RESUMEN
BACKGROUND: Aspirin enhances food allergy symptoms by increasing absorption of ingested allergens. The objective of this study is to elucidate the role of aspirin in facilitating intestinal absorption of the wheat allergen, gliadin, in rats. METHODS: Plasma concentrations of gliadin were determined after oral administration by gavage or administration into a closed intestinal loop in rats. We used an in situ intestinal re-circulating perfusion experiment to examine the effect of pepsin on aspirin-facilitated gliadin absorption. Fluorescein isothiocyanate (FITC)-labeled dextran-40 (FD-40) was used as a marker of non-specific absorption. The molecular size of gliadin and its allergenicity in plasma were examined using immunoblot analysis and intradermal reaction tests with Evans blue dye (EBD) extravasation, respectively. RESULTS: Aspirin increased plasma concentrations of gliadin after oral administration but had no effect in the closed intestinal loop study. An in situ intestinal re-circulating perfusion study showed that FITC-labeled gliadin was absorbed similarly to FD-40. Aspirin increased absorption of both intact and pepsin-digested gliadin, with a more significant effect on absorption of pepsin-treated gliadin. Immunoblotting showed that most gliadin was absorbed in intact form. When the gliadin fraction was extracted from rat plasma after gavage and injected intradermally into gliadin-sensitized rats, EBD extravasation was observed at injection sites in a gliadin dose-dependent manner. CONCLUSIONS: Aspirin increased the absorption of intact and pepsin-digested gliadin via the paracellular pathway, maintaining their allergenicity. Moreover, the effect of aspirin on gliadin absorption was enhanced by modification and digestion of gliadin in the stomach.
Asunto(s)
Alérgenos/farmacocinética , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Gliadina/farmacocinética , Absorción Intestinal/efectos de los fármacos , Administración Oral , Alérgenos/sangre , Alérgenos/química , Animales , Gliadina/sangre , Gliadina/química , Masculino , Pepsina A/química , Ratas Sprague-Dawley , TriticumRESUMEN
SCOPE: Green tea, a polyphenol-rich beverage, has been reported to mitigate a number of inflammatory and hypersensitivity disorders in laboratory models, and has been shown to moderate pathways related to food allergies in vitro. The present study investigates the impact of decaffeinated green tea extract (GTE) on the digestion of gliadin protein in vitro and the effect of physical interactions with GTE on the ability of gliadin to stimulate celiac disease-related symptoms in vitro. METHODS AND RESULTS: Complexation of GTE and gliadin in vitro is confirmed by monitoring increases in turbidity upon titration of GTE into a gliadin solution. This phenomenon is also observed during in vitro digestion when gliadin is exposed to the digestive proteases pepsin and trypsin. SDS-PAGE and enzymatic assays reveal that GTE inhibits digestive protease activity and gliadin digestion. In differentiated Caco-2 cell monolayers as a model of the small intestinal epithelium, complexation of gliadin with GTE reduces gliadin-stimulated monolayer permeability and the release of interleukin (IL)-6 and IL-8. CONCLUSION: There are potential beneficial effects of GTE as an adjuvant therapy for celiac disease through direct interaction between gliadin proteins and green tea polyphenols.
Asunto(s)
Gliadina/farmacocinética , Polifenoles/farmacología , Té/química , Células CACO-2 , Enfermedad Celíaca/etiología , Enteritis/tratamiento farmacológico , Células Epiteliales/efectos de los fármacos , Gliadina/química , Gliadina/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Péptido Hidrolasas/metabolismo , Permeabilidad , Polifenoles/química , Proteolisis , Té/metabolismoRESUMEN
Celiac disease (CD) is an immune-mediated enteropathy triggered by ingested gluten in genetically susceptible individuals and sustained by both adaptive and innate immune responses. Recent studies in murine macrophages demonstrated that the activation of arginase (ARG) metabolic pathway by gluten peptides contributes to the modulation of intestinal permeability in vitro. Here we characterize the effects of gluten on arginine metabolism and cell polarization in human monocytes from both healthy and CD subjects; both a simplified enzymatic digestion of gliadin and a physiological digestion of whole wheat have been tested. Results indicate that gluten digests induce the onset of an M2-like phenotype in activated macrophages; more precisely, both isoforms of arginase, ARG1 and ARG2, are induced likely due to the inhibition of mTOR and the consequent induction of C/EBPß transcription factor. These effects are independent from the origin of gluten as well as from the digestive protocol employed; moreover, no statistical difference can be evidenced between healthy and CD patients, excluding a diverse predisposition of CD monocytes to gluten-triggered polarization with respect to healthy immune cells. Overall, the present findings sustain a role for arginase pathway in the immune response elicited by human monocytes toward ingested gluten that, hence, deserves particular attention when addressing the pathogenesis of CD.
