A few tightly regulated transport proteins mediate iron absorption across the intestinal epithelium. However, at the basolateral border of the intestinal cells there is only one identified transporter, ferroportin, for the transfer of intracellular iron to the vascular system. The aim was to investigate the effect of ascorbate (Vitamin C) on the regulation of ferroportin in human intestinal Caco‐2 cells. We observed increased ferroportin levels in the presence of basolateral ascorbate (150 μM) as measured by Western blot and ELISA. After 2 h of apical iron incubation, the expression of ferroportin protein was dramatically decreased. We also observed that higher ascorbate concentration (300 μM) decreased ferroportin levels. Thus, the effect of ascorbate was dose‐dependent. Interestingly, the level of iron regulatory protein 2 (IRP2) was closely following the levels of ferroportin. In conclusion, our results suggest that the functional protein level of ferroportin is regulated by ascorbate and that IRP2 is involved. In addition, the nature of the IRP2 response suggests that it could be iron‐independent. The Swedish Council for Environment, Agricultural Sciences and Spatial Planning funded this study.

Tissue transglutaminase (tTG) plays an important role in celiac disease development as it generates epitopes from gluten peptides that trigger the immune response that results in the intestinal inflammation (1). We investigated if tTG‐catalyzed modification of gliadin could be reduced in vitro by synthetic peptides selected for high affinity to gliadin.

Background Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products. Results Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 10 11 to 10 12 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro . By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin. Conclusions We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated.

Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions.

Tissue transglutaminase (tTG) plays an important role in celiac disease pathology as it catalyzes deamidation and cross-linking of specific gluten peptides and converts them into potent epitopes recognized by intestinal T-cells. We investigated whether synthetic peptides with high affinity to gliadin could alter tTG activity on gliadin and whole gluten digest. The immobilized substrates were incubated with synthetic peptides identified by the phage display technique and a control peptide with no affinity to gliadin. Transglutaminase activity was measured with time resolved fluorescence. The mean tTG activity, compared to that of the control without the peptides, was reduced by 31, 33, and 36% for three selected gliadin-binding peptides, and 30% for the peptide pool (P < or = 0.001-0.004) when gliadin was the substrate. Finally, substrate specificity experiments suggested that avenin was processed in a manner similar that used for gliadin during in vitro assays with tTG. The results showed that the blocking peptides efficiently reduced tTG processing of gliadin in vitro, and this strategy will be further investigated as an alternative therapy for celiac disease.