Celiac anti-type 2 transglutaminase antibodies induce differential effects in fibroblasts from celiac disease patients and from healthy subjects.

Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the ß-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Anticancer drug-loaded gliadin nanoparticles induce apoptosis in breast cancer cells.

Nanoscale drug carriers play an important role in regulating the delivery, permeability, and retention of the drugs. Although various carriers have been used to encapsulate anticancer drugs, natural biomaterials are of great benefit for delivery and controlled release of drugs. We used the electrospray deposition system to synthesize gliadin and gliadin-gelatin composite nanoparticles for delivery and controlled release of an anticancer drug (e.g., cyclophosphamide). The size profile and synthesis of nanoparticles was characterized by dynamic light scattering and X-ray diffractometry. Cyclophosphamide was gradually released from the gliadin nanoparticles for 48 h. In contrast, the gliadin-gelatin composite nanoparticles released cyclophosphamide in a rapid manner. Furthermore, we demonstrated that breast cancer cells cultured with cyclophosphamide-loaded 7% gliadin nanoparticles for 24 h became apoptotic, confirmed by Western blotting analysis. Therefore, the gliadin-based nanoparticle could be a powerful tool for delivery and controlled release of anticancer drugs.

Gliadins polymerized with cysteine: effects on the physical and water barrier properties of derived films.

To study the effects of disulfide bonds on certain functional properties of films made from the wheat gluten proteins gliadin and glutenin, cysteine was used to promote the formation of interchain disulfide bridges between gliadins in 70% ethanolic solution. Disulfide-mediated polymerization of gliadins was confirmed by means of SDS-PAGE analysis. After chemical treatment of gliadins, films were solution cast and the effects of both glycerol (used as a plasticizer) and relative humidity were studied on water vapor permeability, moisture sorption isotherms at 23 degrees C, and the optical properties of the films. The results were compared with those obtained from analogous films made from untreated glutenin macromolecules. Cysteine-mediated polymerization of gliadins improved the water vapor resistance of films achieving values close to those obtained for glutenin films. Development of intra- and interchain disulfide bonds did not change the moisture sorption capacity of the films but transparency was slightly diminished.

Receptor mediated targeting of lectin conjugated gliadin nanoparticles in the treatment of Helicobacter pylori.

The present work describes the potential for using lectin-conjugated gliadin nanoparticles as a means of locating and anchoring a drug delivery system on the carbohydrate receptors of Helicobacter pylori (H. pylori). Gliadin nanoparticles (GNP) bearing acetohydroxamic acid (AHA) were prepared by a desolvation method. Ulex Europaeus Agglutinin I (UEA I) and Conconavalin A (Con A) lectins were bound to GNP formulations by the two-stage carbodiimide coupling technique. Lectin-agglutination assay was performed to evaluate the binding efficacy of lectin formulations to carbohydrate receptors of H. pylori strains. Strong agglutination patterns were observed with mannose-specific Con A-GNP and alpha(L)-fucose specific UEA-GNP formulations. In situ adherence assay was performed to examine the efficacy of lectin formulations to inhibit the binding of H. pylori strains with human stomach cells. Lectin formulations completely inhibited the H. pylori binding. In addition, the antimicrobial activity of the formulations was evaluated by percent growth inhibition studies (%GI) by using isolated H. pylori strain. The inhibitory efficacy of UEA-GNP and Con A-GNP was approximately two-fold higher compared to GNP. These lectin-conjugated gliadin nanoparticles are found to be potential candidate for targeted drug delivery and are anticipated to be useful in the treatment of H. pylori.

HLA-DQ determines the response to exogenous wheat proteins: a model of gluten sensitivity in transgenic knockout mice.

