Ascorbate-dependent decrease of the mucosal immune inflammatory response to gliadin in coeliac disease patients

Background: The IL-15/NF-KappaB axis has an important role in coeliac disease (CD) and may represent a molecular target for immunomodulation. Ascorbate (vitamin C) is known to show inhibitory effects on NF-KappaB. Therefore, we studied if ascorbate supplementation to gliadin gliadin-stimulated biopsy culture could down-regulate the mucosal immune response to gliadin in CD. Methods: Duodenal biopsy explants from treated CD patients were gliadin challenged in vitro (100ìg/ml) with and without 20mM ascorbate. An extra tissue explant in basal culture was used as internal control. Secretion levels of nitrites (3h), and IFNGamma, TNFalpha, IFNalpha, IL-17, IL-13, and IL-6 (24h) were measured on the supernatants. IL-15 was assayed by western-blot on whole protein duodenal explants. Results: The addition of ascorbate to in vitro culture gliadin-challenged biopsies blocked the secretion of nitrites (p=0.013), IFNGamma (p=0.0207), TNFalpha (p=0.0099), IFNá (p=0.0375), and IL-6 (p=0.0036) compared to samples from non-ascorbate supplemented culture. Cytokine secretion was downregulated by ascorbate even to lower values than those observed in basal cultures (IFNGamma: p=0.0312; TNFalpha: p=0.0312; IFNá: p=0.0312; and IL-6: p=0.0078). Gliadin-challenge induced IL-15 production in biopsies from treated CD patients, while the addition of ascorbate to culture medium completely inhibited IL-15 production. Moreover, the inhibition of IL-15 by ascorbate took place even in the only treated CD-patient who had basal IL-15 production. Conclusions: Ascorbate decreases the mucosal inflammatory response to gluten in an intestinal biopsy culture model, so it might have a role in future supplementary therapy in CD(AU)

Differential effects and transport kinetics of ascorbate derivatives in leukemic cell lines.

In order to investigate the differential effects of ascorbate derivatives on leukemic cell growth, we examined their stabilities and transmembrane transport efficiencies. The growth of HL-60 and U937 cells was dose-dependently inhibited by ascorbic acid and sodium ascorbate, but not by dehydroascorbic acid and magnesium ascorbyl 2-phosphate up to 200 microM. The growth-suppression by ascorbic acid was dependent on its redox state, showing a complete or partial reversion by ascorbate oxidase or FeCl3 addition, respectively. Three different patterns of intracellular ascorbic acid accumulation were observed by HPLC according to the species of ascorbate derivative applied for the incubation. Compared with the reduced form of ascorbic acid, the oxidized forms (dehydroascorbic acid, ascorbic acid plus ascorbate oxidase or FeCl3) were rapidly transported into cells and readily degraded, while magnesium ascorbyl 2-phosphate, a stable derivative of ascorbic acid, slowly elevated the intracellular level of ascorbic acid, reaching a plateau at 24 hours. We also measured the differential kinetics of ascorbic acid levels In culture supernatants following the addition of ascorbate derivatives. Ascorbic acid at 40, 10, or 1 microM was observed 3 hours following treatment with 100 microM of ascorbic acid, ascorbic acid plus FeCl3, or magnesium ascorbyl 2-phosphate, respectively. No ascorbic acid was found in the culture supernatant treated with dehydroascorbic acid. This order of ascorbic acid concentrations in culture supernatant reflects their growth-inhibitory effects. Thus the growth inhibitory effect of ascorbic acid appears to be dependent on its concentration in culture medium rather than its intracellular concentration. In conclusion, the results in this study indicate that the differential effects of ascorbate derivatives appear to be due to the actual concentration differences of the reduced form of ascorbic acid in culture medium following their addition, which is determined by their stability and efficiency of cellular uptake.

[Effect of selected physico-chemical parameters on changes in ascorbate oxidase and ceruloplasmin activities]. / Wplyw wybranych parametrów fizykochemicznych na zmiany aktywnosci oksydazy askorbinianowej i ceruloplazminy.

The paper presents the results of kinetic studies covering the reactions with the participation of two enzymes belonging to the group of "blue oxidases"--ascorbate oxidase and ceruloplasmin. Using variable physico-chemical parameters of reactions such as: temperature, presence of denaturizing factors, various substrates, it was possible to draw conclusions as to the structure and mechanism of reactions catalyzed by these enzymes. The following findings were established: 1. Ceruloplasmin is characterized by the absence of quarternary structure and lesser substrate specificity as compared with ascorbate oxidase. 2. The mechanism of reactions catalyzed by ceruloplasmin and ascorbate oxidase is different, probably due to the fact that there is no typical active centre binding the substrate in ceruloplasmin. 3. The application of variable parameters of physico-chemical reactions in the kinetic studies facilitates the description of the structures of enzymes and the mechanism of reactions being catalyzed by them.