T cell recognition pattern of bovine milk alphaS1-casein and its peptides.

T cell recognition patterns of CAS1_Bovin, its limited hydrolysis, oxidized, reduced/alkylated, cyanogen bromide cleavage fractions and synthetic peptides were examined. Thirteen overlapping peptides covering the intact molecule, with chain lengths varied between 17 and 20 AA, were prepared by f-moc SPPS. In addition, six CNBr-cleavage fragments were obtained and extensively purified using RP/HPLC. Likewise, chemically modified derivatives and limited pepsin hydrolysate, were performed and the specificities were confirmed. Stimulation of PBMC and TCL cultures by the intact CAS1_Bovin molecule, synthetic peptides and modified derivatives were screened by [methyl-3H] thymidine incorporations. PBMC phenotype was performed by flow cytometry and the mean CD4+/CD8+ ratio of freshly prepared PBMC was compared with the ratio following specific CAS1_Bovin stimulation. CD4+ phenotypes (TH1/, TH2 and TH0) were assigned by assay of four marker cytokines IL-4, IL-6, IL-10 and IFN-gamma. Five CNBr fragments and seven of the thirteen tested peptides were recognized by specific TCL. The most reactive epitopes of CAS1_Bovin comprised seven motifs namely: peptides Cas 1-18, Cas 16-35, Cas 67-85, Cas 91-110, Cas 136-155, Cas 152-169 and Cas 166-183. The stimulation range for the seven peptides was 1058-2383 cpm. Stimulation for the CNBr fragments were, respectively, 8670, 5808, 3324, 5465, 2255 and 321 cpm. Cytokine assay showed that CD4+ TH2 phenotype was dominant for half the number of patients, while TH1 solely or combined TH0 were represented in the other four cell culture filtrates. The T cell reactive epitopes described and their antibodies will be useful tools for methods in progress for the detection of masked casein epitopes encompassed in processed food. In conclusion, T cell recognition pattern of CAS1_Bovin was examined using extensively purified synthetic peptides and CNBr fragments. Five large and seven small peptides were clearly recognized. Peptides of chain length less than six AA were left unrecognised. CD4+ TH2 phenotype was the most dominant TCL subpopulations found in atopic patients while CD4+ TH1 was representative in the non-IgE mediated type IV hypersensitivity.

Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.

Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3).