Presence Of Gliadin Research Articles

Advances in gluten detection: A rapid colorimetric approach using core-satellite magnetic particles

Monitoring gluten levels in foods labelled as gluten-free or low-gluten — i.e. containing less than 20 ppm and 100 ppm of gluten, respectively — is crucial for preventing celiac disease-related disorders. Due to their inherent complexity, the standard analytical techniques to assess the gluten content in food are not suitable for on-site measurements, whose need is widely recognized. In this context, we developed a rapid and cost-effective biosensor based on core-satellite magnetic particles (CSMPs) — magnetic cores coated with gold nanoparticles (AuNPs) — further functionalized with anti-gliadin antibodies. The study demonstrates that a 0.016% concentration of the surfactant Tween-20 can induce the spontaneous formation of stable CSMP clusters in dispersion. These clusters, composed of weakly interacting functionalized CSMPs, undergo fragmentation in the presence of gliadin, which specifically binds to the antibodies on the CSMPs. This process results in a colour change, which is measurable by a UV–VIS spectrophotometer. Gliadin extraction was achieved by treating the sample with a non-toxic ethanol-water mixture (60%), sufficient to induce a measurable colour change in the presence of gluten contamination, with a limit of detection (LOD) of 8 ppm, which is lower than low limit established for gluten-free food.

A fluorogenic capped mesoporous aptasensor for gluten detection

Celiac disease is a complex and autoimmune disorder caused by the ingestion of gluten affecting almost 1% of global population. Nowadays an effective treatment does not exist, and the only way to manage the disease is the removal of gluten from the diet. Owing the key role played by gluten, clear and regulated labelling of foodstuff and smart methods for gluten detection are needed to fight frauds on food industry and to avoid the involuntary ingestion of this protein by celiac patients. On that scope, the development of a novel detection system of gluten is here presented. The sensor consists of nanoporous anodic alumina films loaded with a fluorescent dye and capped with an aptamer that recognizes gliadin (gluten’s soluble proteins). In the presence of gliadin, aptamer sequences displace from the surface of anodic alumina resulting in pore opening and dye delivery. The dispositive shows a limit of detection (LOD) of 100 μg kg−1 of gliadin, good selectivity and a detection time of approximately 60 min. Moreover, the sensor is validated in real food samples. This novel probe allows fast gluten detection through a simple signalling process with potential use for food control.

Gliadin Peptide Facilitates FITC Dextran Transport across the Non Everted Gut Sac of Rat Small Intestine.

Superoxide dismutase (SOD) is an antioxidant protein. When administered orally, it has low bioavailability due to its low permeation. In a previous study we fused gliadin peptide P51 (LGQQQPFPPQQPYPQPQPF) and gliadin peptide P61 (QQPYPQPQPF) with SOD Citrus limon (SOD_Cl), namely GliSOD_P51 and GliSOD_P61 to increase permeation of SOD_Cl through intestine. In this work, the permeation of fluorescein isothiocyanate (FITC)-Dextran 10 kDa, FD10 and 40 kDa, FD40 as paracellular transport markers across excised rat intestinal wall was investigated with the presence of GliSOD_P51 and GliSOD_P61. A permeability study was performed using non-everted rat intestine by incubating FD10 or FD40 with SOD_Cl, and GliSOD_P61. The presence of SOD_Cl, GliSOD_P51 or GliSOD_P61 inside intestine (apical) and outside intestine (basolateral) was analyzed by protein electrophoresis. The concentration of FD that penetrated to the basolateral solution was analyzed by spectrofluorometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the presence of GliSOD_P51 and GliSOD_P61 but not SOD_Cl in basolateral compartment. The percentage of FD10 but not FD40 and SOD_Cl that penetrated to the basolateral solution significantly increased with the presence of gliadin in GliSOD_P51 and GliSOD_P61. GliSOD_P51 and GliSOD_P61 are able to penetrate the rat intestinal epithelial membrane and the gliadin peptides facilitate FD10 to penetrate the epithelial.

Modulating the Gut Microbiota Improves Glucose Tolerance, Lipoprotein Profile and Atherosclerotic Plaque Development in ApoE-Deficient Mice.

