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At least 19 recordsLinked to original sources
PubMed1999

Analysis of anti-gliadin antibodies by immunoblot analysis and enzyme-linked immunosorbent assay using gliadin fractions as antigens.

BACKGROUND: Anti-gliadin antibody (AGA) determination has been widely used in the screening test to detect celiac patients in the general population and in risk groups. Serological assays present variable efficiency, probably caused by differences in the antigenic mixtures employed as antigen. The objective of this work is to evaluate the use of purified gliadin fractions in an enzyme-linked immunosorbent assay (ELISA) test. METHODS: Anti-gliadin antibody reactivity was characterized in the sera of patients with celiac disease, and AGA levels were determined by immunoblot analysis using purified gliadin fractions after separation of wheat proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and acid-PAGE and after indirect ELISA. Seven antigenic mixtures were tested: commercial gliadin, ethanolic wheat extract, and five fast protein liquid chromatography-purified fractions (omega-gliadins, two mixtures of alpha-/beta- and beta-/gamma-gliadins). Immunoblot analysis after A-PAGE separation showed that immunoglobulin (Ig)A reactivity was frequently more restricted than that of IgG. Serum IgA in 15 of 23 patients showed intense reactivity against omega-gliadins. RESULTS: In seven cases, only omega-gliadins were detected. To compare the efficiency of ELISA tests, serum samples of 28 patients with celiac disease and 31 control subjects were tested against the seven gliadin fractions. Immunoglobulin G AGAs demonstrated similar levels against the different gliadin fractions, whereas IgA AGAs showed a heterogeneous reactivity that depended on the fraction tested. The lowest number of false-positive and false-negative results was obtained when the omega-gliadin fraction was used. Parameters for ELISA showed that the omega-gliadin fraction elicited the highest assay efficiency for determinations of both IgA and IgG AGAs. A good correlation was found between IgG and IgA anti-omega-gliadin and antiendomysial antibody determinations. Of the 28 biopsy-confirmed patients with celiac disease, 26 samples (23 positive and 3 negative) were found to have concordant results among the three determinations. CONCLUSIONS: In this study, an intense and, in many cases, selective recognition of omega-gliadins was observed. Results suggest that a higher performance in AGA determination could be achieved using omega-gliadin as an antigen in indirect ELISA.

Adolescent↗
PubMed2001

Intranasal administration of one alpha gliadin can downregulate the immune response to whole gliadin in mice.

The mucosal lesion present in coeliac disease is an immune-mediated injury triggered by gliadin and restricted by a particular assortment of major histocompatibility complex genes. In view of this, an immunomodulatory approach that induces tolerance to this antigen appears to be a possible alternative to a strict gluten-free diet in treating coeliac disease. We have shown that intranasal administration of multiple doses of whole gliadin is required to specifically inhibit T helper 1-like T-cell reactivity in BALB/c mice immunized parenterally with whole gliadin. However, T-cell activation to multiple antigens, as a consequence of the chemical complexity shown by the antigen gliadin, could hamper efforts to identify single component(s) useful for tolerance induction. In this study, gliadin fractions were purified and administered intranasally to study their ability to induce tolerance to whole gliadin in our animal model. We found that the alpha fraction was particularly effective in downregulating both the in vitro gliadin-specific T-cell proliferation and interferon-gamma production to whole gliadin. In particular, a purified alpha-gliadin was able to suppress the immune response to the entire gliadin mixture. These results demonstrate how an immune response to a complex antigen may be controlled by treatment with a purified component and specifically indicate alpha-gliadin to be a good candidate for further identification of short peptides to be used as tolerogens in this model.

Administration, Intranasal↗
PubMed1981

An accurate fluorometric method to measure the breakdown of gliadin and gliadin peptides.

