RESUMO
Ligaria cuneifolia has been used in Argentine folk medicine and is currently employed as substitute for the European mistletoe (Viscum album) as hypotensor agent. Extracts from V. album are widely used in cancer therapy and the antineoplasic effect is attributed to their cytostatic/cytotoxic and immunomodulatory actions. When studying immunomodulatory effects of L. cuneifolia extracts (Lc extracts), they inhibited proliferation of murine mitogen-activated lymphocytes, leukaemic lymphocytes (LB) and breast tumour cells (MMT). The aim of this work was to isolate and identify lectins from Lc extracts and investigate their immunobiological actions. A galactoside lectin (L-Lc) of 57 kDa was isolated. A polyclonal antiserum obtained against Lc extract recognised both L-Lc and MLI (V. album lectin), suggesting the possibility of shared epitopes. Treatment of LB tumour cells with L-Lc (0.01 and 0.1 microg/ml) produced up to 40.0+/-6.9% inhibition of cell growth, which seems partly mediated by apoptosis (apoptosis of L-Lc treated cells 58.4+/-10.3% versus non-treated cells 38.1+/-8.8%; P<0.05), analysed by acridine orange and ethidium bromide staining. Inhibitory effect on ConA stimulated splenocyte growth was non-significant, while a mitogenic effect was observed on normal murine splenocytes and MMT cells. L-Lc in non-cytotoxic concentrations (250 ng/ml) modified mRNA expression of IL-10 but neither that of TGF-beta nor of IL-2 produced by LB cells. In addition, 43.9+/-0.5% reduction in NO production by LPS-stimulated murine macrophages was found. Finally, survival rates of LB tumour-bearing mice treated or not with Lc extract or L-Lc failed to show significant differences.
Assuntos
Adjuvantes Imunológicos/farmacologia , Galactosídeos/farmacologia , Loranthaceae , Lectinas de Plantas/farmacologia , Adjuvantes Imunológicos/isolamento & purificação , Animais , Apoptose , Argentina , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Galactosídeos/imunologia , Galactosídeos/isolamento & purificação , Técnicas In Vitro , Loranthaceae/química , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias Experimentais/mortalidade , Óxido Nítrico/biossíntese , Extratos Vegetais/farmacologia , Lectinas de Plantas/imunologia , Lectinas de Plantas/isolamento & purificação , RNA Mensageiro/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
This report describes differences in humoral immune response of acute and chronic phases of human Chagas disease. The reactivities of IgG, IgM, and IgA anti-Trypanosoma cruzi antibodies in serum samples from both groups of patients were compared by enzyme-linked immunosorbent assay (ELISA) employing either one of four antigenic fractions: mouse laminin (LAM), which reacts through Gal alpha 1-3Gal epitopes expressed on trypomastigote surface: whole intact trypomastigotes (TCT); trypomastigotes excreted/secreted antigens (TESA); and epimastigote alkaline extract (EAE). The selection of T. cruzi antigen preparations was based on their relative content of surface and internal antigens found in trypomastigote forms. The proportion of IgG reactive to carbohydrate epitopes was assessed through the decay of IgG reactivity from acute and chronic sera after m-periodate oxidation of solid-phase bound antigens. Trypomastigote and TESA antigens recognized by IgG from acute and chronic sera were also compared by immunoblotting. ELISA and immunoblotting data showed that: (1) the proportion of IgG directed to trypomastigote surface antigens was higher in acute than in chronic sera, whereas the opposite was found for internal antigens, (2) acute sera contained a higher percentage of IgG reactive to trypomastigote carbohydrate epitopes than chronic sera, and (3) anti-T. cruzi IgA was found exclusively in acute sera and led to 100% positivity when LAM, TCT, and TESA were employed as antigens. IgA ELISA with these antigens and IgG immunoblotting pattern with TESA could be useful as serological markers for the acute phase of human Chagas disease.
Assuntos
Anticorpos/imunologia , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos/sangue , Anticorpos/metabolismo , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Brasil , Doença de Chagas/classificação , Dissacarídeos/imunologia , Dissacarídeos/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Galactosídeos/imunologia , Galactosídeos/metabolismo , Humanos , Immunoblotting , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/imunologia , Laminina/química , Laminina/imunologia , Laminina/metabolismo , Camundongos , Ácido Periódico/farmacologia , Trypanosoma cruzi/químicaRESUMO
Antibodies that lyse trypomastigotes in a complement-mediated reaction are believed to be the main participants in the protection against virulent Trypanosoma cruzi. Antibodies with a specificity for alpha-galactosyl-containing determinants--generally called antiGal--were studied to determine their role in the lysis of trypomastigote forms. The titers of antiGal markedly increase in Chagas's disease. In the present study we demonstrate binding of this antibody to T. cruzi and the complement-mediated lysis of trypomastigotes by antiGal. Lysis of metacyclic trypomastigotes by whole Chagasic (Ch) serum or isolated antiGal fractions was equally inhibited by alpha- but not by beta-galactosides. Most of the lytic power of the Ch antiGal as well as of the whole Ch serum was removed by absorption on Synsorb-linked Gal alpha 1, 3Gal beta 1, 4GlcNAc followed by rabbit erythrocyte absorption. The Ch antiGal had a lower affinity for melibiose bound to agarose than for the trisaccharide linked to Synsorb, and was several times more effective in the immunolysis of trypomastigotes than the corresponding antiGal from normal human serum. Lytic antibodies were partly absorbed by Serratia marcescens but not by Escherichia coli O111. A human volunteer immunized with an S. marcescens vaccine elicited a specific antiGal response that was lytic to trypomastigotes (70% lysis). We suggest that in vivo high-affinity antiGal antibody clones, as occur in Ch patients, may significantly contribute to the destruction of the parasite, whereas low-affinity antiGal clones are much less effective in the protection against T. cruzi infection.