Asunto(s)
Enfermedad Celíaca/patología , Glútenes/farmacología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Adolescente , Adulto , Animales , Arginasa/sangre , Arginina/metabolismo , Enfermedad Celíaca/dietoterapia , Polaridad Celular/efectos de los fármacos , Dieta Sin Gluten , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Gliadina/inmunología , Gliadina/farmacocinética , Glútenes/farmacocinética , Humanos , Inmunidad Innata , Masculino , Ratones , Persona de Mediana Edad , Monocitos/patología , Péptidos/inmunología , Péptidos/farmacocinética , Células RAW 264.7 , Granos EnterosRESUMEN
Gluten promotes type 1 diabetes in nonobese diabetic (NOD) mice and likely also in humans. In NOD mice and in non-diabetes-prone mice, it induces inflammation in the pancreatic lymph nodes, suggesting that gluten can initiate inflammation locally. Further, gliadin fragments stimulate insulin secretion from beta cells directly. We hypothesized that gluten fragments may cross the intestinal barrier to be distributed to organs other than the gut. If present in pancreas, gliadin could interact directly with the immune system and the beta cells to initiate diabetes development. We orally and intravenously administered 33-mer and 19-mer gliadin peptide to NOD, BALB/c, and C57BL/6 mice and found that the peptides readily crossed the intestinal barrier in all strains. Several degradation products were found in the pancreas by mass spectroscopy. Notably, the exocrine pancreas incorporated large amounts of radioactive label shortly after administration of the peptides. The study demonstrates that, even in normal animals, large gliadin fragments can reach the pancreas. If applicable to humans, the increased gut permeability in prediabetes and type 1 diabetes patients could expose beta cells directly to gliadin fragments. Here they could initiate inflammation and induce beta cell stress and thus contribute to the development of type 1 diabetes.
Asunto(s)
Gliadina/farmacocinética , Mucosa Intestinal/metabolismo , Páncreas Exocrino/metabolismo , Fragmentos de Péptidos/farmacocinética , Administración Oral , Animales , Cromatografía Liquida , Diabetes Mellitus Tipo 1/inmunología , Electroforesis en Gel de Poliacrilamida , Gliadina/inmunología , Inflamación , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Masculino , Espectrometría de Masas , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Páncreas/metabolismo , Permeabilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
Asunto(s)
Enterocitos/metabolismo , Gliadina/farmacocinética , Gliadina/toxicidad , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/toxicidad , Células CACO-2 , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/metabolismo , Enfermedad Celíaca/patología , Enterocitos/inmunología , Enterocitos/patología , Humanos , Transporte de Proteínas/efectos de los fármacosRESUMEN
Chicken egg yolk immunoglobulin Y (IgY) is a promising alternative for the prevention of enteric gliadin absorption, the predisposing factor of celiac disease (CD). IgY antibody was produced from the egg yolk of Single Comb White Leghorn chickens during the immunization period for the development of an oral immunotherapeutic agent. Here, we report the potential use of spray dried IgY antibody formulation using sugar protectants (mannitol, sorbitol, or microcrystalline cellulose powder (MCCP)). The long-term stability of the spray dried egg yolk powder formulated with 37.5% mannitol (EYP-M) preserved IgY antibody activity at 99.9%, which was significantly higher than that with other protectants (p < 0.05). In a dissolution test, the EYP-M shows 82.4% IgY activity after 2 h in simulated gastric fluid (SGF). A competitive ELISA at 50% inhibition (IC(50)) shows that 1.6 mg/mL EYP-M bound to 7.6 mg/mL and 10.5 mg/mL gliadin in SGF without and with food matrix conditions, respectively, whereas in simulated intestinal fluid, the formulation bound to 10 mg/mL gliadin, regardless of a food matrix. In-vivo study: BALB/c mice fed with EYP-M and gliadin at a ratio of 1:5 (w/w) demonstrated that gliadin absorption in the gastrointestinal tract was minimal at <1%. Thus, EYP-M containing IgY antibody may be used in CD patients to eliminate the effects of ingested toxic gliadin.
