Dataset Information

Epithelial transport of immunogenic and toxic gliadin peptides in vitro.

ABSTRACT:

Scope

Celiac disease is an autoimmune disorder caused by failure of oral tolerance against gluten in genetically predisposed individuals. The epithelial translocation of gluten-derived gliadin peptides is an important pathogenetic step; the underlying mechanisms, however, are poorly understood. Thus, we investigated the degradation and epithelial translocation of two different gliadin peptides, the toxic P31-43 and the immunogenic P56-68. As the size, and hence, the molecular weight of peptides might have an effect on the transport efficiency we chose two peptides of the same, rather short chain length.

Methods and results

Fluorescence labeled P31-43 and P56-68 were synthesized and studied in a transwell system with human enterocytes. Fluorometric measurements were done to reveal antigen translocation and flow cytometry as well as confocal microscopy were used to investigate cellular uptake of peptides. Structural changes of these peptides were analysed by MALDI-TOF-MS. According to fluorescence intensities, significantly more P31-43 compared to P56-68 was transported through the enterocyte layer after 24 h incubation. In contrast to previous reports, however, mass spectrometric data do not only show a time-dependent cleavage of the immunogenic P56-68, but we observed for the first time the degradation of the toxic peptide P31-43 at the apical side of epithelial cells.

Conclusion

Considering the degradation of gliadin peptides by enterocytes, measurement of fluorescence signals do not completely represent translocated intact gliadin peptides. From our experiments it is obvious that even short peptides can be digested prior to the translocation across the epithelial barrier. Thus, the chain length and the sensibility to degradations of gliadin peptides as well as the integrity of the epithelial barrier seem to be critical for the uptake of gliadin peptides and the subsequent inflammatory immune response.

SUBMITTER: Zimmermann C

PROVIDER: S-EPMC4240668 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Epithelial transport of immunogenic and toxic gliadin peptides in vitro.

Zimmermann Christian C Rudloff Silvia S Lochnit Günter G Arampatzi Sevgi S Maison Wolfgang W Zimmer Klaus-Peter KP

PloS one 20141121 11

<h4>Scope</h4>Celiac disease is an autoimmune disorder caused by failure of oral tolerance against gluten in genetically predisposed individuals. The epithelial translocation of gluten-derived gliadin peptides is an important pathogenetic step; the underlying mechanisms, however, are poorly understood. Thus, we investigated the degradation and epithelial translocation of two different gliadin peptides, the toxic P31-43 and the immunogenic P56-68. As the size, and hence, the molecular weight of p ...[more]

PMID: 25415429

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Project description: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.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 1011 to 1012 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.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.

Project description:BackgroundMost methods available to predict protein epitopes are sequence based. There is a need for methods using 3D information for prediction of discontinuous epitopes and derived immunogenic peptides.ResultsPEPOP uses the 3D coordinates of a protein both to predict clusters of surface accessible segments that might correspond to epitopes and to design peptides to be used to raise antibodies that target the cognate antigen at specific sites. To verify the ability of PEPOP to identify epitopes, 13 crystallographically defined epitopes were compared with PEPOP clusters: specificity ranged from 0.75 to 1.00, sensitivity from 0.33 to 1.00, and the positive predictive value from 0.19 to 0.89. Comparison of these results with those obtained with two other prediction algorithms showed comparable specificity and slightly better sensitivity and PPV. To prove the capacity of PEPOP to predict immunogenic peptides that induce protein cross-reactive antibodies, several peptides were designed from the 3D structure of model antigens (IA-2, TPO, and IL8) and chemically synthesized. The reactivity of the resulting anti-peptides antibodies with the cognate antigens was measured. In 80% of the cases (four out of five peptides), the flanking protein sequence process (sequence-based) of PEPOP successfully proposed peptides that elicited antibodies cross-reacting with the parent proteins. Polyclonal antibodies raised against peptides designed from amino acids which are spatially close in the protein, but separated in the sequence, could also be obtained, although they were much less reactive. The capacity of PEPOP to design immunogenic peptides that induce antibodies suitable for a sandwich capture assay was also demonstrated.ConclusionPEPOP has the potential to guide experimentalists that want to localize an epitope or design immunogenic peptides for raising antibodies which target proteins at specific sites. More successful predictions of immunogenic peptides were obtained when a peptide was continuous as compared with peptides corresponding to discontinuous epitopes. PEPOP is available for use at http://diagtools.sysdiag.cnrs.fr/PEPOP/.

Project description:PURPOSE: Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays. METHODS: The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400-425-500 mOsM), in low concentrations of BAK (10(-4)%, 3.10(-4)%, and 5.10(-4)%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells. RESULTS: As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from mitochondria to the nucleus in both conditions. CONCLUSIONS: This study showed increased cytotoxic effects of BAK in hyperosmotic conditions, with characteristic cell death processes, namely caspase-dependent and independent apoptosis and oxidative stress. As BAK is known to disrupt tear film, which could promote evaporative dry eye and tear hyperosmolarity, BAK could promote the conditions enhancing its own cytotoxicity. This in vitro hyperosmolarity model thus highlights the risk of inducing a vicious cycle and the importance of avoiding BAK in patients with dry eye conditions.

Dataset Information

Epithelial transport of immunogenic and toxic gliadin peptides in vitro.

Scope

Methods and results

Conclusion

Publications

Epithelial transport of immunogenic and toxic gliadin peptides in vitro.

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