RESUMEN
BACKGROUND & AIMS: Celiac disease could be treated, and potentially cured, by restoring T-cell tolerance to gliadin. We investigated the safety and efficacy of negatively charged 500-nm poly(lactide-co-glycolide) nanoparticles encapsulating gliadin protein (TIMP-GLIA) in 3 mouse models of celiac disease. Uptake of these nanoparticles by antigen-presenting cells was shown to induce immune tolerance in other animal models of autoimmune disease. METHODS: We performed studies with C57BL/6; RAG1-/- (C57BL/6); and HLA-DQ8, huCD4 transgenic Ab0 NOD mice. Mice were given 1 or 2 tail-vein injections of TIMP-GLIA or control nanoparticles. Some mice were given intradermal injections of gliadin in complete Freund's adjuvant (immunization) or of soluble gliadin or ovalbumin (ear challenge). RAG-/- mice were given intraperitoneal injections of CD4+CD62L-CD44hi T cells from gliadin-immunized C57BL/6 mice and were fed with an AIN-76A-based diet containing wheat gluten (oral challenge) or without gluten. Spleen or lymph node cells were analyzed in proliferation and cytokine secretion assays or by flow cytometry, RNA sequencing, or real-time quantitative polymerase chain reaction. Serum samples were analyzed by gliadin antibody enzyme-linked immunosorbent assay, and intestinal tissues were analyzed by histology. Human peripheral blood mononuclear cells, or immature dendritic cells derived from human peripheral blood mononuclear cells, were cultured in medium containing TIMP-GLIA, anti-CD3 antibody, or lipopolysaccharide (controls) and analyzed in proliferation and cytokine secretion assays or by flow cytometry. Whole blood or plasma from healthy volunteers was incubated with TIMP-GLIA, and hemolysis, platelet activation and aggregation, and complement activation or coagulation were analyzed. RESULTS: TIMP-GLIA did not increase markers of maturation on cultured human dendritic cells or induce activation of T cells from patients with active or treated celiac disease. In the delayed-type hypersensitivity (model 1), the HLA-DQ8 transgenic (model 2), and the gliadin memory T-cell enteropathy (model 3) models of celiac disease, intravenous injections of TIMP-GLIA significantly decreased gliadin-specific T-cell proliferation (in models 1 and 2), inflammatory cytokine secretion (in models 1, 2, and 3), circulating gliadin-specific IgG/IgG2c (in models 1 and 2), ear swelling (in model 1), gluten-dependent enteropathy (in model 3), and body weight loss (in model 3). In model 1, the effects were shown to be dose dependent. Splenocytes from HLA-DQ8 transgenic mice given TIMP-GLIA nanoparticles, but not control nanoparticles, had increased levels of FOXP3 and gene expression signatures associated with tolerance induction. CONCLUSIONS: In mice with gliadin sensitivity, injection of TIMP-GLIA nanoparticles induced unresponsiveness to gliadin and reduced markers of inflammation and enteropathy. This strategy might be developed for the treatment of celiac disease.