Asunto(s)
Yema de Huevo/inmunología , Gliadina/inmunología , Inmunoglobulinas/metabolismo , Animales , Enfermedad Celíaca/prevención & control , Pollos , Desecación/métodos , Estabilidad de Medicamentos , Femenino , Alimentos , Tracto Gastrointestinal/metabolismo , Gliadina/antagonistas & inhibidores , Gliadina/farmacocinética , Inmunización Pasiva , Inmunoglobulinas/administración & dosificación , Absorción Intestinal , Ratones , Ratones Endogámicos BALB C , Preparaciones FarmacéuticasRESUMEN
The intestinal permeability of undegraded α9-gliadin peptide 31-49 (p31-49) and 33-mer gliadin peptides is increased in active celiac disease. Two distinct transport pathways have been proposed: paracellular leakage through epithelial tight junctions and protected transcellular transport. To analyze the relative contribution of these pathways, we compared mucosa-to-serosa permeability of small and large permeability markers [ionic conductance (G), mannitol, 182 Da; horseradish peroxidase, 40 kDa] and gliadin peptides [33-mer (p56-88, 3900 Da), 19-mer (p31-49, 2245 Da; and p202-220, 2127 Da), and 12-mer (p57-68, 1453 Da)] in duodenal biopsy specimens mounted in Ussing chambers. The permeability of intact peptides was much higher for p31-49 or 33-mer than for horseradish peroxidase, p202-220, and p57-68. A positive correlation was observed between G, an index of paracellular diffusion of ions, and mannitol permeability. The absence of correlation between G and permeability to intact 33-mer or p31-49 did not favor paracellular diffusion of the peptides. Immunofluorescence studies indicated that 33-mer enters the early endosome antigen 1-positive compartment but escapes the lysosomal-associated protein 2-positive compartment. The results underline that mannitol and ionic conductance G cannot be considered markers of permeability to gliadin peptides. In active celiac disease, increases in transcellular permeability to intact gliadin peptides might be considered in treatment strategies aimed at controlling epithelial permeability to gluten.
Asunto(s)
Enfermedad Celíaca/metabolismo , Duodeno/metabolismo , Gliadina/farmacocinética , Fragmentos de Péptidos/farmacocinética , Transporte Biológico , Peroxidasa de Rábano Silvestre/farmacocinética , Humanos , Mucosa Intestinal/metabolismo , Manitol/farmacocinética , Permeabilidad , Membrana Serosa/metabolismo , Uniones Estrechas/metabolismoRESUMEN
BACKGROUND AND AIMS: Celiac disease (CD) is a chronic inflammatory disorder of the small intestine that is induced by dietary wheat gluten proteins (gliadins) in genetically predisposed individuals. The overgrowth of potentially pathogenic bacteria and infections has been suggested to contribute to CD pathogenesis. We aimed to study the effects of gliadin and various intestinal bacterial strains on mucosal barrier integrity, gliadin translocation, and cytokine production. METHODOLOGY/PRINCIPAL FINDINGS: Changes in gut mucosa were assessed in the intestinal loops of inbred Wistar-AVN rats that were reared under germ-free conditions in the presence of various intestinal bacteria (enterobacteria and bifidobacteria isolated from CD patients and healthy children, respectively) and CD-triggering agents (gliadin and IFN-γ) by histology, scanning electron microscopy, immunofluorescence, and a rat cytokine antibody array. Adhesion of the bacterial strains to the IEC-6 rat cell line was evaluated in vitro. Gliadin fragments alone or together with the proinflammatory cytokine interferon (IFN)-γ significantly decreased the number of goblet cells in the small intestine; this effect was more pronounced in the presence of Escherichia coli CBL2 and Shigella CBD8. Shigella CBD8 and IFN-γ induced the highest mucin secretion and greatest impairment in tight junctions and, consequently, translocation of gliadin fragments into the lamina propria. Shigella CBD8 and E. coli CBL2 strongly adhered to IEC-6 epithelial cells. The number of goblet cells in small intestine increased by the simultaneous incubation of Bifidobacterium bifidum IATA-ES2 with gliadin, IFN-γ and enterobacteria. B. bifidum IATA-ES2 also enhanced the production of chemotactic factors and inhibitors of metalloproteinases, which can contribute to gut mucosal protection. CONCLUSIONS: Our results suggest that the composition of the intestinal microbiota affects the permeability of the intestinal mucosa and, consequently, could be involved in the early stages of CD pathogenesis.
