Caracterização molecular de micobactérias isoladas de hansenomas /

Orientadora : Vanete Thomaz SoccolCo-orientador : Marcelo Tavora MiraDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba, 2006Inclui bibliografiaÁrea de concentração: Saude humana e anima

Synthetic Peptides as Potential Antigens for Cutaneous Leishmaniosis Diagnosis

This work's goal was to research new candidate antigens for cutaneous leishmaniosis (CL). In order to reach the goal, we used random peptide phage display libraries screened using antibodies from Leishmania braziliensis patients. After selection, three peptides (P1, P2, and P3) were synthesized using Fmoc chemistry. The peptides individually or a mixture of them (MIX) was subsequently emulsified in complete and incomplete Freund's adjuvant and injected subcutaneously in golden hamsters. Sera from the hamsters administered with P1 presented antibodies that recognized proteins between 76 and 150 kDa from L. braziliensis. Sera from hamsters which had peptides P2 and P3, as well as the MIX, administered presented antibodies that recognized proteins between 52 and 76 kDa of L. braziliensis. The research on the similarity of the peptides' sequences in protein databases showed that they match a 63 kDa glycoprotein. The three peptides and the MIX were recognized by the sera from CL patients by immunoassay approach (ELISA). The peptides' MIX showed the best performance (79% sensitivity) followed by the P1 (72% sensitivity), and the AS presented 91% sensitivity. These results show a new route for discovering molecules for diagnosis or for immunoprotection against leishmaniosis

Uso de peptideos para diagnóstico imunológico de hanseníase

Orientadora : Profª Drª Vanete Thomaz SoccolCo_orientadora : Profª. Drª. Juliana Ferreira de MouraTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba, 31/05/2011Bibliografia: f. 110-130Área de concentração: Saúde humana e animalResumo: A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae que acomete principalmente a pele e os nervos periféricos. Apesar da redução da prevalência no mundo, o número de novos casos tem se mantido constante nos últimos anos. Uma das dificuldades encontradas no controle da doença reside na falta de teste de diagnóstico específico de detecção de infecção por M. leprae. O objetivo desse estudo foi identificar antígenos para fins de diagnóstico. Nesse sentido, peptídeos ligantes de anticorpos de pacientes hansenianos foram selecionados de bibliotecas de peptídeos apresentados em fagos. Dentre sete clones de fagos selecionados, três clones se mostraram promissores e suas sequências peptídicas foram sintetizadas quimicamente. O teste ELISA usando pool dos três peptídeos permitiu identificar 52,2% (12/23) dos pacientes hansenianos multibacilares. Cinco de um total de trinta pacientes (16,7%) com tuberculose apresentaram teste positivo frente aos peptídeos. Nenhuma das amostras de pacientes hansenianos paucibacilares, bem como de contatos de pacientes hansenianos e sadios foi positiva pelo ensaio. Os peptídeos não foram reativos frente aos antisoros de diferentes micobactérias produzidos em coelhos. Peptídeos conjugados a proteína carreadora albumina sérica bovina (BSA) induziram a produção de anticorpos em animais. Os anticorpos antipeptídeos foram capazes de reconhecer antígenos de aproximadamente 30 kDa em M. leprae por Western Blotting. Todos os soros antipeptídeos produziram o mesmo perfil de reatividade por análise de Western Blotting a partir de um gel bidimensional de antígenos de M. leprae, indicando que os peptídeos são derivados de uma mesma proteína do bacilo. Dados experimentais associados aos obtidos por bioinformática indicam que a proteína se trata do antígeno 85B. Dois dos três peptídeos mostraram evidências em estimular células T através de teste de reação de hipersensibilidade do tipo tardio. Os resultados indicaram que os peptídeos são reagentes potenciais em ensaios imunológicos, tanto sorológicos, quanto celulares, para detecção da doença. O uso da metodologia descrita aqui permitirá a identificação de outros peptídeos, que combinados aos propostos neste trabalho, possibilitarão aumentar a sensibilidade do diagnóstico de pacientes hansenianos.Abstract: Leprosy is a chronic infectious disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nerves. Despite reductions in prevalence worldwide, the number of new cases has remained constant in recent years. One of the difficulties in controlling the disease is the lack of specific diagnostic test for detecting infection by M. leprae. The aim of this study was to identify antigens for diagnostic purposes. For these purposes, antibody binding peptides of leprosy patients were selected from phage displayed peptide libraries. Among seven clones of selected phages, three clones were promising and their peptides sequences were chemically synthesized. The ELISA test using three peptides pool permitted the identification of 52.2% (12/23) of multibacillary leprosy patients. Five out of thirty patients (16.7%) with tuberculosis had a positive test with the peptides. None of the samples coming from the paucibacillary leprosy patients, contacts of leprosy patients and healthy individuals for the test were positive. The peptides were not reactive to antisera against different mycobacteria produced in rabbits. The peptides conjugated to the carrier protein bovine serum albumin (BSA) induced the production of antibodies in animals. Antipeptides antibodies were able to recognize antigens of approximately 30 kDa in M. leprae by Western blotting. All sera antipeptides produced the same profile of reactivity by Western blot analysis from a two-dimensional gel of M. leprae antigens, indicating that the peptides are derived from the same bacillus protein. Experimental data associated with those obtained by bioinformatics indicated that the protein is the antigen 85B. Two of the three peptides showed evidence of stimulating T cells through delayed-type hypersensitivity reaction. The results indicated that the peptides are potential reagents in immunological assays, both serologic and cellular, to detect the disease. Using the methodology described here will allow the identification of other peptides, that when matched those proposed in this research, will enable the increase of the sensitivity of leprosy patients diagnosis