We have investigated the genetic basis of the immune response to dietary gluten in HCD4/DQ8 and HCD4/DQ6 double transgenic mice. Mice were immunized with gluten i.p. or individual peptides s.c. and spleen or draining lymph node T cells were challenged in vitro. Strong proliferative responses to gluten were seen in the HCD4/DQ8 mice, whereas the HCD4/DQ6 mice responded to gluten poorly. A series of overlapping peptides spanning gliadin were synthesized. The HCD4/DQ8 mice reacted to many of the individual peptides of gliadin, while the HCD4/DQ6 mice were relatively unresponsive. T cells isolated from HCD4/DQ8 mice also responded well to modified (deamidated) versions of the gliadin peptides, whereas HCD4DQ6 mice did not. The T cell response to gluten was CD4 dependent and DQ restricted and led to the production of cytokines IL-6, TGF-beta, and IL-10. Finally, intestinal lymphocytes isolated from gluten-fed HCD4/DQ8 mice displayed an activated phenotype. These data suggest that this HLA class II transgenic murine model of gluten sensitivity may provide insight into the initiation of the MHC class II-restricted gluten sensitivity in celiac disease.

Activation of macrophages by food antigens: enhancing effect of gluten on nitric oxide and cytokine production.

Macrophages play an important role in effector mechanisms of various chronic inflammatory diseases. We studied the effect of gluten, the agent inducing celiac disease, and other food antigens on the activation of macrophages. Nitric oxide (NO) and cytokine production were followed as markers of activation, using cultured murine peritoneal macrophages. None of the food antigens tested caused direct inducible nitric oxide synthase (iNOS) activation in macrophages. Unlike other food antigens gluten, gliadin, and their proteolytic fragments significantly enhanced NO production when applied together with interferon-gamma (IFN-gamma), the most efficient being fragments originating from 25- to 45-min peptic digestion. The activation pathway was mediated via direct stimulation of tumor necrosis factor alpha (TNF-alpha) secretion. The NO-enhancing effect was confirmed at the level of iNOS mRNA transcription. In case of sustained local inflammatory reaction connected with increase of IFN-gamma, gluten and its proteolytic fragments may thus elevate NO production. Increased NO level could consequently participate in the development of mucosal lesions in the gut of celiac patients.

In vitro activities of A-gliadin-related synthetic peptides: damaging effect on the atrophic coeliac mucosa and activation of mucosal immune response in the treated coeliac mucosa.

BACKGROUND: Gliadin amino acid sequence(s) responsible for toxicity in susceptible individuals have not been fully elucidated. Previous in vitro studies have suggested the presence of active sequences in the NH(2)-terminal part of the A-gliadin molecule. In this paper the in vitro activity of A-gliadin synthetic peptides 31-55, 31-43, and 44-55 has been investigated. METHODS: Organ culture of jejunal mucosa from untreated and treated coeliac patients was used. In the first system enterocyte height was used as a measure of peptide toxicity; in the second system evidence of activated mucosal cell-mediated immune response was sought. RESULTS: Peptides 31-55 and 31-43 were active on untreated coeliac mucosa at a concentration of 0.5 mg/ml and peptide 44-55 only at a concentration of 3 mg/ml. In in vitro-cultured treated coeliac mucosa peptides 31-55 and 31-43 at 1 mg/ml and peptide 44-55 at 3 mg/ml were able to induce enhanced epithelial expression of HLA-DR and 4F2 molecules and the appearance of CD25 positive cells. CONCLUSIONS: Our results suggest that 31-43 and 44-55 A-gliadin peptides are both active, even if to different extents. In vitro systems remain essential tools to screen material to be subsequently tested in vivo.

Further studies of the in vitro activity of synthetic gliadin peptides in coeliac disease.

Studies of in vitro activity of synthetic peptides derived from the A-gliadin structure were carried out using assays based on cultures of foetal chick intestinal mucosa and on incubation with rat liver lysosomes. The peptide corresponding to residues 11-19, displayed very high activity in the chick intestinal assay, but was only weakly active in the lysosomal assay. Peptide 9-19 was highly active in the chick intestinal assay but was only mildly active in the lysosomal assay. Peptide 8-19 was still appreciably active in both assays. The results on this group of peptides suggest the importance of residues 8-12 to activity and possibly also of a N-terminal glutamine residue. The peptide 213-227, found in a sub-fraction of fraction 9, was only weakly active in both assays, indicating that the PSQQ motif was not solely responsible for toxicity. Thus, as the peptide 208-219 was shown previously to be active in the chick intestinal assay, it is likely that the 208-212 region of this peptide is of prime importance in conferring activity. The results show, for the first time, that a nonapeptide from the N-terminal region of A-gliadin is very active in an in vitro model of toxicity in coeliac disease.