Asunto(s)
Bacterias/patogenicidad , Gliadina/farmacocinética , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Animales , Toxinas Bacterianas/farmacología , Bifidobacterium/patogenicidad , Enfermedad Celíaca/etiología , Citocinas/biosíntesis , Enterobacteriaceae/patogenicidad , Vida Libre de Gérmenes , Gliadina/farmacología , Células Caliciformes/patología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interferón gamma/farmacología , Mucosa Intestinal/metabolismo , Intestinos/microbiología , Intestinos/patología , Permeabilidad , RatasRESUMEN
Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31-43 (but not peptide 57-68) uptake by cells, thereby impairing the ability of p31-43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31-43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.
Asunto(s)
Autoanticuerpos/farmacología , Enfermedad Celíaca/inmunología , Células Epiteliales/efectos de los fármacos , Gliadina/farmacocinética , Transglutaminasas/antagonistas & inhibidores , Transglutaminasas/inmunología , Células CACO-2 , Enfermedad Celíaca/metabolismo , Antagonismo de Drogas , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Activación Enzimática/efectos de los fármacos , Células Epiteliales/metabolismo , Gliadina/química , Gliadina/farmacología , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/farmacología , Fase S/efectos de los fármacos , Transglutaminasas/metabolismoRESUMEN
INTRODUCTION: Currently the only treatment for celiac disease is a lifelong gluten-free diet. The diet is, however, often burdensome, and thus new treatment options are warranted. We isolated proteases from germinating wheat grain naturally meant for total digestion of wheat storage proteins and investigated whether these enzymes can diminish toxic effects of gluten in vitro and ex vivo. METHODS: Pepsin and trypsin digested (PT) gliadin was pretreated with proteases from germinating wheat, whereafter the degradation was analyzed by HPLC-MS (high-performance liquid chromatography and mass spectroscopy) and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The toxicity of cleaved PT-gliadin products was assessed in Caco-2 epithelial cells, celiac patient-derived T cells, and in human small intestinal mucosal organ culture biopsies. RESULTS: Proteases from germinating wheat degraded gliadin into small peptide fragments, which, unlike unprocessed PT-gliadin, did not increase epithelial permeability, induce cytoskeletal rearrangement or changes in ZO-1 expression in Caco-2 cells. Pretreated gliadin did not stimulate T cell proliferation in vitro or enhance the production of autoantibodies to culture supernatants and the activation of CD25+ lymphocytes in the organ culture to the same extent as unprocessed PT-gliadin. DISCUSSION: Germinating wheat enzymes reduce the toxicity of wheat gliadin in vitro and ex vivo. Further studies are justified to develop an alternative therapy for celiac disease.
Asunto(s)
Enfermedad Celíaca/terapia , Gliadina/farmacocinética , Péptido Hidrolasas/metabolismo , Adolescente , Adulto , Anciano , Biopsia , Células CACO-2 , Enfermedad Celíaca/enzimología , Enfermedad Celíaca/patología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inactivación Metabólica , Intestino Delgado/inmunología , Intestino Delgado/metabolismo , Intestino Delgado/patología , Masculino , Persona de Mediana Edad , Pronóstico , Linfocitos T/inmunología , Triticum/enzimología , Adulto JovenRESUMEN
Because intestinal absorption of food protein can trigger an allergic reaction, the effect of wheat proteins on intestinal epithelial cell permeability was evaluated and the abilities of these proteins in native or pepsin-hydrolyzed state to cross the epithelial cell monolayer were compared. Enterocytic monolayers were established by culturing Caco-2 cells, a model of enterocytes, on permeable supports that separate the apical and basal compartments. Proteins were added into the apical compartment, and the transepithelial resistance (TER) was measured; proteins that crossed the cell monolayer were detected in the basal medium by ELISA. Wheat proteins did not alter the cell monolayer. TER and Caco-2 cell viability were conserved, and the passage of dextran was prevented. Native and pepsin-hydrolyzed forms of omega5-gliadin and lipid transfer proteins were detected in the basal medium. The results suggest that these two major allergens in food allergy to wheat were able to cross the cell monolayer by the transcellular route.