Uso de peptideos para diagnóstico imunológico de hanseníase

Orientadora : Profª Drª Vanete Thomaz SoccolCo_orientadora : Profª. Drª. Juliana Ferreira de MouraTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba, 31/05/2011Bibliografia: f. 110-130Área de concentração: Saúde humana e animalResumo: A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae que acomete principalmente a pele e os nervos periféricos. Apesar da redução da prevalência no mundo, o número de novos casos tem se mantido constante nos últimos anos. Uma das dificuldades encontradas no controle da doença reside na falta de teste de diagnóstico específico de detecção de infecção por M. leprae. O objetivo desse estudo foi identificar antígenos para fins de diagnóstico. Nesse sentido, peptídeos ligantes de anticorpos de pacientes hansenianos foram selecionados de bibliotecas de peptídeos apresentados em fagos. Dentre sete clones de fagos selecionados, três clones se mostraram promissores e suas sequências peptídicas foram sintetizadas quimicamente. O teste ELISA usando pool dos três peptídeos permitiu identificar 52,2% (12/23) dos pacientes hansenianos multibacilares. Cinco de um total de trinta pacientes (16,7%) com tuberculose apresentaram teste positivo frente aos peptídeos. Nenhuma das amostras de pacientes hansenianos paucibacilares, bem como de contatos de pacientes hansenianos e sadios foi positiva pelo ensaio. Os peptídeos não foram reativos frente aos antisoros de diferentes micobactérias produzidos em coelhos. Peptídeos conjugados a proteína carreadora albumina sérica bovina (BSA) induziram a produção de anticorpos em animais. Os anticorpos antipeptídeos foram capazes de reconhecer antígenos de aproximadamente 30 kDa em M. leprae por Western Blotting. Todos os soros antipeptídeos produziram o mesmo perfil de reatividade por análise de Western Blotting a partir de um gel bidimensional de antígenos de M. leprae, indicando que os peptídeos são derivados de uma mesma proteína do bacilo. Dados experimentais associados aos obtidos por bioinformática indicam que a proteína se trata do antígeno 85B. Dois dos três peptídeos mostraram evidências em estimular células T através de teste de reação de hipersensibilidade do tipo tardio. Os resultados indicaram que os peptídeos são reagentes potenciais em ensaios imunológicos, tanto sorológicos, quanto celulares, para detecção da doença. O uso da metodologia descrita aqui permitirá a identificação de outros peptídeos, que combinados aos propostos neste trabalho, possibilitarão aumentar a sensibilidade do diagnóstico de pacientes hansenianos.Abstract: Leprosy is a chronic infectious disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nerves. Despite reductions in prevalence worldwide, the number of new cases has remained constant in recent years. One of the difficulties in controlling the disease is the lack of specific diagnostic test for detecting infection by M. leprae. The aim of this study was to identify antigens for diagnostic purposes. For these purposes, antibody binding peptides of leprosy patients were selected from phage displayed peptide libraries. Among seven clones of selected phages, three clones were promising and their peptides sequences were chemically synthesized. The ELISA test using three peptides pool permitted the identification of 52.2% (12/23) of multibacillary leprosy patients. Five out of thirty patients (16.7%) with tuberculosis had a positive test with the peptides. None of the samples coming from the paucibacillary leprosy patients, contacts of leprosy patients and healthy individuals for the test were positive. The peptides were not reactive to antisera against different mycobacteria produced in rabbits. The peptides conjugated to the carrier protein bovine serum albumin (BSA) induced the production of antibodies in animals. Antipeptides antibodies were able to recognize antigens of approximately 30 kDa in M. leprae by Western blotting. All sera antipeptides produced the same profile of reactivity by Western blot analysis from a two-dimensional gel of M. leprae antigens, indicating that the peptides are derived from the same bacillus protein. Experimental data associated with those obtained by bioinformatics indicated that the protein is the antigen 85B. Two of the three peptides showed evidence of stimulating T cells through delayed-type hypersensitivity reaction. The results indicated that the peptides are potential reagents in immunological assays, both serologic and cellular, to detect the disease. Using the methodology described here will allow the identification of other peptides, that when matched those proposed in this research, will enable the increase of the sensitivity of leprosy patients diagnosis

