Exploring the alpha-gliadin locus: the 33-mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii.

Most alpha-gliadin genes of the Gli-D2 locus on the D genome of hexaploid bread wheat (Triticum aestivum) encode for proteins with epitopes that can trigger coeliac disease (CD), and several contain a 33-mer peptide with six partly overlapping copies of three epitopes, which is regarded as a remarkably potent T-cell stimulator. To increase genetic diversity in the D genome, synthetic hexaploid wheat lines are being made by hybridising accessions of Triticum turgidum (AB genome) and Aegilops tauschii (the progenitor of the D genome). The diversity of alpha-gliadins in A. tauschii has not been studied extensively. We analysed the alpha-gliadin transcriptome of 51 A. tauschii accessions representative of the diversity in A. tauschii. We extracted RNA from developing seeds and performed 454 amplicon sequencing of the first part of the alpha-gliadin genes. The expression profile of allelic variants of the alpha-gliadins was different between accessions, and also between accessions of the Western and Eastern clades of A. tauschii. Generally, both clades expressed many allelic variants not found in bread wheat. In contrast to earlier studies, we detected the 33-mer peptide in some A. tauschii accessions, indicating that it was introduced along with the D genome into bread wheat. In these accessions, transcripts with the 33-mer peptide were present at lower frequencies than in bread wheat varieties. In most A. tauschii accessions, however, the alpha-gliadins do not contain the epitope, and this may be exploited, through synthetic hexaploid wheats, to breed bread wheat varieties with fewer or no coeliac disease epitopes.

CRISPR/Cas9 Gene Editing of Gluten in Wheat to Reduce Gluten Content and Exposure-Reviewing Methods to Screen for Coeliac Safety.

Ingestion of gluten proteins (gliadins and glutenins) from wheat, barley and rye can cause coeliac disease (CD) in genetically predisposed individuals. The only remedy is a strict and lifelong gluten-free diet. There is a growing desire for coeliac-safe, whole-grain wheat-based products, as consumption of whole-grain foods reduces the risk of chronic diseases. However, due to the large number of gluten genes and the complexity of the wheat genome, wheat that is coeliac-safe but retains baking quality cannot be produced by conventional breeding alone. CD is triggered by immunogenic epitopes, notably those present in α-, γ-, and ω-gliadins. RNA interference (RNAi) silencing has been used to down-regulate gliadin families. Recently, targeted gene editing using CRISPR/Cas9 has been applied to gliadins. These methods produce offspring with silenced, deleted, and/or edited gliadins, that overall may reduce the exposure of patients to CD epitopes. Here we review methods to efficiently screen and select the lines from gliadin gene editing programs for CD epitopes at the DNA and protein level, for baking quality, and ultimately in clinical trials. The application of gene editing for the production of coeliac-safe wheat is further considered within the context of food production and in view of current national and international regulatory frameworks.

Food processing and breeding strategies for coeliac-safe and healthy wheat products.

A strict gluten-free diet is currently the only treatment for the 1-2% of the world population who suffer from coeliac disease (CD). However, due to the presence of wheat and wheat derivatives in many food products, avoiding gluten consumption is difficult. Gluten-free products, made without wheat, barley or rye, typically require the inclusion of numerous additives, resulting in products that are often less healthy than gluten-based equivalents. Here, we present and discuss two broad approaches to decrease wheat gluten immunogenicity for CD patients. The first approach is based on food processing strategies, which aim to remove gliadins or all gluten from edible products. We find that several of the candidate food processing techniques to produce low gluten-immunogenic products from wheat already exist. The second approach focuses on wheat breeding strategies to remove immunogenic epitopes from the gluten proteins, while maintaining their food-processing properties. A combination of breeding strategies, including mutation breeding and possibly genome editing, will be necessary to produce coeliac-safe wheat. Individuals suffering from CD and people genetically susceptible who may develop CD after prolonged gluten consumption would benefit from reduced CD-immunogenic wheat. Although the production of healthy and less CD-toxic wheat varieties and food products will be challenging, increasing global demand may require these issues to be addressed in the near future by food processing and cereal breeding companies.

Oats in healthy gluten-free and regular diets: A perspective.

