conventional mice. The initial production of interleukin (IL)-12 and interferon-gamma (IFN-c) in germ-free mice was comparable to the conventional controls. Also, germ-free mice produced elevated levels of IFN-c and lower levels of IL-4 throughout the course of infection, suggesting the development of a Th1 response. Macrophages from germ-free mice exposed to IFN-c and infected with amastigotes in vitro were not as efficient at killing parasites as macrophages from conventional animals. These observations indicate that the microbiota is not essential for the development of Th1 immune responses, but seems to be important for macrophage activation.Item Immunological changes in canine peripheral blood leukocytes triggered by immunization with first or second generation vaccines against canine visceral leishmaniasis.(2011) Araújo, Márcio Sobreira Silva; Andrade, Renata Aline de; Avelar, Renato Sathler; Magalhães, Camila Paula; Carvalho, Andréa Teixeira de; Andrade, Mariléia Chaves; Campolina, Sabrina Sidney; Melo, Maria Norma; Vianna, Leonardo Rocha; Mayrink, Wilson; Reis, Alexandre Barbosa; Malaquias, Luiz Cosme Cotta; Rocha, Luciana Morais; Martins Filho, Olindo AssisIn this study, we summarized the major phenotypic/functional aspects of circulating leuko-cytes following canine immunization with Leishvaccine and Leishmune ®. Our findings showed that Leishvaccine triggered early changes in the innate immunity (neutrophils and eosinophils) with late alterations on monocytes. Conversely, Leishmune ® induced early phenotypic changes in both, neutrophils and monocytes. Moreover, Leishvaccine triggered mixed activation-related phenotypic changes on T-cells (CD4 + and CD8 +) and B-lymphocytes, hereas Leishmune® promoted a selective response, mainly associated with CD8 + T-cell activation. Mixed cytokine profile (IFN- /IL-4) was observed in Leishvaccine immunized dogs whereas a selective pro-inflammatory pattern (IFN- /NO) was induced by Leishmune ® vaccination. The distinct immunological profile triggered by Leishvaccine and Leishmune ® may be a direct consequence of the distinct biochemical composition of these immunobiological, i.e. complex versus purified Leishmaniaantigen along with Bacillus Calmette-Guérin (BCG) versus saponin adjuvant. Both immunobiologicals are able to acti-vate phagocytes and CD8 + T-cells and therefore could be considered as a putative vaccines against canine visceral leishmaniasis (CVL)Item Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment.(2006) Mayrink, Wilson; Botelho, Ana Cristina de Carvalho; Magalhães, Paulo Araújo; Batista, Sebastião Mariano; Lima, Antonio de Oliveira; Genaro, Odair; Costa, Carlos Alberto da; Melo, Maria Norma; Michalick, Marilene Susan Marques; Williams, Paul; Dias, Magno; Caiaffa, Waleska Teixeira; Nascimento, Evaldo do; Coelho, George Luiz Lins MachadoO tratamento de primeira escolha para leishmaniose tegumentar americana é o antimonial pentavalente. Embora este tratamento seja na maioria das vezes efetivo e indicado, devem ser consideradas as desvantagens tais como efeitos colaterais, longa duração do tratamento e contra-indicação para cardiopatas, nefropatas, idosos, grávidas e outras condições. Com o advento da vacina antileishmaniose tegumentar americana para fins profiláticos e terapêuticos, associando-a ou não a outros fármacos, muitas pesquisas têm sido desenvolvidas, sendo a vacina a principal entre os atuais recursos no tratamento e prevenção da leishmaniose tegumentar americana. Em conclusão, a associação do antimônio com a vacina (imunoquimioterapia) apresentou o mesmo índice de cura em relação ao tratamento padrão (100%), e ainda reduziu o volume do sal em 17,9% e o tempo de cura significativamente, de 87 para 62 dias; conseqüentemente, reduzindo os efeitos colaterais.Item Leishmaniose visceral canina : avaliação da metodologia sorológica utilizada em inquéritos epidemiológicos.(1991) Costa, Carlos Alberto da; Genaro, Odair; Lana, Marta de; Magalhães, Paulo Araújo; Dias, Magno; Michalick, Marilene Susan Marques; Melo, Maria Norma; Costa, Roberto Teodoro da; Rocha, Neuza Maria de Magalhaes; Mayrink, WilsonFoi realizado um estudo comparativo da reação de imunofluorescencia indireta em eluatos de sangue de cães infectados experimentalmente com diferentes tripanosomatideos. Utilizaram-se como antigenopromastigotas de L. mexicana, L. braziliensis e L. chagasi. Os resultados mostraram que a sensibilidade do método foi de 87,5% para o diagnostico do calazar canino, independentemente do antígeno empregado; e que ocorre reação cruzada com Leishmaniose tegumentar em 75% dos casos e com doença de Chagas em 83,3%. Levantamento epidemiológico em área de leishmaniose confirma que a reação de imunofluorescencia em eluatos de sangue canino fornece reações cruzadas em cães infectados com Leishmania braziliensis e L. chagasi. Não se verificou reação cruzada pela RFC. Sugere-se a utilização da reação de imunofluorescencia nas campanhas de saúde publica, mas e de se chamar a atenção para o fato de que as taxas de positividade não devem ser utilizadas como indicadores da prevalência do calazar canino.Item Susceptibility to re-infection in C57BL/6 mice with recombinant strains of Toxoplasma gondii.