Caracterização molecular de micobactérias isoladas de hansenomas /

Orientadora : Vanete Thomaz SoccolCo-orientador : Marcelo Tavora MiraDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba, 2006Inclui bibliografiaÁrea de concentração: Saude humana e anima

Synthetic Peptides as Potential Antigens for Cutaneous Leishmaniosis Diagnosis

This work’s goal was to research new candidate antigens for cutaneous leishmaniosis (CL). In order to reach the goal, we used random peptide phage display libraries screened using antibodies from Leishmania braziliensis patients. After selection, three peptides (P1, P2, and P3) were synthesized using Fmoc chemistry. The peptides individually or a mixture of them (MIX) was subsequently emulsified in complete and incomplete Freund’s adjuvant and injected subcutaneously in golden hamsters. Sera from the hamsters administered with P1 presented antibodies that recognized proteins between 76 and 150 kDa from L. braziliensis. Sera from hamsters which had peptides P2 and P3, as well as the MIX, administered presented antibodies that recognized proteins between 52 and 76 kDa of L. braziliensis. The research on the similarity of the peptides’ sequences in protein databases showed that they match a 63 kDa glycoprotein. The three peptides and the MIX were recognized by the sera from CL patients by immunoassay approach (ELISA). The peptides’ MIX showed the best performance (79% sensitivity) followed by the P1 (72% sensitivity), and the AS presented 91% sensitivity. These results show a new route for discovering molecules for diagnosis or for immunoprotection against leishmaniosis

Comparison of conventional serology and PCR methods for the routine diagnosis of Trypanosoma cruzi infection

Introduction Trypanosoma cruzi, a flagellated protozoan, is the etiologic agent of Chagas disease, and it is estimated that approximately 5 million people in Brazil are infected with this parasite. This work aimed to compare the current diagnostic methods for Chagas disease, including conventional serological (IFAT and ELISA) and molecular techniques (PCR), to introduce PCR as an auxiliary technique. Methods A total of 106 chagasic patients were evaluated: 88 from endemic areas of Parana, 6 from São Paulo, 3 from Minas Gerais, 3 from Rio Grande do Sul, 1 from Bahia and 5 from the Santa Catarina T. cruzi outbreak. The samples were analyzed by conventional serological methods (IFAT, ELISA), hemoculture and PCR to confirm Chagas disease. Results When IFAT was used to determine antibody levels, the sensitivity was 81.7% for patients with the cardiac form of the disease and 100% for the other clinical forms. In contrast, ELISA showed 84% sensitivity and 100% specificity. The use of serological and molecular techniques and their implications for the diagnosis of Chagas disease in non-endemics area are discussed. Conclusions PCR constitutes an excellent support methodology for the laboratory diagnosis of Chagas disease due to its high sensitivity and specificity

Variables associated with the prevalence of anti-Leishmania spp. antibodies in dogs on the tri-border of Foz do Iguaçu, Paraná, Brazil

Abstract The aim of this study was to investigate the occurrence of anti-Leishmania spp. antibodies in dogs from localities in the city of Foz do Iguaçu, Paraná state, Brazil, on the border with Argentina and Paraguay. Blood samples dogs were collected to perform the following serologic tests: immunochromatographic DPP® rapid test, indirect immunoenzymatic assay (ELISA) and indirect immunofluorescence assay (IFA). In 2012, 285 dogs were analyzed on Argentina border, and in 2013, serum samples from 396 dogs on the border of Paraguay were collected. Using ELISA for screening and IFA for the confirmatory test, the results showed that the antibody prevalence was 1.8% (5/285) on the border of Argentina and 3.0% (12/396) on Paraguay border. When using the DPP® for screening and ELISA as a confirmatory analysis, we observed a seroreagent prevalence in dogs of 2.5% (7/285) on Argentina border and 5.1% (20/396) on Paraguay border. The non-public collection of domestic waste (p= 0.0004) was shown to be associated with leishmaniasis. This study shows the presence of leishmaniasis and suggest the emergence of canine visceral leishmaniasis in state of Paraná due to the confirmed occurrence of seroreactive dogs on Argentina and Paraguay border, which has environmental and geographical characteristics that favor the spread of the parasite

ELISA reactivities of synthetic peptides with sera from leprosy patients and controls.

<p>Microtiter plates were coated with 1.5 µg/mL of the peptide pool (5A, 6A, and 1B) and incubated with serum that was diluted 1∶50. The detection of the reaction was performed with the anti-human IgG (Fc-specific)-biotin antibody and streptavidin-peroxidase. MB, MB leprosy patients (n = 23); PB, PB leprosy patients (n = 10); TB, TB patients (n = 30); HC, healthy household contacts of MB leprosy patients (n = 26); EC, endemic controls (n = 30). The dotted line represents the cut-off value (as determined using the ROC curve with serum samples from the EC). Each symbol represents the absorbance obtained with a single serum.</p