Changes in gut microbiota due to supplemented fatty acids in diet-induced obese mice.

Consumption of a high-fat diet (HFD), which is associated with chronic 'low-grade' systemic inflammation, alters the gut microbiota (GM). The aim of the present study was to investigate the ability of an oleic acid-derived compound (S1) and a combination of n-3 fatty acids (EPA and DHA, S2) to modulate both body weight and the GM in HFD-induced obese mice. A total of eighty mice were fed either a control diet or a HFD, non-supplemented or supplemented with S1 or S2. At week 19, faeces were collected in order to analyse the GM. Group-specific primers for accurate quantification of several major bacterial groups from faecal samples were assayed using quantitative PCR. The HFD induced an increase in body weight, which was reduced by supplementation with S1. Furthermore, S1 supplementation markedly increased total bacterial density and restored the proportions of bacteria that were increased (i.e. clostridial cluster XIVa and Enterobacteriales) or decreased (i.e. Bifidobacterium spp.) during HFD feeding. S2 supplementation significantly increased the quantities of Firmicutes (especially the Lactobacillus group). Correlation analysis revealed that body weight correlated positively with the phylum Firmicutes and clostridial cluster XIVa, and negatively with the phylum Bacteroidetes. In conclusion, the consumption of a HFD induced changes in the faecal microbiota, which were associated with the appearance of an obese phenotype. Supplementation of the HFD with S1 counteracted HFD-induced gut dysbiosis, together with an improvement in body weight. These data support a role for certain fatty acids as interesting nutrients related to obesity prevention.

Influence of breastfeeding versus formula feeding on lymphocyte subsets in infants at risk of coeliac disease: the PROFICEL study.

PURPOSE: In addition to genetic risk, environmental factors might influence coeliac disease (CD) development. We sought to assess the effect of the interaction between milk-feeding practices and the HLA-DQ genotype on peripheral lymphocyte subsets and their activation markers in infants at familial risk for CD. METHODS: 170 newborns were classified in 3 different genetic risk groups (high risk, HR; intermediate risk, IR; and low risk, LR) after DQB1 and DQA1 typing. Lymphocyte subsets were studied at the age of 4 months by flow cytometry analysis. RESULTS: 79 infants were receiving exclusive breastfeeding (BF) and 91 partial breastfeeding or formula feeding (FF). Regarding genetic risk, 40 infants were classified in HR group, 75 in IR group and 55 in LR group. Two-way ANOVA did not show significant interactions between the type of milk feeding and genetic risk group on the lymphocyte subsets analysed. One-way ANOVA for milk-feeding practice alone showed that the percentage of CD4 + CD25+ cells was significantly higher in BF group than in FF group (BF, 10.92 ± 2.71; FF, 9.94 ± 2.96; p = 0.026), and absolute counts of CD4 + CD38+ cells were significantly higher in FF group than in BF group (FF, 2,881.23 ± 973.48; BF, 2,557.95 ± 977.06; p = 0.038). One-way ANOVA for genetic risk alone showed that absolute counts of NK cells were significantly higher in IR group than HR and LR groups (IR, 539.24 ± 340.63; HR, 405.01 ± 239.53; LR, 419.86 ± 262.85; p = 0.028). CONCLUSION: Lymphocyte subset profiles in the early stages of life could be modulated by milk-feeding practices and genetic risk separately. Breastfeeding might have a positive immunomodulatory effect on lymphocyte subsets in infants at risk of CD.

Immune development and intestinal microbiota in celiac disease.

Celiac disease (CD) is an immune-mediated enteropathy, triggered by dietary wheat gluten and similar proteins of barley and rye in genetically susceptible individuals. The etiology of this disorder is complex, involving both environmental and genetic factors. The major genetic risk factor for CD is represented by HLA-DQ genes, which account for approximately 40% of the genetic risk; however, only a small percentage of carriers develop the disease. Gluten is the main environmental factor responsible for the signs and symptoms of the disease, but exposure to gluten does not fully explain the manifestation of CD. Epidemiological and clinical data suggest that environmental factors other than gluten might play a role in disease development, including early feeding practices (e.g., breast milk versus formula and duration of breastfeeding), infections, and alterations in the intestinal microbiota composition. Herein, we review what is known about the influence of dietary factors, exposure to infectious agents, and intestinal microbiota composition, particularly in early life, on the risk of developing CD, as well as the possible dietary strategies to induce or increase gluten tolerance.

