DEFAR - Departamento de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/530
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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 Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi.(2006) Reis, Alexandre Barbosa; Carvalho, Andréa Teixeira de; Giunchetti, Rodolfo Cordeiro; Guerra, Luanda Liboreiro; Carvalho, Maria das Graças; Mayrink, Wilson; Genaro, Odair; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo AssisCanine visceral leishmaniasis (CVL) manifests itself as a broad clinical spectrum ranging from asymptomatic infection to patent severe disease. Despite relevant findings suggesting changes on lymphocytes subsets regarding the CVL clinical forms, it still remains to be elucidated whether a distinct phenotypic profile would be correlated with degree of tissue parasite density.Herein, we have assessed the correlation between the clinical status as well as the impact of bone marrow parasite density on the phenotypic profile of peripheral blood leucocytes in 40 Brazilian dogs naturally infected by Leishmania chagasi. Our major findings describe the lower frequency of B cells and monocytes as the most important markers of severe CVL. Our main statistically significant findings reveal that the CD8+ T cell subset reflects most accurately both the clinical status and the overall bone marrow parasite density, as increased levels of CD8+ lymphocytes appeared as the major phenotypic feature of asymptomatic disease and dogs bearing a low parasite load. Moreover, enhanced major histocompatibility complex (MHC)-II density as well as a higher CD45RB/CD45RA expression index seems to represent a key element to control disease morbidity. The association between clinical status, bone marrow parasitism and CD8+ T cells re-emphasizes the role of the T cellmediated immune response in the resistance mechanisms during ongoing CVL. Higher levels of circulating T lymphocytes (both CD4+ and CD8+ T cells) and lower MHC-II expression by peripheral blood lymphocytes seem to be the key for the effective immunological response, a hallmark of asymptomatic CVL.Item Antibodies from dogs with canine visceral leishmaniasis (CVL) recognise two proteins from the saliva of lutzomyia longipalpis.(2007) Bahia, Diana; Gontijo, Nelder de Figueiredo; León, Ileana Rodríguez; Perales, Jonas; Pereira, Marcos Horácio; Oliveira, Guilherme Corrêa de; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaThe saliva of the sand fly Lutzomyia longipalpis, a major vector of Leishmania, exhibits pharmacological and immunomodulatory activities that may facilitate entry and establishment of parasites into the vertebrate host. Salivary gland components of the sand fly are, therefore, potential candidates in the development of a vaccine against human leishmaniasis. With the objective of identifying sand fly saliva proteins that could be used to immunise animals against canine visceral leishmaniasis, we have evaluated anti-saliva antibody reactivity using serum samples collected from dogs naturally infected with Leishmania chagasi. Two proteins with molecular weights of 28.6 and 47.3 kDa were recognised by dog antibodies in Western blot assays. Protein bands were excised from an SDS-PAGE gel and the sequences determined by mass spectrometry. The proteins were identified as LuLo-D7 and Lulo YELLOW, respectively. The significance of these findings in the context of the development of multicomponent vaccination experiments is discussed.Item Serological screening confirms the re-emergence of canine leishmaniasis in urban and rural areas in Governador Valadares, Vale do Rio Doce, Minas Gerais, Brazil.(2007) Malaquias, Luiz Cosme Cotta; Romualdo, Rodrigo do Carmo; Anjos Junior, José Batista do; Giunchetti, Rodolfo Cordeiro; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaThis study performed clinical, serological and parasitological assessments in dogs from Vale do Rio Doce, in Minas Gerais State, Brazil, a region considered as a ‘controlled endemic’ area for canine visceral leishmaniosis (CVL). Nevertheless, there are signs that CVL in dogs may be re-emerging as a programme to control the disease was interrupted in the 1990s. The majority of the animals examined presented various symptoms associated with CVL. The enzyme-linked immunosorbent assay test indicated 13.7 and 12.4% of positivity of dogs from the urban and rural areas, respectively. According to indirect immunofluorescence assay test and TRALd tests, 18.2 and 42.2% of dogs in the rural area were seropositive, respectively. Parasitism in seropositive dogs was confirmed by in vitro tissue culture. Sand flies of the genus Lutzomyia, which are able to transmit both cutaneous and visceral leishmaniosis, were found in the area. The results provide a strong evidence of the re-emergence of CVL in this region.