Asunto(s)
Enfermedad Celíaca/tratamiento farmacológico , Gliadina/administración & dosificación , Tolerancia Inmunológica/efectos de los fármacos , Nanopartículas/administración & dosificación , Administración Intravenosa , Animales , Linfocitos T CD4-Positivos , Enfermedad Celíaca/sangre , Enfermedad Celíaca/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Gliadina/inmunología , Gliadina/toxicidad , Glútenes/administración & dosificación , Glútenes/inmunología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Humanos , Mucosa Intestinal , Leucocitos Mononucleares , Ratones , Ratones Transgénicos , Nanopartículas/química , Nanopartículas/toxicidad , Poliglactina 910/química , Cultivo Primario de Células , Pruebas de Toxicidad AgudaRESUMEN
BACKGROUND: The standard method for diagnosing immediate wheat allergy is oral food challenge test (OFC). However, OFC can provoke anaphylaxis during the challenge process. Skin prick test (SPT) using commercial wheat extract yielded unsatisfactory result for diagnosis of wheat allergy. As a result, an in-house, alcohol-dissolved (Coca-10% EtOH) wheat extract was developed to improve accuracy of the SPT. OBJECTIVE: To determine the accuracy of in-house, alcohol-dissolved wheat extract in children with immediate wheat allergy. METHODS: This prospective cross-sectional study included children with history of immediate reaction after wheat ingestion. SPTs with commercial and in-house Coca-10% EtOH wheat extract were performed and wheat and omega-5 (ω-5) gliadin specific IgE (sIgE) were measured. Patients with no history of recent anaphylaxis after wheat ingestion underwent OFC with 31 grams of wheat flour. RESULTS: Thirty children were recruited. Thirteen of those had history of anaphylaxis after wheat ingestion. Eleven of the remaining 17 children (64.7%) had a positive result for wheat challenge test. Wheal size of 3 mm for both in-house and commercial wheat extract yielded the best accuracy for the test. Using these cutoff parameters, in-house Coca-10% EtOH wheat extract yielded 91.7% sensitivity, 66.7% specificity, and 86.7% accuracy. Comparatively, the commercial extract yielded 70.8% sensitivity, 100% specificity, and 76.6% accuracy. CONCLUSIONS: SPT using in-house Coca-10% EtOH wheat extract yielded better accuracy than commercial extract for diagnosing immediate type wheat allergy in children.
Asunto(s)
Alérgenos/inmunología , Gliadina/inmunología , Extractos Vegetales/inmunología , Proteínas de Plantas/inmunología , Hipersensibilidad al Trigo/diagnóstico , Adolescente , Alcoholes/química , Alérgenos/química , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , Hipersensibilidad Inmediata , Inmunoglobulina E/metabolismo , Lactante , Masculino , Extractos Vegetales/química , Proteínas de Plantas/química , Estudios Prospectivos , Autoevaluación , Sensibilidad y Especificidad , Triticum/inmunologíaAsunto(s)
Enfermedad Celíaca/complicaciones , Harina/efectos adversos , Triticum/efectos adversos , Hipersensibilidad al Trigo/complicaciones , Adolescente , Alérgenos/inmunología , Antígenos de Plantas/inmunología , Enfermedad Celíaca/inmunología , Femenino , Gliadina/inmunología , Glútenes/inmunología , Humanos , Inmunoglobulina E/sangre , Persona de Mediana Edad , Extractos Vegetales/inmunología , Pruebas Cutáneas , Triticum/inmunología , Hipersensibilidad al Trigo/diagnóstico , Hipersensibilidad al Trigo/inmunologíaRESUMEN
In celiac disease (CD), an intolerance to dietary gluten/gliadin, antigenic gliadin peptides trigger an HLA-DQ2/DQ8-restricted adaptive Th1 immune response. Epithelial stress, induced by other non-antigenic gliadin peptides, is required for gliadin to become fully immunogenic. We found that cystic-fibrosis-transmembrane-conductance-regulator (CFTR) acts as membrane receptor for gliadin-derived peptide P31-43, as it binds to CFTR and impairs its channel function. P31-43-induced CFTR malfunction generates epithelial stress and intestinal inflammation. Maintaining CFTR in an active open conformation by the CFTR potentiators VX-770 (Ivacaftor) or Vrx-532, prevents P31-43 binding to CFTR and controls gliadin-induced manifestations. Here, we evaluated the possibility that the over-the-counter nutraceutical genistein, known to potentiate CFTR function, would allow to control gliadin-induced alterations. We demonstrated that pre-treatment with genistein prevented P31-43-induced CFTR malfunction and an epithelial stress response in Caco-2 cells. These effects were abrogated when the CFTR gene was knocked out by CRISP/Cas9 technology, indicating that genistein protects intestinal epithelial cells by potentiating CFTR function. Notably, genistein protected gliadin-sensitive mice from intestinal CFTR malfunction and gliadin-induced inflammation as it prevented gliadin-induced IFN-γ production by celiac peripheral-blood-mononuclear-cells (PBMC) cultured ex-vivo in the presence of P31-43-challenged Caco-2 cells. Our results indicate that natural compounds capable to increase CFTR channel gating might be used for the treatment of CD.