During the 20th century, the economic position of oats (Avena sativa L.) decreased strongly in favour of higher yielding crops including winter wheat and maize. Presently, oat represents only ~1.3% of the total world grain production, and its production system is fragmented. Nonetheless, current interest is growing because of recent knowledge on its potential benefits in food, feed and agriculture. This perspective will serve as a further impetus, with special focus on the recently valued advantages of oats in human food and health. Five approved European Food Safety Authority (EFSA) health claims apply to oats. Four relate to the oat-specific soluble fibres, the beta-glucans, and concern the maintenance and reduction of blood cholesterol, better blood glucose balance and increased faecal bulk. The fifth claim concerns the high content of unsaturated fatty acids, especially present in the endosperm, which reduces the risks of heart and vascular diseases. Furthermore, oat starch has a low glycemic index, which is favourable for weight control. Oat-specific polyphenols and avenanthramides have antioxidant and anti-inflammatory properties. Thus, oats can contribute significantly to the presently recommended whole-grain diet. Next to globulins, oats contain a small fraction of prolamin storage proteins, called 'avenins', but at a much lower quantity than gluten proteins in wheat, barley and rye. Oat avenins do not contain any of the known coeliac disease epitopes from gluten of wheat, barley and rye. Long-term food studies confirm the safety of oats for coeliac disease patients and the positive health effects of oat products in a gluten-free diet. These effects are general and independent of oat varieties. In the EU (since 2009), the USA (since 2013) and Canada (since 2015) oat products may be sold as gluten-free provided that any gluten contamination level is below 20ppm. Oats are, however, generally not gluten-free when produced in a conventional production chain, because of regular contamination with wheat, barley or rye. Therefore, establishing a separate gluten-free oat production chain requires controlling all steps in the chain; the strict conditions will be discussed. Genomic tools, including a single nucleotide polymorphism (SNP) marker array and a dense genetic map, have recently been developed and will support marker-assisted breeding. In 2015, the Oat Global initiative emerged enabling a world-wide cooperation starting with a data sharing facility on genotypic, metabolic and phenotypic characteristics. Further, the EU project TRAFOON (Traditional Food Networks) facilitated the transfer of knowledge to small- and medium-sized enterprises (SMEs) to stimulate innovations in oat production, processing, products and marketing, among others with regard to gluten-free. Finally, with focus on counteracting market fragmentation of the global oat market and production chains, interactive innovation strategies between customers (consumers) and companies through co-creation are discussed.

Why Oats Are Safe and Healthy for Celiac Disease Patients.

The water-insoluble storage proteins of cereals (prolamins) are called "gluten" in wheat, barley, and rye, and "avenins" in oat. Gluten can provoke celiac disease (CD) in genetically susceptible individuals (those with human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 serotypes). Avenins are present at a lower concentration (10%-15% of total protein content) in oat as compared to gluten in wheat (80%-85%). The avenins in the genus Avena (cultivated oat as well as various wild species of which gene bank accessions were analyzed) are free of the known CD immunogenic epitopes from wheat, barley, and rye. T cells that recognize avenin-specific epitopes have been found very rarely in CD patients. CD patients that consume oats daily do not show significantly increased levels of intraepithelial lymphocyte (EIL) cells. The safety and the positive health effects of the long-term inclusion of oats in the gluten-free diet have been confirmed in long-term studies. Since 2009 (EC 41/2009) and 2013 (FDA) oat products may be sold as gluten-free in several countries provided a gluten contamination level below 20 ppm. Introduction of oats in the gluten-free diet of celiac patients is advised after the recovery of the intestine. Health effects of oat consumption are reflected in European Food Safety Authority (EFSA)- and Food and Drug Administration (FDA)-approved health claims. Oats can form a healthy, nutritious, fiber-rich, and safe complement to the gluten-free diet.

Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing.

BACKGROUND: Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. RESULTS: A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 'unique deduced protein fragments' of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. CONCLUSIONS: The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.

The tip of the "celiac iceberg" in China: a systematic review and meta-analysis.

OBJECTIVE: Until recently, celiac disease was considered to be rare in China. We aimed to estimate its true status. METHODS: By searching the MEDLINE database and four Chinese full-text databases (CNKI, CBM, VIP and WANFANG) (up to August 2012), as well as two HLA allele frequency net databases and the Chinese Statistics Yearbook databases, we systematically reviewed the literature on definite and suspected cases of celiac disease, the predisposing HLA allele frequencies, and on gluten exposure in China. Meta-analysis was performed by analyzing DQ2, DQ8 and DQB1*0201 gene frequencies and heterogeneity in populations from different geographic regions and ethnicities in China. RESULTS: At present, the number of reported celiac disease cases is extremely low in China. The frequencies of the HLA-DQ2.5 and HLA-DQ8 haplotypes were 3.4% (95% confidence interval 1.3-5.5%) and 2.1% (0.1-4.1%), respectively. HLA-DQ2 and HLA-DQ8 antigen frequencies were 18.4% (15.0-21.7%) and 8.0% (4.5-11.4%), respectively. The frequency of the DQB1*0201 allele was 10.5% (9.3-11.6%) and it was more common in the northern Chinese than in the southern Chinese populations. The chance of being exposed to gluten is rapidly increasing all over China nowadays. CONCLUSION: The data on HLA haplotyping, in conjunction with increasing wheat consumption, strongly suggests that the occurrence of celiac disease is more common in China than currently reported. Coordinated measures by the Chinese government, medical and agricultural research institutions, and food industries, would be justified to create more awareness about celiac disease and to prevent it becoming a medical and societal burden.