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 Evaluation of the influence of tissue parasite density on hematological and phenotypic cellular parameters of circulating leukocytes and splenocytes during ongoing canine visceral leishmaniasis.(2009) Guerra, Luanda Liboreiro; Carvalho, Andréa Teixeira de; Giunchetti, Rodolfo Cordeiro; Martins Filho, Olindo Assis; Reis, Alexandre Barbosa; Oliveira, Rodrigo Corrêa deDuring Leishmania infection, tissue parasitism at different sites may differ and imply distinct immunopathological patterns during canine visceral leishmaniasis (CVL). For this reason, we have assessed by flow cytometry the impact of spleen and skin parasite density on the phenotypic profile of splenocytes and circulating leukocytes of 40 Brazilian dogs naturally infected by Leishmania chagasi categorized according to splenic and cutaneous parasite load. Our major statistically significant findings demonstrated that dogs with splenic high parasitism presented a significant decrease in absolute counts of CD5+ T lymphocytes in comparison with dogs presenting splenic medium parasitism. Moreover, a decrease in the absolute number of circulating monocytes was observed as a hallmark of high parasitism. The increased frequency of CD8+ T cells is associated with low splenic parasitism during CVL. Although we did not found any significant differences between the immunophenotypic analysis performed in circulating lymphocytes according to cutaneous parasite load, there were negative correlations between CD5+ and CD8+ T cells and cutaneous parasite density reemphasizes the role of T cell-mediated immune response in resistance mechanisms during ongoing CVL. These results add new insights about the pathogenesis of CVL and may help in the establishment of additional tools for future studies on drugs and vaccine approaches.Item Qualitative and quantitative immunohistochemical evaluation of iNOS expression in the spleen of dogs naturally infected with leishmania chagasi.(2011) Santos, Fernando Rocha dos; Vieira, Paula Melo de Abreu; Oliveira, Rodrigo Corrêa de; Giunchetti, Rodolfo Cordeiro; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa; Malaquias, Luiz Cosme CottaNitric oxide (NO), the product of the nitric oxide synthase enzymes has been detected in Leishmania-infected animals. Besides its role on the immunity to infection, the role of NO and the inducible nitric oxide synthase (iNOS) in the pathogenesis of canine visceral leishmaniasis (CVL) is not well understood. This study aimed at evaluating immunohistochemically the iNOS expression in the spleen of dogs naturally infected (ID) with Leishmania (L.) chagasi compared with non-infected dogs (NID). The ID was grouped according to the clinical form and the parasite load. Symptomatic dogs (SD) presented higher parasite load in relation to oligosymptomatic (OD) and asymptomatic (AD). The qualitative expression of iNOS was observed only in ID. SD presented strong and prominent labeling of iNOS, followed by OD and AD. Quantitatively, the results showed that the median expression of iNOS was higher in SD and OD compared to NID. Also, dog spleens with high parasitism load showed marked iNOS expression. Taken together, the results suggest that the expression of iNOS in the spleen of infected dogs with CVL was associated with clinical worsening of the disease and with high parasitism.Item An antigenic domain within a catalytically active leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.(2013) Maia, Ana Carolina Ribeiro Gomes; Porcino, Gabriane Nascimento; Detoni, Michelle de Lima; Emídio, Nayara Braga; Marconato, Danielle Gomes; Pinto, Priscila de Faria; Fessel, Melissa Regina; Reis, Alexandre Barbosa; Juliano Neto, Luiz; Juliano, Maria Aparecida; Marques, Marcos José; Vasconcelos, Eveline GomesWeidentified a shared B domainwithin nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50 kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived fromthe potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived fromthe Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83–122). By immunoprecipitation assays andWestern blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87–99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.Item Immunotherapy and immunochemotherapy in visceral leishmaniasis : promising treatments for this neglected disease.