The importance of the gut microbiota (GM) in disease development has recently received increased attention, and numerous approaches have been made to better understand this important interplay. For example, metabolites derived from the GM have been shown to promote atherosclerosis, the underlying cause of cardiovascular disease (CVD), and to increase CVD risk factors. Popular interest in the role of the intestine in a variety of disease states has now resulted in a significant proportion of individuals without coeliac disease switching to gluten-free diets. The effect of gluten-free diets on atherosclerosis and cardiovascular risk factors is largely unknown. We therefore investigated the effect of a gluten-free high-fat cholesterol-rich diet, as compared to the same diet in which the gluten peptide gliadin had been added back, on atherosclerosis and several cardiovascular risk factors in apolipoprotein E-deficient (Apoe-/-) mice. The gluten-free diet transiently altered GM composition in these mice, as compared to the gliadin-supplemented diet, but did not alter body weights, glucose tolerance, insulin levels, plasma lipids, or atherosclerosis. In parallel, other Apoe-/- mice fed the same diets were treated with ampicillin, a broad-spectrum antibiotic known to affect GM composition. Ampicillin-treatment had a marked and sustained effect on GM composition, as expected. Furthermore, although ampicillin-treated mice were slightly heavier than controls, ampicillin-treatment transiently improved glucose tolerance both in the absence or presence of gliadin, reduced plasma LDL and VLDL cholesterol levels, and reduced aortic atherosclerotic lesion area. These results demonstrate that a gluten-free diet does not seem to have beneficial effects on atherosclerosis or several CVD risk factors in this mouse model, but that sustained alteration of GM composition with a broad-spectrum antibiotic has beneficial effects on CVD risk factors and atherosclerosis. These findings support the concept that altering the microbiota might provide novel treatment strategies for CVD.

Role of tissue transglutaminase in celiac disease.

Role of tissue transglutaminase in celiac disease.

Production of antiendomysial antibodies after in-vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease

Diagnosis of many immune-mediated diseases is helped by detection of antibodies directed against the pathogenetic (self or foreign) antigen. In coeliac disease (CD), we have a situation in which antiendomysial antibodies (EMA), which are not specific for the pathogenetic antigen, reach a specificity and sensitivity of detection of CD approaching 100%, whereas detection of antibodies against gliadin (AGA), the pathogenetic antigen, is far less specific and sensitive in diagnosis. No direct evidence of a relation between gluten/gliadin challenge and EMA production exists. We tried to establish whether the small intestine of CD patients is the site of EMA production and whether gliadin challenge could induce their release. Small intestine biopsy samples from treated (23) and untreated (16) CD patients and controls (18) were cultured in vitro for 24-48 h in the presence of gliadin, another alimentary antigen, or medium. EMA and AGA were detected in the supernatants of these organ culture biopsy samples by ELISA and immunofluorescence technique, respectively. No EMA were found in the culture supernatants of biopsy samples of 18 controls, whereas they were detected in the culture supernatants of all 16 untreated CD patients irrespective of gliadin challenge. Conversely, EMA were not detected in supernatants of biopsy samples cultured in medium only from 23 treated CD patients, but were detected in 17 of the 23 biopsy samples challenged with gliadin. Our results suggest that a more complex pathogenetic mechanism than normally accepted is involved in CD. Furthermore, our findings raise the possibility that EMA, or the antigen recognised by them, are involved directly in the pathogenesis of CD.

Size Characterisation of Wheat Proteins, Particularly Glutenin, by Asymmetrical Flow Field-Flow Fractionation

Asymmetrical flow field-flow fractionation was demonstrated to fractionate the most high molecular weight proteins of glutenin found in flour from wheat cultivars with excellent bread-making performances. The fractionation occurs in a flow of liquid on the basis of differences in diffusion coefficients, and therefore hydrodynamic diameters, and applies to large molecules and colloidal particles. Microgram quantities of glutenin were analysed and shown to have hydrodynamic diameters of 5 to 45 nm. Much larger sizes can be analysed because the method does not have an upper size limit as do gel electrophoresis and gel filtration. Fractionations can often be done in less than 5 minutes. Sonication in combination with a surfactant, SDS, dissolved proteins from freeze-dried extracts of flour obtained by sequential extraction using progressively increasing concentrations of dilute HCl. An early fraction showed a response around 8 nm hydrodynamic diameter, which corresponds to a molecular weight range of 23 000–128 000. This indicated the presence of gliadins. Later fractions gave responses up to 35 nm, which translated to the molecular weight range 440 000–11 million, which is consistent with the fact that these fractions contain glutenin. Reduction of a late-fraction sample shifted the band to 8 nm, which corresponds to glutenin subunits. The method appears to be a viable procedure to obtain rapid size fractionations of unreduced glutenin.

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.