Expansion of the gamma-gliadin gene family in Aegilops and Triticum.

BACKGROUND: The gamma-gliadins are considered to be the oldest of the gliadin family of storage proteins in Aegilops/Triticum. However, the expansion of this multigene family has not been studied in an evolutionary perspective. RESULTS: We have cloned 59 gamma-gliadin genes from Aegilops and Triticum species (Aegilops caudata L., Aegilops comosa Sm. in Sibth. & Sm., Aegilops mutica Boiss., Aegilops speltoides Tausch, Aegilops tauschii Coss., Aegilops umbellulata Zhuk., Aegilops uniaristata Vis., and Triticum monococcum L.) representing eight different genomes: Am, B/S, C, D, M, N, T and U. Overall, 15% of the sequences contained internal stop codons resulting in pseudogenes, but this percentage was variable among genomes, up to over 50% in Ae. umbellulata. The most common length of the deduced protein, including the signal peptide, was 302 amino acids, but the length varied from 215 to 362 amino acids, both obtained from Ae. speltoides. Most genes encoded proteins with eight cysteines. However, all Aegilops species had genes that encoded a gamma-gliadin protein of 302 amino acids with an additional cysteine. These conserved nine-cysteine gamma-gliadins may perform a specific function, possibly as chain terminators in gluten network formation in protein bodies during endosperm development. A phylogenetic analysis of gamma-gliadins derived from Aegilops and Triticum species and the related genera Lophopyrum, Crithopsis, and Dasypyrum showed six groups of genes. Most Aegilops species contained gamma-gliadin genes from several of these groups, which also included sequences from the genera Lophopyrum, Crithopsis, and Dasypyrum. Hordein and secalin sequences formed separate groups. CONCLUSIONS: We present a model for the evolution of the gamma-gliadins from which we deduce that the most recent common ancestor (MRCA) of Aegilops/Triticum-Dasypyrum-Lophopyrum-Crithopsis already had four groups of gamma-gliadin sequences, presumably the result of two rounds of duplication of the locus.

Celiac disease T-cell epitopes from gamma-gliadins: immunoreactivity depends on the genome of origin, transcript frequency, and flanking protein variation.

BACKGROUND: Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains. RESULTS: A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone. CONCLUSIONS: Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).

Proteomic analysis of the major birch allergen Bet v 1 predicts allergenicity for 15 birch species.

Pollen of the European and Asian white birch (Betula pendula and B. platyphylla) causes hay fever in humans. The allergenic potency of other birch species is largely unknown. To identify birch trees with a reduced allergenicity, we assessed the immunochemical characteristics of 15 species and two hybrids, representing four subgenera within the genus Betula, while focusing on the major pollen allergen Bet v 1. Antigenic and allergenic profiles of pollen extracts from these species were evaluated by SDS-PAGE and Western blot using pooled sera of birch-allergic individuals. Tryptic digests of the Bet v 1 bands were analyzed by LC-MS(E) to determine the abundance of various Bet v 1 isoforms. Bet v 1 was the most abundant pollen protein across all birch species. LC-MS(E) confirmed that pollen of all species contained a mixture of multiple Bet v 1 isoforms. Considerable differences in Bet v 1 isoform composition exist between birch species. However, isoforms that are predicted to have a high IgE-reactivity prevailed in pollen of all species. Immunoblotting confirmed that all pollen extracts were similar in immune-reactivity, implying that pollen of all birch species is likely to evoke strong allergic reactions.

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.

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen.