Asunto(s)
Antígenos de Plantas/metabolismo , Gliadina/farmacocinética , Glútenes/farmacocinética , Intestino Delgado/metabolismo , Células CACO-2 , Impedancia Eléctrica , Humanos , Hipersensibilidad al Trigo/metabolismoRESUMEN
BACKGROUND & AIMS: The resistance of prolamines to digestive enzymes is thought to be a key contributor to the pathogenesis of celiac disease by promoting the intestinal entrance of peptides able to trigger inflammation in at-risk individuals. Oral administration of a bacterial prolyl-endopeptidase (PEP) therefore was proposed as a treatment for celiac disease. To delineate the feasibility of this treatment, the effect of PEP on gliadin peptides was assessed in vitro, and ex vivo during their transport across intestinal biopsy specimens of active celiac disease patients. METHODS: In vitro degradation by PEP of 3H-labeled gliadin peptides 56-88 (33-mer) and 31-49, was analyzed by radio-reverse-phase high-performance liquid chromatography and mass spectrometry. For ex vivo studies, PEP and 3H-peptides were added together onto the mucosal side of duodenal biopsy specimens mounted in Ussing chambers, and peptide transport and digestion were assessed by radio-reverse-phase high-performance liquid chromatography. RESULTS: Gliadin peptides were degraded partly by 20 mU/mL PEP both in vitro and ex vivo. This concentration of PEP decreased the amount of intact peptides 31-49 and 56-88 crossing the intestinal biopsy specimens of celiac disease patients, but could not prevent the intestinal passage of toxic or immunostimulatory metabolites. PEP concentrations of at least 500 mU/mL for 3 hours were required to achieve full detoxification of peptides and to prevent intestinal transport of active fragments. CONCLUSIONS: After prolonged exposure to high concentrations of PEP, the amount of immunostimulatory gliadin peptides reaching the local immune system in celiac patients is decreased. These results provide a basis to establish whether such conditions are achievable in vivo.
Asunto(s)
Enfermedad Celíaca/tratamiento farmacológico , Gliadina/toxicidad , Serina Endopeptidasas/uso terapéutico , Biopsia , Enfermedad Celíaca/patología , Cromatografía Líquida de Alta Presión , Gliadina/farmacocinética , Inactivación Metabólica , Marcaje Isotópico , Espectrometría de Masas , Oligopéptidos/farmacocinética , Prolil Oligopeptidasas , TritioRESUMEN
The objective of this work was to prepare, characterise and evaluate the adhesive potential of gliadin nanoparticulate carriers. Firstly, lectin-nanoparticle conjugates were obtained by the carbodiimide (CDI) covalent binding of Dolichos biflorus lectin (DBA) to the surface of gliadin nanoparticles (NP) containing carbazole (as a model lipophilic drug). The DBA binding efficiency was favoured in mild acidic conditions. Similarly, a CDI concentration of about 0.63 mg/mg nanoparticles, acting during at least 1 h, provided binding efficiencies of about 50% bulk lectin. Under optimised experimental conditions, the DBA conjugates showed a size of around 500 nm and the amount of loaded carbazole and the DBA content were calculated to be around 15 and 23.5 microg/mg, respectively. The bioadhesive activity of NP and DBA conjugates was determined in samples of small and large rat intestinal mucosa. The amount of adsorbed NP was calculated to be around 8 and 4 g/m(2) in the small and large intestine, respectively. This high capacity to interact with the mucosa may be explained by gliadin composition. In fact, gliadin is rich in neutral and lipophilic residues. Neutral amino acids can promote hydrogen bonding interactions with the mucosa, while the lipophilic components can interact with the biological tissue by hydrophobic interactions. The bioadhesive activity of DBA conjugates was calculated to be about 2 g/m(2) in the small intestine and greater than 4 g/m(2) in the caecum and distal colon. These degrees of interaction were always significantly higher than those obtained with controls. Finally, DBA did not provide the specificity for interaction with Peyer's patches. In summary, gliadin nanoparticles show a high capacity of non-specific interaction with the intestine, whereas DBA binding to the surface of these carriers provided a greater specificity for colonic mucosa.