T cells recognize a peptide derived from alpha-gliadin presented by the celiac disease-associated HLA-DQ (alpha 1*0501, beta 1*0201) heterodimer.

CD is unique among the HLA-associated diseases since (a) the disease-promoting agent (gliadin) is known and (b) the disease is precipitated mainly in individuals carrying a particular cis- or trans-encoded HLA-DQ heterodimer; i.e., DQ(alpha 1*0501, beta 1*0201). Further, a preponderance of gliadin-specific T cells derived from the small intestinal mucosa of CD patients are restricted by this DQ heterodimer. T-cell recognition of gliadin peptides presented by the DQ(alpha 1*0501, beta 1*0201) heterodimer may thus be of importance in CD. Here we report that a T-cell clone from a patient with CD recognizes a synthetic alpha-gliadin peptide, when presented by the cis- or trans-encoded CD-associated DQ(alpha 1*0501, beta 1*0201) heterodimer. The minimal peptide recognized by the T-cell clone corresponds to residues 31-47 of alpha-gliadin, which is included in the part of alpha-gliadin previously shown to have disease-promoting activity. When testing analogue peptides derived from other alpha-gliadin sequences, one peptide differing by one amino acid was recognized by the T-cell clone, whereas the other peptide differing by two amino acids was not recognized. Our findings demonstrate that the CD-associated DQ(alpha 1*0501, beta 1*0201) heterodimer may serve as an antigen-presenting molecule to T cells for certain gliadin peptides.

Wheat peptide challenge in coeliac disease.

The exact nature of the cereal moiety that exacerbates coeliac disease is unknown. In-vitro studies have implicated both the N-terminal and far C-terminal domains of one of the wheat prolamins, A-gliadin. Peptides within these regions may act as epitopes that trigger immune events leading to enteropathy. We synthesized three peptides corresponding to amino-acids 3-21, 31-49, and 202-220 of A-gliadin. Four patients with coeliac disease were challenged by intraduodenal infusion of 1 g of gliadin or 200 mg of the synthetic peptides. Jejunal biopsies were taken before and at hourly intervals for 6 h after the infusion. Morphometric variables were measured and intraepithelial lymphocytes counted. Significant histological changes occurred in the small intestinal mucosa after challenge with a synthetic peptide corresponding to amino acids 31-49 of A-gliadin. The N-terminal peptide, residues 3-21 of A-gliadin, did not cause histological changes in any of the patients. In one of the four patients, minor histological changes following challenge with the peptide corresponding to residues 202-220 of A-gliadin were seen. Our results suggest that the oligopeptide corresponding to aminoacids 31-49 of A-gliadin is toxic in vivo, but there is no evidence of toxicity of the far N-terminal peptide, residues 3-21. The C-terminal peptide 202-220 may contain an epitope to which patients with coeliac disease display variable sensitivity. Since the oligopeptide corresponding to amino-acids 31-49 of A-gliadin is recognised by HLA DQ2-restricted T cells, the observed effects may be due to immune activation within the intestinal mucosa.

Continuous-flow synthesis of alpha-gliadin peptides in an ultrasonic field and assay of their inhibition of intestinal sucrase activity.

Three dodecapeptide amides derived from the amino acid sequence of alpha-gliadin and four of their analogues were synthesized by continuous-flow solid-phase multiple peptide synthesis. Ultrasonic field conditions accelerated the coupling reaction without affecting purity. Biological tests of the synthetic fragments showed the relevance of toxicity prediction for preselection of immunogenic alpha-gliadin fragments.

Cell-mediated immunity to a synthetic gliadin peptide resembling a sequence from adenovirus 12.

Cell-mediated immunity to a synthetic peptide, which has a 12-residue sequence from A-gliadin analogous to part of an early-region protein (Elb) from adenovirus 12, was investigated in patients with coeliac disease, healthy subjects, and disease controls by means of an indirect leucocyte-migration-inhibition assay. Patients with coeliac disease being treated with a gluten-free diet showed a significantly greater response than healthy subjects (p less than 0.001) or patients with inflammatory bowel disease. This cellular immune response was dependent on antigen concentration and was not present in untreated coeliac patients.