A simple and accurate method is described to measure the breakdown of gliadin and gliadin peptides. It involves measuring the release of the predominant amino acids glutamine and glutamic acid using a fluorometric double enzyme assay and contains none of the problems normally associated with previously used techniques. The assay is highly accurate in that small concentrations of the free amino acids can be measured with no interference from the peptide bound amino acids. The assay system was used to study the breakdown of: whole gliadin, a peptic/tryptic digest of gliadin (PT gliadin), and a peptic/tryptic/chymotryptic digest of gliadin (PTC gliadin) using a rat intestinal brush border fraction. Both PT and PTC gliadin are hydrolysed at higher rates than is whole gliadin. Exhaustive hydrolysis shows that a brush border fraction can totally break down PT gliadin while an initial linear rate of breakdown is observed (up to 60 min).

Animals↗
PubMed1979

Failure to detect gliadin or gliadin binding sites in the skin of patients with dermatitis herpetiformis: immunofluorescence, organ culture and autoradiographic studies.

Recent investigations indicate an abnormal binding of gluten or gliadin to lymphocytes or intestinal mucosa cells in gluten sensitive enteropathy. Since dermatitis herpetiformis is closely associated to gluten sensitive enteropathy, similar receptors could also exist in the skin of patients with dermatitis herpetiformis. To prove this hypothesis, skin of normal volunteers and uninvolved skin of 3 patients with dermatitis herpetiformis was investigated for the presence of gliadin and gliadin binding sites. In vivo bound gliadin was not found by direct immunofluorescence using 3 different rabbit antigliadin antisera. In order to test skin for gliadin binding sites, normal sera and autologous dermatitis herpetiformis sera containing 25 mg% gliadin and tritium labeled gliadin, respectively, were used for incubation of normal and dermatitis herpetiformis skin cryocut sections and of normal and dermatitis herpetiformis skin specimens, grown under organ culture conditions. As checked by direct immunofluorescence and autoradiography, there was no specific in vitro binding of gliadin, indicating that gliadin does not fix to normal human or dermatitis herpetiformis skin. Thus, the role of gliadin in the fixation in vivo, of antibodies or immune complexes to skin in dermatitis herpetiformis, remains obscure.

Antibodies↗
PubMed2003

Intranasal administration of a recombinant alpha-gliadin down-regulates the immune response to wheat gliadin in DQ8 transgenic mice.

The mucosal lesion in coeliac disease (CD) is an immune-mediated injury triggered by gliadin and associated with HLA-DQ2 and HLA-DQ8. In view of this, an approach that re-induces tolerance to this antigen should be considered as possible alternative to a strict gluten-free diet in treating CD. However, T-cell activation to multiple antigens, as a consequence of the chemical complexity shown by the antigen gliadin, could hamper the efforts to identify single component(s) useful for tolerance induction. To address this issue, a recombinant alpha-gliadin was tested in tolerance experiments in HLA-DQ8 transgenic mice. As tissue transglutaminase (tTG) treatment of gliadin, previously reported to enhance its stimulatory activity in CD, did not increase its immunogenicity when parenterally administered in these mice, untreated gliadin was used as immunogen. A decrease in systemic T cell responses to the recombinant alpha-gliadin was found after nasal administration of antigen, reflected by lymphocytes proliferation assay. Interestingly, while the immunisation protocol induced transcription of both Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, the tolerisation protocol down-regulated significantly only the IFN-gamma mRNA expression. More important, the recombinant alpha-gliadin induced a similar down-regulatory effect in mice immunised with a commercial preparation of wheat gliadin, that is a mixture of many different gliadin components. As the Th1 phenotype and the HLA-DQ8 molecule play a role in the pathogenesis of CD, our data underlined the potential usefulness of this recombinant protein for the immunomodulation of this disease.

Administration, Intranasal↗
PubMedDate not supplied

Food proteins and maturation of small intestinal microvillus membranes (MVM). II. Binding of gliadin hydrolysate fractions and of the gliadin peptide B3142.