BACKGROUND: Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MSE was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome. RESULTS: All examined birch species contained several PR-10 genes. In total, 134 unique sequences were recovered. Sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. Five subfamilies were common to all birch species. Genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. Isoforms that were similar to isoforms with a high IgE-reactivity (Bet v 1a = PR-10.01A01) were abundant in all species except B. lenta, while the hypoallergenic isoform Bet v 1d (= PR-10.01B01) was only found in B. pendula and its closest relatives. CONCLUSION: Q-TOF LC-MSE allows efficient screening of Bet v 1 isoforms by determining the presence and relative abundance of these isoforms in pollen. B. pendula contains a Bet v 1-mixture in which isoforms with a high and low IgE-reactivity are both abundant. With the possible exception of B. lenta, isoforms identical or very similar to those with a high IgE-reactivity were found in the pollen proteome of all examined birch species. Consequently, these species are also predicted to be allergenic with regard to Bet v 1 related allergies.

Tetraploid and hexaploid wheat varieties reveal large differences in expression of alpha-gliadins from homoeologous Gli-2 loci.

BACKGROUND: Alpha-gliadins form a multigene protein family encoded by multiple alpha-gliadin (Gli-2) genes at three genomic loci, Gli-A2, Gli-B2 and Gli-D2, respectively located on the homoeologous wheat chromosomes 6AS, 6BS, and 6DS. These proteins contain a number of important celiac disease (CD)-immunogenic domains. The alpha-gliadins expressed from the Gli-B2 locus harbour fewer conserved CD-epitopes than those from Gli-A2, whereas the Gli-D2 gliadins have the highest CD-immunogenic potential. In order to detect differences in the highly CD-immunogenic alpha-gliadin fraction we determined the relative expression level from the homoeologous Gli-2 loci in various tetraploid and hexaploid wheat genotypes by using a quantitative pyrosequencing method and by analyzing expressed sequence tag (EST) sequences. RESULTS: We detected large differences in relative expression levels of alpha-gliadin genes from the three homoeologous loci among wheat genotypes, both as relative numbers of expressed sequence tag (EST) sequences from specific varieties and when using a quantitative pyrosequencing assay specific for Gli-A2 genes. The relative Gli-A2 expression level in a tetraploid durum wheat cultivar ('Probstdorfer Pandur') was 41%. In genotypes derived from landraces, the Gli-A2 frequency varied between 12% and 58%. In some advanced hexaploid bread wheat cultivars the genes from locus Gli-B2 were hardly expressed (e.g., less than 5% in 'Lavett') but in others they made up more than 40% (e.g., in 'Baldus'). CONCLUSION: Here, we have shown that large differences exist in relative expression levels of alpha-gliadins from the homoeologous Gli-2 loci among wheat genotypes. Since the homoelogous genes differ in the amount of conserved CD-epitopes, screening for differential expression from the homoeologous Gli-2 loci can be employed for the pre-selection of wheat varieties in the search for varieties with very low CD-immunogenic potential. Pyrosequencing is a method that can be employed for such a 'gene family-specific quantitative transcriptome profiling'.

Assessment of allelic diversity in intron-containing Mal d 1 genes and their association to apple allergenicity.

BACKGROUND: Mal d 1 is a major apple allergen causing food allergic symptoms of the oral allergy syndrome (OAS) in birch-pollen sensitised patients. The Mal d 1 gene family is known to have at least 7 intron-containing and 11 intronless members that have been mapped in clusters on three linkage groups. In this study, the allelic diversity of the seven intron-containing Mal d 1 genes was assessed among a set of apple cultivars by sequencing or indirectly through pedigree genotyping. Protein variant constitutions were subsequently compared with Skin Prick Test (SPT) responses to study the association of deduced protein variants with allergenicity in a set of 14 cultivars. RESULTS: From the seven intron-containing Mal d 1 genes investigated, Mal d 1.01 and Mal d 1.02 were highly conserved, as nine out of ten cultivars coded for the same protein variant, while only one cultivar coded for a second variant. Mal d 1.04, Mal d 1.05 and Mal d 1.06 A, B and C were more variable, coding for three to six different protein variants. Comparison of Mal d 1 allelic composition between the high-allergenic cultivar Golden Delicious and the low-allergenic cultivars Santana and Priscilla, which are linked in pedigree, showed an association between the protein variants coded by the Mal d 1.04 and -1.06A genes (both located on linkage group 16) with allergenicity. This association was confirmed in 10 other cultivars. In addition, Mal d 1.06A allele dosage effects associated with the degree of allergenicity based on prick to prick testing. Conversely, no associations were observed for the protein variants coded by the Mal d 1.01 (on linkage group 13), -1.02, -1.06B, -1.06C genes (all on linkage group 16), nor by the Mal d 1.05 gene (on linkage group 6). CONCLUSION: Protein variant compositions of Mal d 1.04 and -1.06A and, in case of Mal d 1.06A, allele doses are associated with the differences in allergenicity among fourteen apple cultivars. This information indicates the involvement of qualitative as well as quantitative factors in allergenicity and warrants further research in the relative importance of quantitative and qualitative aspects of Mal d 1 gene expression on allergenicity. Results from this study have implications for medical diagnostics, immunotherapy, clinical research and breeding schemes for new hypo-allergenic cultivars.

