RESUMO
Proteins stored in insect hemolymph may serve as a source of amino acids and energy for metabolism and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCR and Western blot. After ensuring that the immune system had been activated by measuring the ensuing expression of the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (proPO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLp-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLp-III transcripts. Our findings are consistent with a down-regulation of the expression and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon represents a strategy to redirect resources to combat injury or infection.
Assuntos
Infecções Bacterianas/imunologia , Abelhas/genética , Hemolinfa/imunologia , Proteínas de Insetos/genética , Animais , Apolipoproteínas/genética , Apolipoproteínas/imunologia , Apolipoproteínas/metabolismo , Infecções Bacterianas/metabolismo , Abelhas/imunologia , Abelhas/metabolismo , Catecol Oxidase/genética , Catecol Oxidase/imunologia , Catecol Oxidase/metabolismo , Defensinas/imunologia , Defensinas/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/imunologia , Precursores Enzimáticos/metabolismo , Feminino , Regulação da Expressão Gênica , Hemolinfa/metabolismo , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , RNA/análise , RNA Mensageiro/análise , Especificidade da Espécie , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Vitelogeninas/genética , Vitelogeninas/imunologia , Vitelogeninas/metabolismoRESUMO
We produced, selected and cloned hybridomas that secrete monoclonal antibodies against human apolipoprotein (apo) A-I. All of the antibodies corresponded to the IgG(1) subclass and were named 1C11, 2B4, 2C10, 7C5, 8A4 and 8A5. The antibodies were characterized by their reactivity with whole lipoproteins, apolipoproteins, synthetic peptides and fragments generated by cleavage of the apo A-I. Three of the monoclonal antibodies studied (2B4, 2C10 and 7C5) were similarly inhibited by an amino-terminal peptide (amino acid sequence 1-20) of apo A-I, whereas antibodies 1C11, 8A4 and 8A5 had no reaction. Other results show that monoclonal antibody 1C11 recognizes an epitope located between amino acids 135-148. We evaluated the monoclonal antibody 8A4 against different HDL subpopulations by competitive displacement analysis and it showed a similar reactivity with the HDL particles: LpA-I and LpA-I:A-II. This antibody was used to standardize a sandwich ELISA to quantitate LpA-I in plasma. We conclude that these monoclonal antibodies are relevant for the study of apo A-I epitope expression and for quantitating apo A-I containing lipoparticles.
Assuntos
Anticorpos Monoclonais , Apolipoproteínas/análise , Apolipoproteínas/imunologia , Lipoproteínas HDL/análise , Animais , Ligação Competitiva , Western Blotting , Células Cultivadas , Brometo de Cianogênio , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Indicadores e Reagentes , Lipoproteína(a)/análise , Lipossomos/química , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Baço/citologiaRESUMO
The lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are clinically relevant because of their association with thrombosis and pregnancy loss. The group of antiphospholipid antibodies (aPL) includes antibodies primarily directed against various phospholipid-binding proteins, mainly beta2-glycoprotein I (beta2GPI) and prothrombin. Some studies suggest that there is an association between the presence of anti beta2GPI antibodies (alphabeta2GPI) of IgG isotype and thrombosis. Therefore, aPL defined according to the plasma protein to which they are directed appear to be more appropriate for the evaluation of their clinical importance. Using home-made ELISAs we evaluated the presence of alphabeta2GPI and antiprothrombin antibodies (anti-II) of both isotypes (IgG and IgM) in a group of 233 patients with LA and/or aCL. Forty-four women had a history of pregnancy loss, 45 patients had a history of venous thrombosis (VT) and 32 of arterial thrombosis (AT). Patients from the autoimmune group (systemic lupus erythematosus and antiphospholipid syndrome) had a higher prevalence of alphabeta2GPI and/or anti-II than those from the miscellaneous group. In the univariate analysis, a significant association was shown between the presence of alphabeta2GPI-IgG (OR 3.2; 95% CI 1.5-6.6) and previous VT, but not AT. Anti-II were related to VT but the multivariate analysis showed that alphabeta2GPI-IgG are the only independent risk factor for VT (OR 3.0; 95% CI 1.3-6.2). The presence of alphabeta2GPI-IgM correlates well with a history of pregnancy loss (OR 2.6; 95% CI 1.1-6.1). The coagulation tests profile showed that the clotting assays were more prolonged in patients having aCL, alphabeta2GPI or anti-II. But a higher prevalence of abnormal results was only found for the dilute Russell viper venom time in patients with VT, as compared to those without thrombosis (94.4% vs. 58.7%, p <0.02). The measurement of alphabeta2GPI of both isotypes could help to identify aPL-positive patients with a higher risk for thrombosis and pregnancy loss, although this association should be confirmed by prospective studies.