Asunto(s)
Adhesivos/farmacocinética , Gliadina/farmacocinética , Mucosa Intestinal/metabolismo , Lectinas/farmacocinética , Animales , Portadores de Fármacos , Concentración de Iones de Hidrógeno , Masculino , Ratas , Ratas WistarRESUMEN
BACKGROUND: Secretion of dietary antigens into breast milk has been extensively documented. The presence of these antigens is of relevance because they could be involved in the modulation of the immune response in neonates. The objective of this study is to determine the gliadin concentration in milk, colostrum, and serum samples from healthy lactating mothers on a normal diet. Gliadin levels in milk samples from a group of six mothers after a brief period of gluten restriction were also determined. The molecular weight of secreted gliadins was also analysed. METHODS: Gliadin concentration was determined with a highly sensitive competitive enzyme-linked immunosorbent assay, modified so as to eliminate anti-gliadin antibody interference. The level of gliadin/IgA anti-gliadin immune complexes in milk, colostrum, and serum samples was determined. RESULTS: Gliadin was detected in all 49 milk samples. Its concentration varied between 5 and 1200 ng/ml (mean, 178 ng/ml). In colostrum (n = 14) gliadin levels were higher (range, 28-9000 ng/ml; mean, 883 ng/ml), not being detectable in one case. Gliadin was detectable in 14 of 31 serum samples, in which levels were lower than in milk and colostrum samples (mean, 41 ng/ml). Neither a correlation between gliadin levels in milk, colostrum, and serum samples from the same subject nor a relation between gluten intake and gliadin concentration in milk samples from six subjects under a 3-day gluten-free diet could be found. Higher levels of immune complexes were observed in colostrum samples than in milk and serum samples. No correlation was detected between gliadin concentration and the level of immune complexes. The analysis of milk and colostrum samples by immunoblotting showed bands of immunoreactive gliadin presenting Mr similar to those of native proteins from wheat extracts. CONCLUSIONS: Very high levels of gliadin were detected in milk samples from healthy mothers on an unrestricted diet. Gliadin levels were higher than those reported for dietary antigens in other studies. Breast milk contained non-degraded gliadins and gliadin/anti-gliadin IgA immune complexes.
Asunto(s)
Gliadina/análisis , Leche Humana/química , Complejo Antígeno-Anticuerpo/análisis , Calostro/química , Dieta con Restricción de Proteínas , Ensayo de Inmunoadsorción Enzimática , Femenino , Gliadina/inmunología , Gliadina/farmacocinética , Glútenes/administración & dosificación , Humanos , Immunoblotting , Inmunoglobulina A/análisis , Peso MolecularRESUMEN
Association of different psychological and neurological disturbances with gluten intake in coeliac patients was repeatedly described. In the present study gluten-induced enteropathy was elicited in rats by prolonged intragastric administration of gliadin from birth to 10 weeks. Various neurological (contact and visual placing reactions, equilibrium on horizontal bar) and behavioral tests (open field and Morris water maze task) were used to assess the possible deficits. No substantial differences were found in the behavior of rats fed with gliadin compared with those fed with bovine serum albumin (control group). The only difference found between control and experimental rats was that gliadin-fed rats showed slightly higher emotionality in the open field test. It is concluded that prolonged application of gliadin to young rats at enteropathy-inducing dosages does not modify their behavior.