Validation of a new enzyme-linked immunosorbent assay to detect the triggering proteins and peptides for celiac disease: interlaboratory study.

The performance of Gluten-Tec (EuroProxima, Arnhem, The Netherlands) was tested through an interlaboratory study in accordance with AOAC guidelines. Gluten-Tec is a competitive ELISA that detects an immunostimulatory epitope of a-gliadin in dietary food for celiacs. Fifteen laboratories, representing 14 different countries, announced their interest in taking part in this study. Of the 12 laboratories that sent the results within the established timeframe, two submitted inappropriate standard curves and were excluded from the statistical analysis. Four different food matrixes (rice-based baby food, maize bread, chocolate cake mix, and beer) were selected for preparing the test samples. Two gliadin extraction procedures were used: the conventional 60% ethanol, and a new method based on the reducing reagent dithiothreitol. The 38 samples (19 blind duplicates) tested in this study were prepared by diluting the different extracts in order to cover a wide range of gliadin levels. Both sample extraction and dilution were performed by EuroProxima; the present interlaboratory study was focused only on testing the ELISA part of the Gluten-Tec kit protocol. Repeatability values (within-laboratory variance), expressed as RSD(r) ranged from 6.2 to 25.7%, while reproducibility values (interlaboratory variance), expressed as RSD(R), ranged from 10.6 to 45.9%. Both statistical parameters were in the acceptable range of ELISAs under these conditions, and the method will be presented to the Codex Alimentarius as a preferred method for gluten analysis.

Sourdough Biotechnology Applied to Gluten-Free Baked Goods: Rescuing the Tradition.

Recent studies suggest that the beneficial properties provided by sourdough fermentation may be translated to the development of new GF products that could improve their technological and nutritional properties. The main objective of this manuscript is to review the current evidence regarding the elaboration of GF baked goods, and to present the latest knowledge about the so-called sourdough biotechnology. A bibliographic search of articles published in the last 12 years has been carried out. It is common to use additives, such as hydrocolloids, proteins, enzymes, and emulsifiers, to technologically improve GF products. Sourdough is a mixture of flour and water fermented by an ecosystem of lactic acid bacteria (LAB) and yeasts that provide technological and nutritional improvements to the bakery products. LAB-synthesized biopolymers can mimic gluten molecules. Sourdough biotechnology is an ecological and cost-effective technology with great potential in the field of GF products. Further research is necessary to optimize the process and select species of microorganisms robust enough to be competitive in any circumstance.

Measurement of wheat gluten and barley hordeins in contaminated oats from Europe, the United States and Canada by Sandwich R5 ELISA.

OBJECTIVES: We have investigated the extent of contamination with wheat, barley, rye or a mixture of these cereals in a large number of grains and commercial oats. We have also attempted to identify the type of cereal contaminant. METHODS: Sandwich R5 ELISA (using either gliadins or hordeins as standards), western blot, matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and quantitative real-time PCR (Q-PCR) techniques have been used to analyze a total of 134 oats, comprising grains and commercial oat products collected from Europe, the United States and Canada. RESULTS: Twenty-five of the 134 pure, uncontaminated oat varieties were found to have undetectable levels of gluten, whereas most of the 109 grains and commercial oat products were mainly contaminated with mixtures of wheat, barley and rye, barley being the predominant contaminant. The percentages of these cereals in the oat samples have been calculated by specific wheat, barley and rye Q-PCR systems. The oat samples were grouped according to the avenin spectra determined by the mass spectrometric technique. The data confirmed that contaminated oat foods, based on the same variety, could have different levels of wheat, barley and rye contamination. CONCLUSION: This study has verified that contamination with wheat gliadins or barley hordeins in oat samples can be measured by the Sandwich R5 ELISA, using either gliadins or hordeins as standards, and also the importance of using confirmatory techniques (such as western blot, Q-PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) to confirm that most oats are contaminated with mixtures of wheat, barley and rye.

T-cell receptor recognition of HLA-DQ2-gliadin complexes associated with celiac disease.

Celiac disease is a T cell-mediated disease induced by dietary gluten, a component of which is gliadin. 95% of individuals with celiac disease carry the HLA (human leukocyte antigen)-DQ2 locus. Here we determined the T-cell receptor (TCR) usage and fine specificity of patient-derived T-cell clones specific for two epitopes from wheat gliadin, DQ2.5-glia-α1a and DQ2.5-glia-α2. We determined the ternary structures of four distinct biased TCRs specific for those epitopes. All three TCRs specific for DQ2.5-glia-α2 docked centrally above HLA-DQ2, which together with mutagenesis and affinity measurements provided a basis for the biased TCR usage. A non-germline encoded arginine residue within the CDR3ß loop acted as the lynchpin within this common docking footprint. Although the TCRs specific for DQ2.5-glia-α1a and DQ2.5-glia-α2 docked similarly, their interactions with the respective gliadin determinants differed markedly, thereby providing a basis for epitope specificity.

