IL-5 and IL-17A are critical for the chronic IgE response and differentiation of long-lived antibody-secreting cells in inflamed tissues.

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220(neg)CD43(high) phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220(high) and B220(low)), whereas the largest pool of ASC into in the BM was constituted by the B220(high)CD43(low) phenotype; and finally, terminally differentiated cells (B220(neg)CD43(high)) were only maintained in the inflamed peritoneal cavity in late phase. After 120d a sustained production of cytokines (KC, IL-5, TNF-α, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220(neg) in the peritoneal cavity and negatively regulate the number of ASC B220(pos), confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A.

Staurosporine induces tyrosine phosphorylation in Dictyostelium discoideum proteins.

The treatment of cells with staurosporine results in inhibition and less frequently activation of protein kinases, in a cell-type specific manner. In the social amoeba Dictyostelium discoideum, staurosporine induces marked changes in cell morphology affecting growth and development. Here we describe that incubation of D. discoideum growing or starved cells with staurosporine results in a rapid and unexpected tyrosine phosphorylation on two polypeptides of approximately 64 and approximately 62 kDa. These proteins emerge as novel substrates for tyrosine phosphorylation opening up new perspectives for the study of cell signalling in D. discoideum.

Cholesterol sulphate-reactive autoantibodies are specifically increased in chronic chagasic human patients.

An antibody reactive with cholesterol sulphate (CS) was characterized in human sera by ELISA, erythrocyte and liposome absorption. This antibody was found evenly distributed between the IgA and IgM classes, and whilst this was present at low titres in the serum of 16% of healthy individuals studied, it was significantly elevated in 78% of Trypanosoma cruzi-infected subjects. No association was found between antibody levels and the degree of myocardial damage. No significant difference in immunoreactivity was found between healthy and chagasic subjects using dehydro-epiandrosterone sulphate and pregnenolone sulphate and cholesterol, ergosterol, lanosterol, stigmastanol, beta-stigmasterol, pregnenolone, prednisolone and dehydroepiandrosterone as antigens, suggesting that in chagasic sera the whole sterol molecule is important for optimal antibody binding. CS-reactive antibodies were easily purified by absorption either with CS-bearing liposomes or with dextran sulphate gel and further elution with 1.5 M NaCl. The optimal pH of CS-antibody interaction was 4.0 with 85% binding at pH 7.0. Polylysine strongly decreased the binding of these antibodies to the corresponding antigen. Furthermore, these antibodies were strongly absorbed by rabbit and guinea pig erythrocyte but not by rat or human erythrocyte. In contrast with anti-sulphatide antibodies, no significant increase in CS-reactive antibodies was found in dilated cardiomyopathies. Whilst CS itself was not detected in T. cruzi lipid extracts, there is an unidentified sulphated sterol, which migrates close to standard CS and which strongly binds chagasic but not control sera. This latter sterol might be acting in chagasic patients as a powerful antigen, triggering specific autoantibody production.

Increasing the efficiency of lipid-conjugated antibodies incorporation into the membrane of antigen presenting B cells.

We have introduced some modifications in the technique called "cell decoration" in order to increase the amount of lipid-conjugated antibodies which can be incorporated into the membrane of B cells. As shown by FACS analysis, we have obtained an approximately 4-fold increment in the amount of specific antibodies incorporated into the cell membrane. The procedure, which consists of successive changes of the medium that contains the lipid-conjugated antibodies, avoided changes on parameters that interfere with cell viability. The proposed modification resulted in an approximately 2-fold enhancement of the ability of decorated B cells to act as antigen presenting cells for specific T hybridomas.