Asunto(s)
Conducta Animal/efectos de los fármacos , Enfermedad Celíaca/inducido químicamente , Gliadina/efectos adversos , Gliadina/farmacología , Animales , Femenino , Gliadina/farmacocinética , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Masculino , Ratas , Albúmina Sérica Bovina/farmacocinética , Albúmina Sérica Bovina/farmacologíaRESUMEN
Two quick, sensitive and direct sandwich enzyme-linked immunosorbent assays (ELISAs) for the measurement of gliadin and ovalbumin in serum have been developed. The ovalbumin assay has a sensitivity of 0.14 ng/ml (2 SD from zero). Cross-reactivities with ovomucoid and conalbumin were 58% and 0.2%, respectively. No cross-reactivity was observed with bovine serum albumin (BSA), human serum albumin (HSA), gliadin and beta-lactoglobulin. The gliadin assay did not cross-react with BSA, HSA, ovalbumin and beta-lactoglobulin and had a sensitivity of 7 pg/ml. In an acute feeding study, of 6 h duration, serum ovalbumin levels were measured in five non-food-allergic subjects who had been given raw eggs (5 g ovalbumin) after an overnight fast. Following a period of at least 1 week the same subjects were given 132 g wholemeal bread (5 g gliadin) after an overnight fast, and serum gliadin levels were measured. After the consumption of raw eggs ovalbumin levels peaked between 210 and 300 min with maximum serum concentration between 0.52 and 31.08 ng/ml. After the consumption of bread, gliadin levels reached a maximum between 180 and 300 min, reaching peak levels between 1.12 and 12.2 ng/ml. We conclude that levels of detected ovalbumin were higher than those for gliadin in this group of individuals.
Asunto(s)
Digestión , Ensayo de Inmunoadsorción Enzimática , Gliadina/sangre , Absorción Intestinal , Ovalbúmina/sangre , Adulto , Albúminas/inmunología , Pan/análisis , Conalbúmina/inmunología , Reacciones Cruzadas , Huevos/análisis , Metabolismo Energético , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Evaluación como Asunto , Femenino , Análisis de los Alimentos , Gliadina/inmunología , Gliadina/farmacocinética , Humanos , Lactoglobulinas/inmunología , Masculino , Ovalbúmina/inmunología , Ovalbúmina/farmacocinética , Ovomucina/inmunología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Albúmina Sérica Bovina/inmunologíaRESUMEN
The pepsin trypsin digest of the wheat prolamin gliadin (PT-gliadin) is deleterious to the small intestinal mucosa of coeliac patients. The handling of PT-gliadin by the intestinal epithelium in coeliac patients in remission and control individuals was investigated by in vivo instillation of PT-gliadin. The uptake of PT-gliadin was monitored by immunofluorescence microscopy of intestinal biopsy specimens, using affinity purified PT-gliadin antibodies. Control individuals show weak staining in the apical region of the enterocytes thereby showing an uptake of PT-gliadin. Coeliac patients have a conspicuous fluorescence in relation to the lateral membrane/intercellular space of enterocytes and intense staining intracellularly in the apical region. There is only weak staining in the enterocytes after the instillation was terminated, indicating an intracellular clearance. The study shows that normal enterocytes are able to take up PT-gliadin. The increased uptake in coeliac patients might be of importance for the pathogenesis either by direct toxicity or by presentation to immunocompetent cells. Furthermore, the results are in agreement with the suggestion of a functional alteration in the zonula occludens in the intestinal epithelium of coeliac patients.
Asunto(s)
Enfermedad Celíaca/metabolismo , Gliadina/farmacocinética , Yeyuno/metabolismo , Adulto , Epitelio/metabolismo , Humanos , Inmunohistoquímica , Microscopía FluorescenteRESUMEN
The rat everted gut sac was used as a model of intestinal protein and peptide handling to study small intestinal binding and uptake of different gliadin peptide fractions (B1, B2, B3, B4) in comparison with bovine serum albumin (BSA), alpha-lactalbumin (alpha-LA), and a BSA peptide fraction (BSA-P). Not only binding curves, but also uptake curves of BSA, alpha-LA, and BSA-P were run in parallel between 1 and 30 min. Binding and uptake obviously depended on molecular weight. BSA-P binding was found to be highest of all. Binding of BSA was significantly lower. After 20 and 30 min, significantly more alpha-LA and BSA-P were taken up than was BSA. There was no difference in handling of the gliadin peptide fractions tested by the gut mucosal surface. Binding of B1-B4 was significantly higher than binding of BSA and was significantly lower than binding of BSA-P. No difference was found between binding of B1-B4 and alpha-LA. There was a consistently higher uptake of all gliadin peptide fractions, independent of molecular weight. Slopes for uptake curves of B1-B4 were steeper than those of BSA, alpha-LA, and BSA-P. Crossing of uptake over binding curves was consistently observed with gliadin fractions, but never with cow's milk proteins/peptides. Differences in uptake between gliadin peptide fractions and cow's milk proteins/peptides might be based on a specific interaction between gliadin peptides and the gut surface.