No abstract

High in vitro lymphoproliferative responses were induced in humans and dogs by a recombinant Leishmania (Leishmania) chagasi cysteine proteinase, with secretion of IFN-␥ in asymptomatic subjects or of IFN-␥, interleukin 4 (IL-4), and IL-10 in oligosymptomatic subjects. In contrast, responses of symptomatic patients and dogs were lower, with production of IL-4 and IL-10.Visceral leishmaniasis (VL) is caused by Leishmania (Leishmania) chagasi in South America, and dogs are the main reservoir of the disease. Among the immunological changes found with VL, those involving T cells and interleukin 10 (IL-10) production have been correlated with pathology, whereas control of the infection has been associated with the production of gamma interferon (IFN-␥) (3,4,5,8,13). Leishmania (L.) infantum cysteine proteinases have been used for the evaluation of humoral and cellular immune responses in human and canine VL (9, 12). A recombinant cysteine proteinase from L. (L.) chagasi, rLdccys1, expressed by the Ldccys1 gene (10) in Escherichia coli, was shown to be a suitable tool for the diagnosis of American VL (6). The present study demonstrates that rLdccys1 elicits cellular immune responses in naturally infected humans and dogs in different stages of VL.L. (L.) chagasi (MHOM/BR/1972/LD) amastigotes were isolated from spleens of infected hamsters, as previously described (2). The L. (L.) chagasi Ldccys1 was cloned and expressed as described elsewhere (6). The study population included human and canine blood samples obtained from subjects living in Teresina, Piauí State, Brazil, an area where VL is endemic, and were classified as shown in Table 1. The human and dog procedures were approved by the Ethical Committee for Human and Animal Care at the Universidade Federal de São Paulo, Escola Paulista de Medicina. The lymphoproliferative assays were performed with peripheral blood mononuclear cells (PBMC) purified by Ficoll-Hypaque density gradient centrifugation. For in vitro proliferation assays, the cells were cultured into 96-wells plates (1 ϫ 10 6 cells/ml) in RPMI 1640 medium with 10% pooled human serum (R10) in the presence of rLdccys1 (2.5 g/ml) or L. (L.) chagasi amastigote extracts (AM) (equivalent to 1 ϫ 10 7 amastigotes). After 96 h at 37°C in 5% CO 2 , the cells were pulsed with 1 Ci of [ 3 H]thymidine/ well for 18 h, and [ 3 H]thymidine uptake was determined after filtration on glass fiber filters. Lymphoproliferative responses were expressed as stimulation indexes (SI) determined by dividing the mean counts per minute for antigen-stimulated cells, in triplicate, by the mean counts for the control medium. Student's t test was used with SigmaStat software to evaluate the significance of the data (P Ͻ 0.05). After stimulation with rLdccys1, PBMC from asymptomatic, oligosymptomatic, symptomatic, and treated patients proliferated with mean stimulation indexes of 8.0, 6.25, 2.45, and 4.1, respectively, whereas healthy individuals showed basal levels of lymphoproliferation. Lower stimulation was obtained for PBMC from the sa...

A recombinant protein, rLdccys1, which was produced by expression of the gene encoding a 30 kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used for detection of antibodies in sera from patients with active visceral leishmaniasis (VL) in enzyme-linked immunosorbent assays. Analysis of the predicted amino acid sequence of rLdccys1 showed that it contains all the characteristics of a cysteine proteinase. The ability of the protein to react with sera from humans with VL was also shown by Western blotting. The sensitivity for detection of specific antibodies to L. (L.) chagasi bodies using rLdccys1, L. (L.) chagasi promastigote lysates, and amastigote lysates was 80%, 98%, and 99%, respectively. No cross-reactivity between rLdccys1 and Chagas disease was observed, and there was little positive reactivity with sera from patients with cutaneous leishmaniasis and tuberculosis, compared with promastigote and amastigote extracts. Our findings indicate that rLdccys1 from L. (L.) chagasi constitutes a potential tool for the diagnosis of American VL.

No abstract

Direitos para esta edição cedidos à Atena Editora pelos autores. Todo o conteúdo deste livro está licenciado sob uma Licença de Atribuição Creative Commons. Atribuição-Não-Comercial-NãoDerivativos 4.0 Internacional (CC BY-NC-ND 4.0). O conteúdo dos artigos e seus dados em sua forma, correção e confiabilidade são de responsabilidade exclusiva dos autores, inclusive não representam necessariamente a posição oficial da Atena Editora. Permitido o download da obra e o compartilhamento desde que sejam atribuídos créditos aos autores, mas sem a possibilidade de alterá-la de nenhuma forma ou utilizá-la para fins comerciais. A Atena Editora não se responsabiliza por eventuais mudanças ocorridas nos endereços convencionais ou eletrônicos citados nesta obra. Todos os manuscritos foram previamente submetidos à avaliação cega pelos pares, membros do Conselho Editorial desta Editora, tendo sido aprovados para a publicação.