Asunto(s)
Enfermedad Celíaca/prevención & control , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Genisteína/farmacología , Gliadina/toxicidad , Fragmentos de Péptidos/toxicidad , Animales , Células CACO-2 , Enfermedad Celíaca/etiología , Enfermedad Celíaca/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Suplementos Dietéticos , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Gliadina/inmunología , Humanos , Interferón gamma/biosíntesis , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Mucosa Intestinal/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Fragmentos de Péptidos/inmunología , Unión ProteicaRESUMEN
Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.
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Proteínas Arqueales/genética , Proteínas Bacterianas/genética , Glútenes/metabolismo , Péptido Hidrolasas/genética , Proteínas de Plantas/genética , Triticum/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Biolística , Enfermedad Celíaca/dietoterapia , Enfermedad Celíaca/inmunología , Chryseobacterium/enzimología , Chryseobacterium/genética , Expresión Génica , Ingeniería Genética/métodos , Gliadina/inmunología , Gliadina/aislamiento & purificación , Gliadina/metabolismo , Gliadina/farmacología , Glútenes/química , Glútenes/inmunología , Hordeum/enzimología , Hordeum/genética , Humanos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Péptido Hidrolasas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteolisis , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/genética , Transgenes , Triticum/enzimologíaAsunto(s)
Enfermedad Celíaca/diagnóstico , Errores Diagnósticos/prevención & control , Proteínas de Unión al GTP/sangre , Hemólisis , Inmunoensayo/normas , Inmunoglobulina G/sangre , Transglutaminasas/sangre , Anticuerpos Antiproteína Citrulinada/sangre , Enfermedad Celíaca/sangre , Enfermedad Celíaca/inmunología , Errores Diagnósticos/estadística & datos numéricos , Emulsiones , Reacciones Falso Negativas , Proteínas de Unión al GTP/inmunología , Gliadina/sangre , Gliadina/inmunología , Humanos , Hiperlipidemias/sangre , Hiperlipidemias/inmunología , Inmunoglobulina A/sangre , Fosfolípidos/sangre , Proteína Glutamina Gamma Glutamiltransferasa 2 , Aceite de Soja/sangre , Transglutaminasas/inmunologíaRESUMEN
Initiation of celiac disease is triggered in the gastrointestinal tract by transglutaminase 2 (TG2) assisted deamidation of gluten peptides. Deamidation is a side-reaction to transamidation and occurs if primary amines are absent. In contrast to deamidation, transamidation does not trigger an immune response. The aim of the study was to identify a suitable food additive that interacts with TG2 binding motives in gluten-derived peptides to prevent deamidation/transamidation. Homology modelling of α2-gliadin and computational screening of compounds for their binding affinity to a common TG2 binding motive (P)QLP were done by using computational approaches followed by experimental testing of TG2 activity. A database containing 1174 potential food grade ligands was screened against the model of α2-gliadin (27 out of 33 aa). Out of the five best ligands, ascorbyl palmitate, was observed to decrease TG2 transamidation of gliadin by 82% ± 2%. To completely silence the transamidation, we added zinc chloride (ZnCl2), and thereby reached a 99% ± 1% inhibition of TG2 activity. In addition, we conducted a pilot experiment in which ascorbyl palmitate was observed to decrease TG2 deamidation of gliadin completely. We propose ascorbyl palmitate in combination with ZnCl2 with the future perspective to become an additive in celiac-safe foods.
