Navegando por Autor "Genaro, Odair"
Agora exibindo 1 - 15 de 15
Resultados por página
Opções de Ordenação
Item Comparison of polymerase chain reaction with other laboratory methods for the diagnosis of American cutaneous leishmaniasis Diagnosis of cutaneous leishmaniasis by polymerase chain reaction.(2006) Marques, Marcos José; Volpini, Ângela Cristina; Coelho, George Luiz Lins Machado; Pinto, Jackson Machado; Costa, Carlos Alberto da; Mayrink, Wilson; Genaro, Odair; Romanha, Alvaro JoséAn evaluation of 5 laboratory methods for diagnosing American cutaneous leishmaniasis (ACL) was carried out on patients from an endemic area of Brazil. From 164 patients presenting cutaneous lesions, and suspected to have ACL, 133 (81.1%) were confirmed for the disease by Montenegro skin test (MST) and/or parasitologic examination (PE). In both groups of patients, the positivity of polymerase chain reaction (PCR) was similar to that of immunofluorescence assay and enzyme-linked immunosorbent assay, and higher than that of MST and PE ( P b .05). In the group of patients suspected to have ACL, PCR presented the same positivity as PE and MST together. No correlation between positivity of the laboratory methods and clinical or epidemiologic aspects was observed. Our data confirmed the value of PCR as an alternative laboratory method for diagnosing ACL, especially for those patients with negative PE and MST.Item Effective immunotherapy against canine visceral leishmaniasis with the FML-vaccine.(2004) Borja Cabrera, Gulnara Patricia; Mendes, Amanda Cruz; Souza, Edilma Paraguai de; Okada, Lilian Y. Hashimoto; Trivellato, Fernando Antonio de Assis; Kawasaki, Jarbas Kiyoshi Alves; Costa, Andreia Cerqueira; Reis, Alexandre Barbosa; Genaro, Odair; Batista, Leopoldina Maria Melo; Palatnik, Marcos; Souza, Clarisa Beatriz Palatnik deThe potential effect of the fucose mannose ligand (FML)-vaccine on immunotherapy of canine visceral leishmaniasis was assayed on five mongrel dogs experimentally infected withLeishmania donovani and on 21 Leishmania chagasi naturally infected dogs when seropositive to FML but completely asymptomatic. The clinical signs of the experimentally infected, symptomatic dogs only disappeared after the complete vaccination. Protection was obtained in 3/5 animals that remained asymptomatic, IDR positive and parasite free, 1 year after infection. Furthermore, the asymptomatic, FML-vaccine treated dogs showed stable anti-FML IgG1 levels, increasing IgG2 levels and 79–95% of positive DTH response, during the whole experiment. Twenty-two months after complete vaccination, no obits due to visceral leishmaniasis were recorded and 90% of these dogs were still asymptomatic, healthy and parasite free. On the other hand, 37% (17/46 dogs) kala-azar obits were recorded in a control group that received no treatment during the same period, and that was FML-seropositive and asymtpomatic at the beginning of the assay. Our results indicate that the FML-vaccine was effective in the immunotherapy against visceral leishmaniasis of asymptomatic infected dogs. Normal proportions of CD4 and CD21 lymphocytes were detected in PBMC by FACS analysis, in dogs submitted to immunotherapy, suggesting their non-infectious condition. All animals showed as well significantly increased percents of CD8 lymphocytes as expected for Quillajasaponin (QuilA) vaccine treatments.Item Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes.(2005) Reis, Alexandre Barbosa; Carneiro, Cláudia Martins; Carvalho, Maria das Graças; Carvalho, Andréa Teixeira de; Giunchetti, Rodolfo Cordeiro; Mayrink, Wilson; Genaro, Odair; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo AssisThe effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1 + /CD5 + /CD4 + /CD8 + /CD21 + and CD14 + ) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5 + -T-cells within the lymphocyte-gate and increased percentage of CD21 + -B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1 + , CD4 + , CD8 + and CD21 + lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/ CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process.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 Immunochemotherapy in american cutaneous leishmaniasis : immunological aspects before and after treatment.(2001) Toledo, Vicente de Paulo Coelho Peixoto de; Mayrink, Wilson; Gollob, Kenneth John; Oliveira, Milton Adriano Pelli de; Costa, Carlos Alberto da; Genaro, Odair; Pinto, J. A.; Afonso, Luís Carlos CroccoIn this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.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 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 Leishmania major-like antigen for specific and sensitive serodiagnosis of human and canine visceral leishmaniasis.(2002) Deus, Rosângela Barbosa de; Guia, Marcos Luíz dos Mares; Nunes, Adriane Zacarias; Costa, Kátia Morais; Junqueira, Roberto Gonçalves; Mayrink, Wilson; Genaro, Odair; Tavares, Carlos Alberto PereiraAn antigen (LMS) prepared from Leishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1,822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas’ disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas’ disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested.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 Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis.(2006) Reis, Alexandre Barbosa; Martins Filho, Olindo Assis; Carvalho, Andréa Teixeira de; Carvalho, Maria das Graças; Mayrink, Wilson; Silva, João Carlos França da; Giunchetti, Rodolfo Cordeiro; Genaro, Odair; Oliveira, Rodrigo Corrêa deWe have performed a detailed investigation in 40 dogs naturally infected withLeishmania infantum ( syn. chagasi), subdivided into three groups: asymptomatic (AD = 12), oligosymptomatic (OD = 12) and symptomatic (SD = 16), based on their clinical features. Twenty non-infected dogs (CD) were included as control group. Serological analysis, performed by IFAT and ELISA, demonstrated higher antibodies titers in SD in comparison to the AD. A positive correlation was found between parasite density in the spleen and skin smears as well as the bone marrow parasitism with clinical status of the infection. We observed that the progression of the disease from asymptomatic to symptomatic clinical form was accompanied by intense parasitism in the bone marrow. It is likely that this led to the impaired biochemical/hematological status observed. Finally, we believe that the follow-up of these parameters could be a relevant approach to be used as markers during therapeutic and vaccine evaluations.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 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 Risk factors for mucosal manifestation of American cutaneous leishmaniasis.(2003) Coelho, George Luiz Lins Machado; Caiaffa, Waleska Teixeira; Genaro, Odair; Magalhães, Paulo Araújo; Mayrink, WilsonA case-comparison study was carried out to identify risk factors for mucosal manifestations of American cutaneous leishmaniasis (ACL) in southeast Brazil, using a series of 2820 patients, diagnosed with ACL between 1966 and 1999. The significant factors independently associated with mucosal leishmaniasis were: gender, age, nutritional status and length of disease. Mucosal leishmaniasis occurred 1.7 times more frequently among males than females; twice as often in individuals older than 22 years compared with the younger group; almost four times as often in individuals with severe malnutrition compared with those who were well nourished; and almost four times more frequently in individuals reporting the disease for more than 4 months compared with those reporting a shorter duration of the disease. Among individuals older than 22 years the risk of mucosal leishmaniasis increased significantly (from 1.9 to 9.6) as the nutritional status decreased, when compared with younger and well-nourished patients. The characteristics herein described and correlated with severe forms could be used as diagnostic markers as part of clinical screening in areas endemic for ACL.Item Vaccination of C57BL/10 mice against cutaneous leishmaniasis using killed promastigotes of different strains and species of Leishmania.(2002) Mayrink, Wilson; Santos, Gilmara Cristina dos; Toledo, Vicente de Paulo Coelho Peixoto de; Guimarães, Tânia Mara Pinto Dabés; Coelho, George Luiz Lins Machado; Genaro, Odair; Costa, Carlos Alberto daAntigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacinand PH8 monovalent vaccine yielded the highest IFN-levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection.Item Vaccine for prophylaxis and immunotherapy, Brazil.(1996) Genaro, Odair; Toledo, Vicente de Paulo Coelho Peixoto de; Costa, Carlos Alberto da; Hermeto, Marco Victor; Afonso, Luís Carlos Crocco; Mayrink, Wilson