(2014) Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Vital, Wendel Coura; Ker, Henrique Gama; Moreira, Nádia das Dores; Souza, Juliana Vitoriano de; Giunchetti, Rodolfo Cordeiro; Carneiro, Cláudia Martins; Reis, Alexandre BarbosaLeishmaniasis has several clinical forms: self-healing or chronic cutaneous leishmaniasis or post-kala-azar dermal leishmaniasis; mucosal leishmaniasis; visceral leishmaniasis (VL), which is fatal if left untreated.The epidemiology and clinical features of VL vary greatly due to the interaction of multiple factors including parasite strains, vectors, host genetics, and the environment. Human immunodeficiency virus infection augments the severity of VL increasing the risk of developing active disease by 100–2320 times. An effective vaccine for humans is not yet available. Resistance to chemotherapy is a growing problem in many regions, and the costs associated with drug identification and development, make commercial production for leishmaniasis, unattractive.The toxicity of currently drugs, their long treatment course, and limited efficacy are significant concerns. For cutaneous disease, many studies have shown promising results with immunotherapy/immunochemotherapy, aimed to modulate and activate the immune response to obtain a therapeutic cure. Nowadays, the focus of many groups centers on treating canine VL by using vaccines and immunomodulators with or without chemotherapy. In human disease, the use of cytokines like interferon-g associated with pentavalent antimonials demonstrated promising results in patients that did not respond to conventional treatment. In mice, immunomodulation based on monoclonal antibodies to remove endogenous immunosuppressive cytokines (interleukin-10) or block their receptors, antigen-pulsed syngeneic dendritic cells, or biological products like Pam3Cys (TLR ligand) has already been shown as a prospective treatment of the disease. This review addresses VL treatment, particularly immunotherapy and/or immunochemotherapy as an alternative to conventional drug treatment in experimental models, canine VL, and human disease.Item An assessment on epitope prediction methods for protozoa genomes.(2012) Resende, Daniela de Melo; Rezende, Antonio Mauro; Oliveira, Nesley Jesus Daher de; Batista, Izabella Cristina Andrade; Oliveira, Rodrigo Corrêa de; Reis, Alexandre Barbosa; Ruiz, Jeronimo ConceiçãoBackground: Epitope prediction using computational methods represents one of the most promising approaches to vaccine development. Reduction of time, cost, and the availability of completely sequenced genomes are key points and highly motivating regarding the use of reverse vaccinology. Parasites of genus Leishmania are widely spread and they are the etiologic agents of leishmaniasis. Currently, there is no efficient vaccine against this pathogen and the drug treatment is highly toxic. The lack of sufficiently large datasets of experimentally validated parasites epitopes represents a serious limitation, especially for trypanomatids genomes. In this work we highlight the predictive performances of several algorithms that were evaluated through the development of a MySQL database built with the purpose of: a) evaluating individual algorithms prediction performances and their combination for CD8+ T cell epitopes, B-cell epitopes and subcellular localization by means of AUC (Area Under Curve) performance and a threshold dependent method that employs a confusion matrix; b) integrating data from experimentally validated and in silico predicted epitopes; and c) integrating the subcellular localization predictions and experimental data. NetCTL, NetMHC, BepiPred, BCPred12, and AAP12 algorithms were used for in silico epitope prediction and WoLF PSORT, Sigcleave and TargetP for in silico subcellular localization prediction against trypanosomatid genomes. Results: A database-driven epitope prediction method was developed with built-in functions that were capable of: a) removing experimental data redundancy; b) parsing algorithms predictions and storage experimental validated and predict data; and c) evaluating algorithm performances. Results show that a better performance is achieved when the combined prediction is considered. This is particularly true for B cell epitope predictors, where the combined prediction of AAP12 and BCPred12 reached an AUC value of 0.77. For T CD8+ epitope predictors, the combined prediction of NetCTL and NetMHC reached an AUC value of 0.64. Finally, regarding the subcellular localization prediction, the best performance is achieved when the combined prediction of Sigcleave, TargetP and WoLF PSORT is used. Conclusions: Our study indicates that the combination of B cells epitope predictors is the best tool for predicting epitopes on protozoan parasites proteins. Regarding subcellular localization, the best result was obtained when the three algorithms predictions were combined. The developed pipeline is available upon request to authors.