Asunto(s)
Ácido Ascórbico/análogos & derivados , Enfermedad Celíaca/inmunología , Cloruros/farmacología , Proteínas de Unión al GTP/metabolismo , Gliadina/química , Transglutaminasas/metabolismo , Compuestos de Zinc/farmacología , Ácido Ascórbico/farmacología , Enfermedad Celíaca/enzimología , Evaluación Preclínica de Medicamentos , Epítopos de Linfocito T/inmunología , Aditivos Alimentarios/farmacología , Proteínas de Unión al GTP/química , Predisposición Genética a la Enfermedad , Gliadina/inmunología , Humanos , Activación de Linfocitos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Proyectos Piloto , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/químicaRESUMEN
Wheat allergy is an IgE-mediated disorder. Polyphenols, which are known to interact with certain proteins, could be used to reduce allergic reactions. This study screened several polyphenol sources for their ability to interact with gliadins, mask epitopes, and affect basophil degranulation. Polyphenol extracts from artichoke leaves, cranberries, apples, and green tea leaves were examined. Of these extracts, the first three formed insoluble complexes with gliadins. Only the cranberry and apple extracts masked epitopes in dot blot assays using anti-gliadin IgG and IgE antibodies from patients with wheat allergies. The cranberry and artichoke extracts limited cellular degranulation by reducing mouse anti-gliadin IgE recognition. In conclusion, the cranberry extract is the most effective polyphenol source at reducing the immunogenicity and allergenicity of wheat gliadins.
Asunto(s)
Alérgenos/inmunología , Gliadina/inmunología , Extractos Vegetales/química , Polifenoles/química , Hipersensibilidad al Trigo/inmunología , Alérgenos/química , Animales , Basófilos/inmunología , Gliadina/química , Humanos , Inmunoglobulina E/inmunología , Espectrometría de Masas , Unión Proteica , RatasRESUMEN
BACKGROUND: Identification of wheat sensitization by a skin prick test (SPT) is essential for children with wheat-induced anaphylaxis, since oral food challenge can cause serious adverse effects. Wheat allergens are both water/salt and alcohol soluble. The preparation of wheat extract for SPT containing both water/salt and alcohol soluble allergen is needed. OBJECTIVE: To determine if a wheat extract using Coca's solution containing 10% alcohol (Coca-10% EtOH), prepared in-house, contians both water/salt and alcohol soluble allergens. METHODS: Serum of children with a history of anaphylaxis after wheat ingestion was used. Wheat flour was extracted in Coca-10% alcohol solution. An SPT with both commercial and in-house wheat extracts was performed as well as specific IgE (sIgE) for wheat and omega-5 gliadin. Direct and IgE inhibition immunoblots were performed to determine serum sIgE levels against water/salt as well as alcohol soluble (gliadins and glutenins) allergens in the extracts. RESULTS: Six children with history of wheat anaphylaxis had positive SPT to both commercial and in-house extracts. They also had different levels of sIgE against wheat and omega-5 gliadin allergens. The results of direct immunoblotting showed all tested sera had sIgE bound to ~35 kDa wheat protein. Further IgE inhibition immunoblotting identified the ~35 kDa wheat protein as gliadin but not gluten allergen. CONCLUSION: The in-house prepared Coca-10% EtOH solution could extract both water/salt and alcohol soluble allergens. The ~35 kDa gliadin appears to be a major wheat allergen among tested individuals.
Asunto(s)
Alérgenos/inmunología , Anafilaxia/diagnóstico , Gliadina/inmunología , Extractos Vegetales/inmunología , Pruebas Cutáneas , Triticum/inmunología , Hipersensibilidad al Trigo/diagnóstico , Alérgenos/administración & dosificación , Alérgenos/química , Anafilaxia/sangre , Anafilaxia/inducido químicamente , Anafilaxia/inmunología , Biomarcadores/sangre , Western Blotting , Etanol/química , Femenino , Gliadina/administración & dosificación , Gliadina/química , Humanos , Inmunoglobulina E/sangre , Lactante , Inyecciones Intradérmicas , Masculino , Peso Molecular , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Valor Predictivo de las Pruebas , Solubilidad , Solventes/química , Triticum/química , Agua/química , Hipersensibilidad al Trigo/sangre , Hipersensibilidad al Trigo/inmunologíaRESUMEN
BACKGROUND: Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). METHODS: Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. FINDINGS: The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. CONCLUSION: Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes.
Asunto(s)
Suplementos Dietéticos , Epítopos de Linfocito T/química , Gliadina/química , Péptido Hidrolasas/química , Proteolisis , Células Cultivadas , Epítopos de Linfocito T/inmunología , Gliadina/inmunología , Humanos , Péptido Hidrolasas/inmunologíaRESUMEN
Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-co-styrene sulfonate) (P(HEMA-co-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated in vivo (in rats) and in vitro (genetic toxicity studies). In vitro binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.
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Enfermedad Celíaca/inmunología , Gliadina/inmunología , Polihidroxietil Metacrilato/análogos & derivados , Poliestirenos/farmacología , Animales , Enfermedad Celíaca/tratamiento farmacológico , Enfermedad Celíaca/patología , Modelos Animales de Enfermedad , Femenino , Gliadina/metabolismo , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Transgénicos , Permeabilidad , Polihidroxietil Metacrilato/síntesis química , Polihidroxietil Metacrilato/metabolismo , Polihidroxietil Metacrilato/farmacología , Poliestirenos/síntesis química , Poliestirenos/metabolismo , Unión Proteica , Ratas , Pruebas de ToxicidadAsunto(s)
Enfermedad Celíaca/inmunología , Toxina del Cólera/metabolismo , Triticum/efectos adversos , Secuencia de Aminoácidos , Enfermedades Autoinmunes , Enfermedad Celíaca/metabolismo , Enfermedad Celíaca/prevención & control , Toxina del Cólera/inmunología , Gliadina/inmunología , Gliadina/metabolismo , Glútenes/inmunología , Haptoglobinas/inmunología , Haptoglobinas/metabolismo , Humanos , Datos de Secuencia Molecular , Precursores de ProteínasRESUMEN
BACKGROUND: The IL-15/NF-κB axis has an important role in coeliac disease (CD) and may represent a molecular target for immunomodulation. Ascorbate (vitamin C) is known to show inhibitory effects on NF-κB. Therefore, we studied if ascorbate supplementation to gliadin gliadin-stimulated biopsy culture could down-regulate the mucosal immune response to gliadin in CD. METHODS: Duodenal biopsy explants from treated CD patients were gliadin challenged in vitro (100 µg/ml) with and without 20mM ascorbate. An extra tissue explant in basal culture was used as internal control. Secretion levels of nitrites (3h), and IFNγ, TNFα, IFNα, IL-17, IL-13, and IL-6 (24h) were measured on the supernatants. IL-15 was assayed by western-blot on whole protein duodenal explants. RESULTS: The addition of ascorbate to in vitro culture gliadin-challenged biopsies blocked the secretion of nitrites (p=0.013), IFNγ (p=0.0207), TNFα (p=0.0099), IFNα (p=0.0375), and IL-6 (p=0.0036) compared to samples from non-ascorbate supplemented culture. Cytokine secretion was downregulated by ascorbate even to lower values than those observed in basal cultures (IFNγ: p=0.0312; TNFα: p=0.0312; IFNα: p=0.0312; and IL-6: p=0.0078). Gliadin-challenge induced IL-15 production in biopsies from treated CD patients, while the addition of ascorbate to culture medium completely inhibited IL-15 production. Moreover, the inhibition of IL-15 by ascorbate took place even in the only treated CD-patient who had basal IL-15 production. CONCLUSIONS: Ascorbate decreases the mucosal inflammatory response to gluten in an intestinal biopsy culture model, so it might have a role in future supplementary therapy in CD.
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Ácido Ascórbico/farmacología , Enfermedad Celíaca/tratamiento farmacológico , Gliadina/inmunología , Inflamación/prevención & control , Adulto , Anciano , Biopsia , Enfermedad Celíaca/inmunología , Citocinas/biosíntesis , Femenino , Humanos , Inmunidad Mucosa/efectos de los fármacos , Interleucina-15/antagonistas & inhibidores , Interleucina-15/fisiología , Masculino , Persona de Mediana EdadRESUMEN
Under physiological conditions the gut-associated lymphoid tissues not only prevent the induction of a local inflammatory immune response, but also induce systemic tolerance to fed antigens. A notable exception is coeliac disease, where genetically susceptible individuals expressing human leukocyte antigen (HLA) HLA-DQ2 or HLA-DQ8 molecules develop inflammatory T-cell and antibody responses against dietary gluten, a protein present in wheat. The mechanisms underlying this dysregulated mucosal immune response to a soluble antigen have not been identified. Retinoic acid, a metabolite of vitamin A, has been shown to have a critical role in the induction of intestinal regulatory responses. Here we find in mice that in conjunction with IL-15, a cytokine greatly upregulated in the gut of coeliac disease patients, retinoic acid rapidly activates dendritic cells to induce JNK (also known as MAPK8) phosphorylation and release the proinflammatory cytokines IL-12p70 and IL-23. As a result, in a stressed intestinal environment, retinoic acid acted as an adjuvant that promoted rather than prevented inflammatory cellular and humoral responses to fed antigen. Altogether, these findings reveal an unexpected role for retinoic acid and IL-15 in the abrogation of tolerance to dietary antigens.
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Adyuvantes Inmunológicos/farmacología , Enfermedad Celíaca/inmunología , Glútenes/inmunología , Interleucina-15/inmunología , Tretinoina/farmacología , Administración Oral , Adolescente , Adulto , Animales , Enfermedad Celíaca/inducido químicamente , Enfermedad Celíaca/etiología , Células Cultivadas , Niño , Preescolar , Técnicas de Cocultivo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Dieta , Factores de Transcripción Forkhead/metabolismo , Gliadina/administración & dosificación , Gliadina/inmunología , Glútenes/administración & dosificación , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Inflamación/inmunología , Interleucina-12/biosíntesis , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-15/genética , Interleucina-23/inmunología , Interleucina-23/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Receptores de Interleucina-12/deficiencia , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Tretinoina/inmunología , Adulto JovenRESUMEN
BACKGROUND: Food allergy to wheat-derived foodstuffs is on the rise. Tri a 14, a wheat flour lipid transfer protein (LTP) allergen, has been described as a major allergen associated with baker's asthma and wheat food allergy. Cross-reactivity among LTP allergens leads to the so-called 'LTP syndrome'. METHODS: Eight adult patients showing anaphylaxis after ingestion of wheat-derived foodstuffs were selected. A homemade wheat extract, purified natural (n) and recombinant (r) Tri a 14, and peach fruit and Artemisia pollen LTP allergens Pru p 3 and Art v 3 were subjected to skin prick test, specific IgE determination (ELISA) and IgE immunodetection assays. RESULTS: All tests were positive in the 8 selected patients with the homemade extract. Positive skin prick test responses to nTri a 14, Pru p 3 and Art v 3 were found in 5/8, 6/8 and 4/4 patients, respectively. Specific IgE determined by ELISA assays was detected in 6 to nTri a 14 and rTri a 14, in 4 to Pru p 3 and in 3 to Art v 3 out of 8 individual sera tested, whereas all these sera showed IgE binding to nTri a 14 and Pru p 3 in immunodetection after SDS-PAGE separation. CONCLUSIONS: Tri a 14 seems to be a relevant allergen in patients with anaphylaxis after ingestion of wheat flour foodstuffs, according to in vitro and in vivo results. Clinical history of the analyzed patients, together with sensitization to peach Pru p 3 and Artemisia pollen Art v 3, suggests that 6 of them suffer from LTP syndrome.
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Anafilaxia/inmunología , Antígenos de Plantas/inmunología , Proteínas Portadoras/inmunología , Proteínas de Plantas/inmunología , Hipersensibilidad al Trigo/inmunología , Adulto , Alérgenos/inmunología , Antígenos de Plantas/biosíntesis , Antígenos de Plantas/genética , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Femenino , Hipersensibilidad a los Alimentos/inmunología , Gliadina/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Persona de Mediana Edad , Extractos Vegetales/inmunología , Proteínas Recombinantes/inmunología , Pruebas Cutáneas , Síndrome , Triticum/química , Triticum/inmunología , Adulto JovenRESUMEN
BACKGROUND: For in vitro diagnosis of wheat allergy, specific IgE to wheat is known to be a poor predictive marker. Oral food challenge, the gold standard for the diagnosis, is accompanied by a risk of severe induced reactions. Reliable in vitro tests are needed to be developed for safe indication for oral challenge. OBJECTIVE: We examined the utility of a basophil activation marker, CD203c, for the diagnosis of IgE-mediated wheat allergy. METHODS: Fifty-eight children with suspected wheat allergy with positive CAP-FEIA to wheat were enrolled. On 70 occasions, the clinical distinction between patients with wheat allergy (WA) and patients tolerant to wheat (TW) was made by means of an oral food challenge test or recent history of immediate allergic reactions or tolerance after ingestion of wheat. Twelve replicate evaluations were performed in 9 patients over more than a 6-month interval. Thirty two patients on 43 occasions were diagnosed with WA and 27 were confirmed to be TW. One patient had both diagnoses 18 months apart. Peripheral blood was incubated with fractionated wheat extracts, purified native omega-5 gliadin (nOG5) and recombinant omega-5 gliadin (rOG5). Expression of CD203c on basophils was then analyzed by flow cytometry using a commercial kit. RESULTS: All wheat proteins induced concentration-dependent enhancement of CD203c expression in WA, but did not in TW. The analysis of receiver operating characteristics (ROC) showed that nOG5-induced CD203c(high)% values provided the best power for discriminating between WA and TW, with a sensitivity of 85.0% and specificity of 77.0% at the cut-off level of 14.4%. AUC for CD203c with nOG5 were significantly higher than that for conventional CAP-FEIA, 0.89 and 0.73, respectively (p < 0.01). CONCLUSIONS: Measurement of nOG-induced enhancement of CD203c on basophils is useful for the diagnosis of immediate wheat allergy in children.
Asunto(s)
Antígenos de Plantas/inmunología , Basófilos/metabolismo , Inmunoglobulina E/inmunología , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Hipersensibilidad al Trigo/diagnóstico , Alérgenos/genética , Alérgenos/inmunología , Antígenos de Plantas/genética , Área Bajo la Curva , Basófilos/inmunología , Preescolar , Femenino , Gliadina/genética , Gliadina/inmunología , Humanos , Inmunoglobulina E/sangre , Masculino , Extractos Vegetales/inmunología , Proteínas de Plantas/inmunología , Valor Predictivo de las Pruebas , Curva ROC , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Hipersensibilidad al Trigo/inmunologíaRESUMEN
Celiac disease (CD) is a T helper 1-driven autoimmune permanent enteropathy, triggered in susceptible individuals by the ingestion of gluten, the alcohol-soluble protein fraction of some cereals, such as wheat, rye, and barley. The only available treatment for CD is the life-long withdrawal of gluten-containing foods from the diet. Complying with gluten-free diet is difficult and affects the quality of life. Therefore, alternative therapies are being investigated. In this paper, we review a new therapeutic strategy for CD, relying upon peptides that are analogs of gliadin T-cell epitopes that show the ability to down-modulate the immune response pathogenic of CD. These peptides have been obtained artificially by amino acids substitution of gliadin T-cell stimulatory sequences and an immunomodulatory sequence has been identified in the alcohol-soluble protein fraction of cultivars of durum wheat.
Asunto(s)
Enfermedad Celíaca/inmunología , Enfermedad Celíaca/terapia , Epítopos de Linfocito T/química , Gliadina/química , Activación de Linfocitos/inmunología , Péptidos , Enfermedad Celíaca/fisiopatología , Epítopos de Linfocito T/efectos de los fármacos , Epítopos de Linfocito T/inmunología , Gliadina/genética , Gliadina/inmunología , Humanos , Inmunoterapia , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Péptidos/uso terapéutico , Linfocitos T/inmunología , Triticum/químicaRESUMEN
Wheat proteins are involved in respiratory allergy, contact allergy and food allergy. Wheat allergens involve in these pathologies are well-known. However, establishment of wheat allergy diagnostic can be sometimes difficult on account of the complex allergenic composition of skin prick test (SPT) solutions of wheat flour. Therefore, we have studied specific IgE reactivity from patient sera with wheat food allergy, and characterized allergenic composition of wheat SPT solutions by specific antibodies directed to wheat allergens. The results showed that 20 of the 25 sera analyzed contained specific IgE to at least one wheat protein fraction. Among positive sera, 75% have specific IgE to water/salt soluble fraction, 85% to native gluten fractions and 65% to wheat isolate fraction. The results showed also that SPT solutions of wheat flour contained major food allergens from each allergenic fraction. These results highlighted the importance of using fractions, which constitute the whole wheat allergenic pattern, during specific IgE reactivity analyses. Moreover, we have observed that wheat isolate extract (results of food industrial process) contained not only modified allergens (neo-allergens) involve of specific food allergy to wheat isolate but also some native allergens involve in wheat food allergy. Thus, these results showed the importance to use, for wheat in vivo diagnosis together wheat SPT solutions (gluten extract and wheat isolate) in order to differentiate wheat food allergy to specific wheat isolate allergy.
Asunto(s)
Alérgenos/efectos adversos , Antígenos de Plantas/efectos adversos , Harina/efectos adversos , Inmunoglobulina E/sangre , Extractos Vegetales , Proteínas de Plantas/efectos adversos , Triticum/efectos adversos , Hipersensibilidad al Trigo/diagnóstico , Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Especificidad de Anticuerpos , Antígenos de Plantas/inmunología , Antígenos de Plantas/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Harina/análisis , Industria de Alimentos/métodos , Gliadina/efectos adversos , Gliadina/inmunología , Glútenes/efectos adversos , Glútenes/inmunología , Glútenes/aislamiento & purificación , Humanos , Inmunoglobulina E/inmunología , Peso Molecular , Extractos Vegetales/inmunología , Extractos Vegetales/aislamiento & purificación , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Cloruro de Sodio , Solubilidad , Triticum/química , Agua , Hipersensibilidad al Trigo/sangre , Hipersensibilidad al Trigo/etiología , Hipersensibilidad al Trigo/inmunologíaRESUMEN
Coeliac disease is a common condition and its prevalence in UK is now thought to be approximately 1:100. It is being diagnosed and treated more frequently as awareness at the primary care level has increased. Coeliac disease is a complex disorder and is frequently associated with other disease processes. The management of these patients needs to take on a holistic approach, whilst the physician needs to be aware of the rare complications. This article gives an up-to-date review of the literature written on the pathogenesis of coeliac disease. We have attempted to paint a picture from beginning to end, whilst clarifying the grey areas in between. General epidemiological factors are reviewed before looking at genetic risk factors. We assess the sensitivity and specificity of the investigative modalities available for clinical use and comment on optimum management of these patients thereafter. The future of coeliac disease looks promising for patients with several novel therapies on the horizon. Whilst further work is still needed to breed out the toxic epitopes from wheat, novel therapies may come from other areas such as the work aimed at restoring normal tolerance to gluten.