Presence of celiac disease epitopes in modern and old hexaploid wheat varieties: wheat breeding may have contributed to increased prevalence of celiac disease.

Gluten proteins from wheat can induce celiac disease (CD) in genetically susceptible individuals. Specific gluten peptides can be presented by antigen presenting cells to gluten-sensitive T-cell lymphocytes leading to CD. During the last decades, a significant increase has been observed in the prevalence of CD. This may partly be attributed to an increase in awareness and to improved diagnostic techniques, but increased wheat and gluten consumption is also considered a major cause. To analyze whether wheat breeding contributed to the increase of the prevalence of CD, we have compared the genetic diversity of gluten proteins for the presence of two CD epitopes (Glia-α9 and Glia-α20) in 36 modern European wheat varieties and in 50 landraces representing the wheat varieties grown up to around a century ago. Glia-α9 is a major (immunodominant) epitope that is recognized by the majority of CD patients. The minor Glia-α20 was included as a technical reference. Overall, the presence of the Glia-α9 epitope was higher in the modern varieties, whereas the presence of the Glia-α20 epitope was lower, as compared to the landraces. This suggests that modern wheat breeding practices may have led to an increased exposure to CD epitopes. On the other hand, some modern varieties and landraces have been identified that have relatively low contents of both epitopes. Such selected lines may serve as a start to breed wheat for the introduction of 'low CD toxic' as a new breeding trait. Large-scale culture and consumption of such varieties would considerably aid in decreasing the prevalence of CD.

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines.

BACKGROUND: Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4+ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD). RESULTS: The effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of Triticum aestivum cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the alpha-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the omega-gliadin, gamma-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties. CONCLUSION: The consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties.

Staining efficiency of specific proteins depends on the staining method: wheat gluten proteins.

To analyze gluten proteins involved in celiac disease (CD) by proteomic analysis, prolamins extracted from hexaploid wheat varieties were analyzed by SDS-PAGE and 2-DE. Differences between staining methods (CBB, silver nitrate, SYPRO Ruby, and CyDye) were analyzed in comparison to immunoblotting. Staining efficiency varied per protein across methods, and complete staining of all gluten proteins could not be achieved by one of these methods. Care should be taken in the selection of staining method especially if one wants to relate the results to data obtained by immunoblotting.

Seven different genes encode a diverse mixture of isoforms of Bet v 1, the major birch pollen allergen.

BACKGROUND: Pollen of the European white birch (Betula pendula, syn. B. verrucosa) is an important cause of hay fever. The main allergen is Bet v 1, member of the pathogenesis-related class 10 (PR-10) multigene family. To establish the number of PR-10/Bet v 1 genes and the isoform diversity within a single tree, PCR amplification, cloning and sequencing of PR-10 genes was performed on two diploid B. pendula cultivars and one interspecific tetraploid Betula hybrid. Sequences were attributed to putative genes based on sequence identity and intron length. Information on transcription was derived by comparison with homologous cDNA sequences available in GenBank/EMBL/DDJB. PCR-cloning of multigene families is accompanied by a high risk for the occurrence of PCR recombination artifacts. We screened for and excluded these artifacts, and also detected putative artifact sequences among database sequences. RESULTS: Forty-four different PR-10 sequences were recovered from B. pendula and assigned to thirteen putative genes. Sequence homology suggests that three genes were transcribed in somatic tissue and seven genes in pollen. The transcription of three other genes remains unknown. In total, fourteen different Bet v 1-type isoforms were identified in the three cultivars, of which nine isoforms were entirely new. Isoforms with high and low IgE-reactivity are encoded by different genes and one birch pollen grain has the genetic background to produce a mixture of isoforms with varying IgE-reactivity. Allergen diversity is even higher in the interspecific tetraploid hybrid, consistent with the presence of two genomes. CONCLUSION: Isoforms of the major birch allergen Bet v 1 are encoded by multiple genes, and we propose to name them accordingly. The present characterization of the Bet v 1 genes provides a framework for the screening of specific Bet v 1 genes among other B. pendula cultivars or Betula species, and for future breeding for trees with a reduced allergenicity. Investigations towards sensitization and immunotherapy should anticipate that patients are exposed to a mixture of Bet v 1 isoforms of different IgE-reactivity, even if pollen originates from a single birch tree.

Profiling of Nutritional and Health-Related Compounds in Oat Varieties.

The use of oats in the human diet has decreased over the past 70 years. This is an unfortunate development from the perspective of human health because oats have a high nutritional value and contain many compounds, including ß-glucan, polyphenols, vitamins, and unsaturated fatty acids that are able to maintain or may even improve consumer's health. In addition, oats fit into a gluten-free diet of celiac disease patients because they lack the T-cell stimulating epitopes from wheat, rye, and barley. We focused on the presence of health-related compounds in oats and how their levels vary among varieties in response to the type of soil. Ten oat varieties were grown in the Netherlands in sandy and clay soil and were analyzed for the presence and concentration of healthy compounds (ß-glucan, fatty acids, vitamin E, and antioxidant activity), avenin composition, total protein and starch content, and agronomical characteristics. Principal component analysis showed that genetic background influenced the levels of all analyzed components. Protein, starch, ß-glucan, and antioxidants were also affected by the type of soil. The obtained results showed that this kind of analysis can be used to profile oat varieties in general and enables the selection of specific varieties with specific compound characteristics.

In search of tetraploid wheat accessions reduced in celiac disease-related gluten epitopes.

Tetraploid wheat (durum wheat) is mainly used for the preparation of pasta. As a result of breeding, thousands of tetraploid wheat varieties exist, but also tetraploid landraces are still maintained and used for local food preparations. Gluten proteins present in wheat can induce celiac disease, a T-cell mediated auto-immune disorder, in genetically predisposed individuals after ingestion. Compared to hexaploid wheat, tetraploid wheat might be reduced in T-cell stimulatory epitopes that cause celiac disease because of the absence of the D-genome. We tested gluten protein extracts from 103 tetraploid wheat accessions (obtained from the Dutch CGN genebank and from the French INRA collection) including landraces, old, modern, and domesticated accessions of various tetraploid species and subspecies from many geographic origins. Those accessions were typed for their level of T-cell stimulatory epitopes by immunoblotting with monoclonal antibodies against the α-gliadin epitopes Glia-α9 and Glia-α20. In the first selection, we found 8 CGN and 6 INRA accessions with reduced epitope staining. Fourteen of the 57 CGN accessions turned out to be mixed with hexaploid wheat, and 5 out of the 8 selected CGN accessions were mixtures of two or more different gluten protein chemotypes. Based on single seed analysis, lines from two CGN accessions and one INRA accession were obtained with significantly reduced levels of Glia-α9 and Glia-α20 epitopes. These lines will be further tested for industrial quality and may contribute to the development of safer foods for celiac patients.

A modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheat.

The detection, analysis, and quantification of individual celiac disease (CD) immune responsive gluten proteins in wheat and related cereals (barley, rye) require an adequate and reliable extraction protocol. Because different types of gluten proteins behave differently in terms of solubility, currently different extraction protocols exist. The performance of various documented gluten extraction protocols is evaluated for specificity and completeness by gel electrophoresis (SDS-PAGE), immunoblotting and RIDASCREEN Gliadin competitive ELISA. Based on these results, an optimized, two-step extraction protocol has been developed.