Influence of early environmental factors on lymphocyte subsets and gut microbiota in infants at risk of celiac disease; the PROFICEL study.

INTRODUCTION: It is known that the HLA genotype can explain about a 40% of the genetic risk of celiac disease (CD), thus, other genetic predisposing factors as well as factors that subtly modulate T cell activation and differentiation need to be studied. This includes environmental factors that are currently believed to impact on the immune system and gut microbiota development. AIM: To assess the associations between early environmental factors (EEF), lymphocyte subsets, and intestinal microbiota composition in infants at familial risk for CD. STUDY DESIGN: Prospective observational study. SUBJECTS: Fifty-five 4 month-old infants with at least a first-degree relative suffering CD. Infants were classified according to type of delivery, mother's antibiotic intake during pregnancy and during labor, milk-feeding practices, early infections and antibiotic intake, rotavirus vaccine administration, and allergy incidence within the first 18 months of life. METHODS: Lymphocyte subsets and gut microbiota composition were studied at the age of 4 months. RESULTS: Formula feeding and infant's infections were associated with higher CD3+, CD4+, CD4+CD38+, CD4+CD28+ and CD3+CD4+CD45RO+ counts (P0.01). Infant s infections were also associated with higher CD4+CD25+, CD4+HLA-DR+ and NK cell counts (P0.01). Cesarean delivery and rotavirus vaccine administration were associated with lower percentage of CD4+CD25+ cells. Infant's antibiotic intake was associated and correlated with lower counts of Bifidobacterium longum and higher counts of Bacteroides fragilis group. CONCLUSIONS: Infant s infections and antibiotic intake in the first 4 months of life are the EEF more strongly and/or frequently associated to lymphocyte subpopulations and microbiota composition, respectively, in infants at risk of CD.

Introducción: Es bien sabido que el genotipo HLA puede explicar un 40% del riesgo genético de enfermedad celíaca, por lo que otros factores de predisposición genéticos así como factores que sutilmente modulen la activación y diferenciación de células T necesitan ser estudiados. Esto incluye factores ambientales, de los que se cree actualmente que ejercen un efecto sobre el desarrollo del sistema inmune y la microbiota intestinal. Objetivo: Evaluar las asociaciones entre factores ambientales tempranos, las subpoblaciones de linfocitos y la composición de la microbiota intestinal en niños con riesgo familiar de enfermedad celíaca. Diseño del estudio: Estudio prospectivo observacional Sujetos: 55 niños de 4 meses de edad con al menos un familiar celíaco de primer grado. Los niños fueron clasificados de acuerdo al tipo de parto, ingesta materna de antibióticos durante el embarazo y durante el parto, tipo de lactancia, infecciones tempranas y toma de antibióticos, administración de la vacuna de rotavirus, y incidencia de alergias dentro de los 18 primeros meses de vida. Métodos: Las subpoblaciones de linfocitos y la composición de la microbiota intestinal fueron estudiadas a la edad de 4 meses. Resultados: La lactancia de fórmula y las infecciones tempranas se asociaron con un mayor número absoluto de células CD3+, CD4+, CD4+CD38+, CD4+CD28+ y CD3+CD4+CD45RO+ (P0.01). El parto por cesárea y la administración de la vacuna de rotavirus se asociaron a un menor porcentaje de células CD4+CD25+. La toma temprana de antibióticos se asoció y correlacionó con menor número de Bifidobacterium longum y mayor número de Bacteroides fragilis. Conclusiones: Las infecciones y la toma de antibióticos en los primeros 4 meses de edad son los factores ambientales tempranos más fuertemente y/o frecuentemente asociados a las subpoblaciones de linfocitos y la composición de la microbiota, respectivamente, en niños con riesgo de enfermedad celíaca.

A universal approach to eliminate antigenic properties of alpha-gliadin peptides in celiac disease.

Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the α-gliadins. It has been shown that α-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from α-gliadins. We have analyzed over 3,000 expressed α-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived α-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the α-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations α-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients.