RESUMO
Binding of a fluorescent-labelled soluble immune complex to different types of Atlantic salmon leucocytes was investigated using flow cytometry. Peripheral blood leucocytes (PBL) were separated into sIg+ and sIg enriched populations by magnetic activated cell sorting, blood neutrophils were identified by electronic gating, and kidney macrophages were selected by plastic-adherence. About 60% of both sIg+ and sIg- enriched PBL, 44% of neutrophils and 34% of macrophages bound the soluble immune complexes.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Leucócitos/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Salmo salar/imunologia , Animais , Linfócitos B/imunologia , Separação Celular , Citometria de Fluxo , Imunoglobulinas/imunologia , Rim/citologia , SolubilidadeRESUMO
To facilitate site-directed chemical coupling of antigens to the heat labile enterotoxin B subunit of Escherichia coli (LTB), a series of genetically modified fusion proteins of LTB were constructed. The LTB fusion proteins had modified versions of a short (14 amino acid) spacer epitope called the Pk tag attached at their C termini. The LTB-Pk.cys mutants differed from each other in the position of a single cysteine residue within the Pk tag. The presence of a cysteine residue at any position within the Pk spacer tag did not prevent the LTB-Pk.cys proteins from forming pentamers or binding to GM1 gangliosides, but the position of the cysteine had variable impact on the yield of the fusion proteins. Following site-directed chemical coupling of antigens to the cysteine residue within the Pk tag, the LTB antigen conjugates retained their ability to bind GM1 on the surface of eukaryotic cells. Intranasal immunisation of mice with an experimental antigen (HRP) chemically linked to LTB-Pk.cys induced high levels of anti-HRP antibodies that could be detected in the serum, saliva and nasal and lung washes. No antibody responses to HRP were detected when HRP was co-administered with, but not linked to, LTB-Pk.cys.
Assuntos
Adjuvantes Imunológicos , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Enterotoxinas/química , Enterotoxinas/imunologia , Proteínas de Escherichia coli , Fragmentos de Peptídeos/imunologia , Vacinas Sintéticas , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Toxinas Bacterianas/administração & dosagem , Cisteína/imunologia , Enterotoxinas/administração & dosagem , Epitopos/imunologia , Escherichia coli , Gangliosídeo G(M1)/metabolismo , Peroxidase do Rábano Silvestre/imunologia , Imunidade nas Mucosas , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Relação Estrutura-AtividadeRESUMO
The ability of peripheral blood leucocytes (PBL) of Atlantic salmon to bind immune complexes in an antibody-dependent fashion was investigated. Immune complexes were labelled with fluorescein isothiocyanate and binding of these complexes to isolated PBL was determined by flow cytometry. The data show that a high proportion (up to 65%) of PBL were capable of binding immune complexes, and this binding did not occur when immune serum was replaced with normal serum. The presence of fresh normal serum inhibited or abrogated immune complex-binding of PBL. This is the first report of high levels of immune complex receptors on leucocytes in fish, and the dependence of complex binding on the presence of antibody suggests that these receptors may be similar to Fc receptors which are widely distributed on immunocytes of mammals.