Measurement of lipid transfer protein in 88 apple cultivars.

BACKGROUND: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars. OBJECTIVE: Determination of the levels of LTP in a wide range of apple cultivars. METHODS: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC). RESULTS: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson's correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4-253.2 (sELISA), 2.7-120.2 (cELISA) and 0.4-47.3 microg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels. CONCLUSIONS: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes.

BACKGROUND: Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the alpha-gliadins contain several peptides that are associated to the disease. RESULTS: We obtained 230 distinct alpha-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All alpha-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, alpha-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-alpha9 and glia-alpha20, but never the intact epitopes glia-alpha and glia-alpha2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific. CONCLUSION: Our analysis shows that alpha-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxicity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.

In vivo assessment with prick-to-prick testing and double-blind, placebo-controlled food challenge of allergenicity of apple cultivars.

BACKGROUND: Apple cultivars have been reported to differ in allergenicity on the basis of in vitro and skin prick tests with apple extracts. OBJECTIVES: We sought to evaluate the efficacy of the prick-to-prick method in assessing differences in allergenicity of apple cultivars and to confirm differences by means of double-blind, placebo-controlled food challenge (DBPCFC). METHODS: Intra-assay and intracultivar variation of prick-to-prick test results were determined in 6 Dutch and 8 Spanish patients with apple allergy by using 5 apples of the cultivars Golden Delicious, Fuji, and Ecolette in duplicate. In addition, 21 cultivars were screened for allergenicity in 15 Dutch patients with birch pollen and apple allergy. Two selected cultivars (Golden Delicious and Santana) were tested with DBPCFCs. The influence of storage conditions on allergenicity was assessed in 5 cultivars. RESULTS: Intra-assay variation of skin prick testing was 3.9%, and intracultivar variation was 4.1%. A ranking of 21 cultivars was made on the basis of prick-to-prick tests in 9 patients. Apple cultivars were classified as of low, intermediate, and high allergenicity, with a significant difference between low and high allergenicity (P < .001). A significant difference in allergenicity determined between Golden Delicious and Santana cultivars (P < .05) was confirmed by means of DBPCFC. With 5 cultivars, controlled atmosphere (2.5% oxygen/1% carbon dioxide) was shown to reduce allergenicity (P < .001) by 15% compared with storage at 2 degrees C. CONCLUSIONS: Prick-to-prick testing with fresh apples is a reproducible method of assessing allergenicity. Apples can be classified as of low or high allergenicity for the majority of patients. This was confirmed by using DBPCFCs. Selection of cultivars and control of storage conditions are both viable strategies for reduction of symptoms in patients with apple allergy.

Natural variation in toxicity of wheat: potential for selection of nontoxic varieties for celiac disease patients.

BACKGROUND & AIMS: Celiac disease (CD) is an intestinal disorder caused by T-cell responses to peptides derived from the gluten proteins present in wheat. Such peptides have been found both in the gliadin and glutenin proteins in gluten. The only cure for CD is a lifelong gluten-free diet. It is unknown, however, if all wheat varieties are equally harmful for patients. We investigated whether wheat varieties exist with a natural low number of T-cell-stimulatory epitopes. METHODS: Gluten proteins present in public databases were analyzed for the presence of T-cell-stimulatory sequences. In addition, wheat accessions from diploid (AA, SS/BB, and DD genomes), tetraploid (AABB), and hexaploid (AABBDD) Triticum species were tested for the presence of T-cell-stimulatory epitopes in gliadins and glutenins by both T-cell and monoclonal antibody-based assays. RESULTS: The database analysis readily identified gluten proteins that lack 1 or more of the known T-cell-stimulatory sequences. Moreover, both the T-cell- and antibody-based assays showed that a large variation exists in the amount of T-cell-stimulatory peptides present in the wheat accessions. CONCLUSIONS: Sufficient genetic variation is present to endeavor the selection of wheat accessions that contain low amounts of T-cell-stimulatory sequences. Such materials may be used to select and breed wheat varieties suitable for consumption by CD patients, contributing to a well-balanced diet and an increase in their quality of life. Such varieties also may be useful for disease prevention in individuals at risk.

Silencing the major apple allergen Mal d 1 by using the RNA interference approach.

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.