To investigate in vitro interactions between gliadin peptide fractions that have been shown to be toxic to celiac small intestinal mucosa in humans and small intestinal microvillus membranes (MVM) from rats during postnatal maturation, MVM were prepared from newborn, 18-day-old preweanling, and adult rats. Partially hydrolyzed gliadin peptide fractions B1-B4, and the pure gliadin peptide B3142 were radioiodinated and used for binding assays. Miniature ultracentrifugation was used for separation of unbound material. Binding of gliadin fractions to MVM was weak and nonspecific in terms of lacking saturation and inhibition. There was no inhibition of binding by mannan. Enzyme pretreatment of MVM (trypsin, neuraminidase, phospholipase C) did not result in any significant change of binding. Compared with peptides prepared from bovine serum albumin as a control, there was no significant difference in binding of gliadin peptide fractions to MVM. Thus, a lectin-like effect of gliadin peptides toward MVM, or the existence of a specific intestinal surface receptor for gliadin peptides appeared improbable. There were, however, consistent maturational changes in MVM binding in that newborn MVM bound more B1-B4 and B3142 compared with adult controls (p less than 0.001). Nonspecific binding of gliadin fractions to MVM might be related to the initiation of nonspecific in vitro effects of gliadin, particularly toward the immature small intestine. The MVM binding model in the rat clearly does not provide a system for studying celiac disease pathogenesis, but it might help clarify basic processes in the interaction between food-derived substances and elements of the gastrointestinal mucosal barrier.

Animals↗
PubMed1992

Specific circulating anti-gliadin IgG-class antibody does not mediate intestinal enteropathy in gliadin-fed mice.

The effects of specific circulating IgG antibody on the uptake of dietary antigen and in the generation of intestinal enteropathy have been investigated in Balb/c mice bred on a gluten-free diet. A monoclonal IgG1 antibody (GD3) was prepared against gliadin. After adoptive transfer into mice, this antibody was capable of mediating a type III hypersensitivity response in vivo to footpad challenge with gliadin. The titres of circulating GD3, as estimated in vitro by ELISA, correlated well with the degree of inflammation at sites of type III responses in vivo. Following footpad challenge with gliadin, titres of circulating GD3 antibody were reduced. GD3 antibody was tested for its ability to mediate inflammatory responses in vivo in the intestinal mucosa of mice fed with gliadin. Circulating GD3 antibody was removed selectively and specifically by dietary gliadin, compared to feeding with bovine serum albumin or maintenance on a gliadin-free diet only. However, we were unable to demonstrate any pathological changes in the intestine as a result of possible local antigen-antibody complex formation or deposition. Using radio-iodinated gliadin as a trace marker, no significant retention of gliadin in the intestinal mucosa was found in mice pre-injected with GD3 antibody. These data suggest that circulating IgG antibody has little effect on dietary antigen uptake in the gut, and alone is insufficient to mediate an enteropathy.

Animals↗
PubMed1998

Anti-alpha-gliadin antibodies (AGA) in the serum of coeliac children and controls recognize an identical collection of linear epitopes of alpha-gliadin.

Anti-gliadin antibodies can be found in the serum of patients with overt and subclinical coeliac disease, but also in that of some controls. The aim of the present study was to identify the linear epitopes of the alpha-gliadin molecule to which the humoral response is directed. Therefore, the IgG and IgA antibody reactivity against an overlapping set of synthetic peptides covering the entire sequence of alpha-gliadin was measured in the sera from patients with coeliac disease, from controls with elevated titres of anti-gliadin antibodies and from healthy children using an ELISA technique. The antibodies mainly recognize peptides derived from the N-terminal region of alpha-gliadin, containing the motif QPFXXQXPY. Reactivity was also detected against two other synthetic peptides, which do not contain this motif and represent a sequence encoded further to the C-terminal region of alpha-gliadin. Anti-gliadin antibodies in sera from patients with coeliac disease and from controls recognize the same linear epitopes. Thus, serological investigation of the specificity of these antibodies using a peptide ELISA does not allow discrimination between patients and controls.

Adolescent↗
PubMed1998

Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition.

Coeliac disease probably results from a T-cell response to wheat gliadin and is associated to HLA-DQ2. No gliadin epitopes recognized by intestinal T cells have yet been identified, limiting our understanding of the pathogenesis. Gut-lesion-derived DQ2-restricted T cells from coeliac disease patients were used to identify an epitope within a purified gamma-type gliadin. The structure of the epitope was characterized by mass spectrometry and verified by synthesis. The epitope (QPQQSFPEQQ) results from deamidation of a distinct glutamine in the native structure. This deamidation is important for binding to DQ2 and T-cell recognition. Other gut-derived T cells fail to recognize the epitope, although deamidation of unfractionated gliadin enhances the response of all gut-derived DQ2-restricted T cells isolated from several patients. Several DQ2-restricted T-cell epitopes exist, but for all of them deamidation of glutamine residues appears to be critical for creation of active epitopes. Native gliadin has few negatively charged residues but is very rich in glutamine. After deamidation gliadin becomes a rich source of DQ2 epitopes thus providing a link between DQ2, gliadin and coeliac disease. The necessity for modification may have general immunological relevance.

Amides↗
PubMed2003

New strategy for the determination of gliadins in maize- or rice-based foods matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: fractionation of gliadins from maize or rice prolamins by acidic treatment.

A procedure for determining small quantities of gliadins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) in gluten-free foods containing relatively large amounts of prolamin proteins from maize or rice is described. We report for the first time that gliadins, the ethanol-soluble wheat prolamin fraction, can be quantitatively solubilized in 1.0 M acetic acid, while the corresponding ethanol-soluble maize or rice prolamin fraction remains insoluble in acetic acid. We describe a methodology for the detection of gliadins in maize and rice foods based on a two-step procedure of extraction (60% aqueous ethanol followed by 1 M acetic acid). Subsequent MALDI-TOFMS analysis of the resulting acidic extract from these gluten-free foods clearly confirms the presence of a typical mass pattern corresponding to gliadin components, ranging from 30 to 45 kDa. Depending on the percentages of maize or rice flours employed in the elaboration of these foods, the combined procedure enables levels of gliadins from 100 to 400 ppm to be detected. The efficiency of this combined procedure corroborates enzyme-linked immunosorbent assay data for a large number of maize/rice gluten-free foods by means of direct visualization of the characteristic gliadin mass pattern in maize or rice foods.

Food Analysis↗
PubMed1979

Immunofluorescent serum gliadin antibodies in children with coeliac disease and various malabsorptive disorders. I. Technique, clinical evaluation and diagnostic use of a gliadin antibody assay using pyruvic aldehyde-treated human red cells.

An immunofluorescent gliadin antibody assay is described using pyruvic aldehyde-stabilized human erythrocytes coated with gliadin. Fifty coeliac children all had high serum IgG-antigliadin titres during a normal diet or a challenge with gluten. On a gluten-free diet (30 children), titres were much lower. In patients followed-up for one year on a gluten-free diet, an initial rise in titres was followed by a slow decline. On challenge, IgG-antigliadin titres showed a slow rise or persistence at the same level in most patients. Fifty-two percent of control children with malabosrptive disorders, but without the typical "flat" mucosal lesion on jejunal biopsy, were shown to have positive titres in their sera, as were 6% of normal children and 4% of adult blood donors. The fluorescent antibody technique was compared with methods commonly used to detect wheat-protein antibodies, and was found to be superior to all of them. The immunofluorescent gliadin antibody assay appears to be useful in following-up children with coeliac disease, and in selecting patients for jejunal biopsy, although it does not replace biopsy.

Antibodies↗
PubMed2006

Glutamine-binding protein from Escherichia coli specifically binds a wheat gliadin peptide. 2. Resonance energy transfer studies suggest a new sensing approach for an easy detection of wheat gliadin.

In this work is presented the first attempt to develop a fluorescence assay for detection of traces of gluten in food by utilizing the recombinant glutamine-binding protein (GlnBP) from E. coli. We found that GlnBP specifically binds the sequence of amino acids present both in gliadin and other prolamines classified as toxic for celiac patients. Affinity chromatography experiments together with mass spectrometry experiments demonstrated that GlnBP can bind the following amino acid sequence XXQPQPQQQQQQQQQQQQL. Sequence alignment experiments pointed out that this sequence is exclusively representative of the gliadin and the other prolamines considered toxic for celiac patients. These findings suggest the development of a competitive resonance energy transfer (RET) assay for an easy and rapid detection of this sequence in raw and cooked food.

Amino Acid Sequence↗
PubMed1980

The lymphocyte transformation test in coeliac disease: effect of gliadin and detoxified gliadin.

Following in vitro stimulation with phytohaemagglutinin, lymphocytes from coeliac patients transformed less than those from control subjects. Neither gliadin nor detoxified gliadin stimulated lymphocyte transformation in patients with adult coeliac disease, but depressed transformation in lymphocytes from normal subjects and from patients on a gluten-free diet.

Antigen-Antibody Reactions↗
PubMedDate not supplied

Gliadin characterization by SANS and gliadin nanoparticle growth modelization.

Nanosized colloidal carriers can ensure a controlled and targeted therapeutic substances delivery. The original contribution of this work was to use biopolymers of vegetable source, which are an interesting alternative to synthetic polymers. The aim of this study was to prepare submicronic particles from wheat proteins: Gliadins extracted from gluten. The carrier preparation was based on the desolvatation of the macromolecules by a couple solvent/non-solvent of the proteins. In a first step, it was of interest to elucidate the gliadin macromolecular conformation in order to understand the mechanism of nanoparticle formation. The experimental work was based on SANS experiments. Because the size of the colloidal particle suspension is an important parameter to monitor, the modelization of the particle growth was thoroughly studied. Furthermore, it was observed that the determination of the solubility parameters of the proteins allowed optimization of the size of the particles. From those previous experimental results it can be concluded that there is a correlation between the protein conformation in the solvent and the size of the nanoparticles (NP).

Computer Simulation↗
PubMed1997

Frequent occurrence of non-specific gliadin antibodies in chronic liver disease. Endomysial but not gliadin antibodies predict coeliac disease in patients with chronic liver disease.

BACKGROUND: The frequency of gliadin antibody (GA) positivity has been found to be increased among patients with chronic liver disease, as has that of coeliac disease (CD). CD has also been found to be increased among patients with primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC). METHODS: To investigate these relationships further, a micro-enzyme-linked immunosorbent assay and immunofluorescence tests for GAs and endomysial antibodies (EMAs) were performed in large subgroups of patients representing various chronic liver diseases and in healthy blood donors. RESULTS: As compared with blood donors (among whom it was 5%) the frequency of IgA GA positivity was higher in all patient subgroups: alcoholic liver disease, 20% (22 of 110, P < 0.001); PBC, 16% (16 of 101, P < 0.001); PSC, 24% (19 of 80, P < 0.001); chronic hepatitis, 19% (13 of 70, P < 0.001); and hepatitis C virus infection, 11% (11 of 104, P < 0.01). Two patients with autoimmune chronic hepatitis were EMA-positive, and in both cases the presence of CD was verified by small-bowel biopsy. CONCLUSIONS: IgA GA positivity generally occurs at increased frequency among patients with chronic liver disease and may represent non-specific immune activation. In liver disease GA testing is not useful in screening for CD, whereas the EMA test seems to be highly specific. CD is more prevalent than expected among patients with autoimmune chronic hepatitis but not among those with PBC or PSC.

Adult↗