(2011) Brandão, Geane Peroni; Melo, Maria Norma; Caetano, Bráulia Costa; Carneiro, Cláudia Martins; Silva, Letícia de Azevedo; Vitor, Ricardo Wagner de AlmeidaThis work reports results of re-infection of BALB/c and C57BL/6 mice with different recombinant strains of Toxoplasma gondii. Mice were prime-infected with the non-virulent D8 strain and challenged with virulent strains. PCR–RFLP of cS10-A6 genetic marker of T. gondii demonstrated that BALB/c mice were reinfected with the EGS strain, while C57BL/6 mice were re-infected with the EGS and CH3 strains. Levels of IFN-c and IL-10 after D8 prime-infection were lower in C57BL/6 than in BALB/c mice. Brain inflammation after D8 prime-infection was more intense in C57BL/6 than in BALB/c mice. It was shown that reinfection depends on mice lineage and genotype of the strain used in the challenge.Item T-cell-derived cytokines, nitric oxide production by peripheral blood monocytes and seric anti- Leishmania (Leishmania) chagasi IgG subclass patterns following immunization against canine visceral leishmaniasis using Leishvaccine and Leishmune ®.(2009) Araújo, Márcio Sobreira Silva; Andrade, Renata Aline de; Avelar, Renato Sathler; Carvalho, Andréa Teixeira de; Andrade, Mariléia Chaves; Vianna, Leonardo Rocha; Mayrink, Wilson; Reis, Alexandre Barbosa; Malaquias, Luiz Cosme Cotta; Melo, Maria Norma; Martins Filho, Olindo AssisIt is generally accepted that distinct cytokine expression by the cellular immune response plays a critica role during the outcome of experimental as well as natural canine visceral Leishmaniasis (CVL). Despite the fact that immunoprophylaxis of CVL has become an important control strategy and protective immunity has been reported upon immunization with whole as well as purifiedLeishmaniaantigens, the cytokine profile of T-cells triggered by anti-CVL vaccines still remain to be determined. Herein, we have developed a cross-sectional analysis of German Shepherd dogs submitted to vaccination protocols with Leishvaccine (n = 6) and Leishmune ® (n = 6). Our data identified distinct immunological profiles elicited by Leishvaccine and Leishmune ® , with the Leishvaccine triggering a mixed, IFN- and IL-4, cytokine pattern in addition to high levels of anti- LeishmaniaIgG1, whereas the Leishmune ® induced an immunological pattern char- acterized by enhanced levels of IFN- , NO and anti- Leishmania chagasi IgG2. It was important to notice that despite the distinct immunological patterns triggered by Leishvaccine and Leishmune ® , the ability of both immunobiologicals to activate T-cell-derived IFN- synthesis further suggesting their immunogenic potential against CVL. These findings added support to our hypothesis that both antigenic composition (whole antigen in Leishvaccine versus purified antigen in Leishmune ® ) as well as the adjuvant nature (BGC and saponin) used for the vaccine formulation may count for the distinct activation pattern observed.Item The influence of ecto-nucleotidases on Leishmania amazonensis infection and immune response in C57B/6 mice.(2010) Testasicca, Miriam Conceição de Souza; Assis, Elisângela Aparecida de; Gomes, Rodrigo Saar; Silva, Eduardo de Almeida Marques da; Melo, Maria Norma; Fietto, Juliana Lopes Rangel; Afonso, Luís Carlos CroccoPrevious results from our laboratory and from the literature have implicated the expression of ectonucleotidases in the establishment of Leishmania infection. In the present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed the immune response associated with the infection by these parasites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-_ and have smaller parasite load, after 8 weeks of infection. Furthermore, after 1 week of infection, there is increased expression of the chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infection of peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role in infection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.