M.F. Schenk

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

BackgroundBread 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 α-gliadins contain several peptides that are associated to the disease.ResultsWe obtained 230 distinct α-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 α-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, α-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-α9 and glia-α20, but never the intact epitopes glia-α and glia-α2. 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.ConclusionOur analysis shows that α-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.

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

BackgroundPollen 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.ResultsForty-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.ConclusionIsoforms 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.

Phylogenetic relationships in Betula (Betulaceae) based on AFLP markers

The genus Betula comprises various species in boreal and temperate climate zones of the Northern Hemisphere. The taxonomy of Betula is controversial and complicated by parallel evolution of morphological traits, polyploidization events, and extensive hybridization and introgression among species. Multilocus molecular data from AFLPs were used to provide phylogenetic information. A large number of polymorphic markers (321 variable bands) were produced in 107 Betula accessions from 23 species and 11 hybrids. The AFLP results were largely congruent with the results from previously examined nuclear DNA markers. Four distinct subgenera were identified within the genus Betula. These subgenera were partly in disagreement with the traditional (but disputed) division of the genus. In addition, the results indicated several groups of conspecific taxa. The majority of the species fell within subgenus Betula and shared a high degree of similarity with B. pendula. All hybrids were associated with this group, and the AFLP data contained signals on putative parents for some of the interspecific hybrids. Subgenus Chamaebetula and part of the Neurobetula species should be merged with Betula. The subgenera Betulenta, Betulaster, and the remaining part of Neurobetula are distinct and well supported. Although our results indicate that four major taxonomic groups can be recognized within the genus Betula, the relationship between them remains unclear. This may be due to the occurrence of hybridization and introgression, which would have a homogenizing effect on the relationships between species. Naturally occurring Betula species of hybrid origin may explain the low bootstrap values within the Betula clade.

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

BackgroundBet 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.ResultsAll 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.ConclusionQ-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.

Impact of Urbanization on the Proteome of Birch Pollen and Its Chemotactic Activity on Human Granulocytes

Background: Epidemiologic studies reveal a dramatic increase in allergies in the last decades. Air pollution is considered to be one of the factors responsible for this augmentation. The aim of this study was to analyze the impact of urbanization on birch pollen. The birch pollen proteome was investigated in order to identify differences in protein abundance between pollen from rural and urban areas. The allergenicity of birch pollen from both areas was evaluated by assessing its chemotactic potency as well as its protein and allergen contents. Methods: Difference gel electrophoresis (DIGE) was used to analyze the pollen proteome. The chemotactic activity of aqueous pollen extracts was determined by migration assays of human neutrophils. Results: DIGE revealed 26 differences in protein spot intensity between pollen from urban and rural areas. One of these proteins was identified by de novo sequencing as the 14-3-3 protein, which resembles a stress-induced factor in other plant species. Furthermore, extracts from pollen collected in urban areas had higher chemotactic activity on human neutrophils compared to pollen from rural sites. Conclusions: The present study points to an impact of air pollution on allergen carrier proteome and release of chemotactic substances. The increment in proinflammatory substances such as pollen-associated lipid mediators might contribute to the described urban-rural gradient of allergy prevalence. Furthermore, our study suggests that allergenicity is determined by more than the sole allergen content.

The influence of perceived benefits on acceptance of GM applications for allergy prevention

Allergic diseases, such as hay fever and food allergy, affect an increasing part of the population in Westernized countries and have a negative impact on the patients quality of life. Allergy prevention measures that focus on reducing the allergenic load are currently developed, and these may include the use of genetic modification of allergenic plants. Such developments should take societal concerns about genetic modification into account. We examined the attitude of allergic and non-allergic respondents towards applications of genetic modification for allergy prevention in one food allergy application (apple) and two hay fever applications (birch, grass). Attitude towards genetic modification was described in terms of ‘benefits’ and ‘rejection factors.’ We found that respondents suffering from self-reported allergy perceived greater benefits associated with the birch application as compared to non-sufferers. The perceived benefits increased with an increasing impact of allergic complaints on quality of life. No differences were found between sufferers and non-sufferers for the food allergy application. The impact of perceived benefits on acceptance was larger than that of rejection factors. This supports the idea that acceptance of genetic modification is primarily a function of perceived personal benefit. Novel genetically modified products that are perceived to be beneficial by some consumers may consequently experience an increased consumer acceptance.

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.

The use of attenuated isolates of Pepino mosaic virus for cross-protection

Pepino mosaic virus (PepMV) has recently emerged as a highly infectious viral pathogen in tomato crops. Greenhouse trials were conducted under conditions similar to commercial tomato production. These trials examined whether tomato plants can be protected against PepMV by a preceding infection with an attenuated isolate of this virus. Two potential attenuated isolates that displayed mild leaf symptoms were selected from field isolates. Two PepMV isolates that displayed severe leaf symptoms were also selected from field isolates to challenge the attenuated isolates. The isolates with aggressive symptoms were found to reduce bulk yields by 8 and 24% in single infections, respectively. Yield losses were reduced to a 0–3% loss in plants that were treated with either one of the attenuated isolates, while no effects were observed on the quality of the fruits. After the challenge infection, virus accumulation levels and symptom severity of the isolates with aggressive symptoms were also reduced by cross-protection. Infection with the attenuated isolates alone did neither affect bulk yield, nor quality of the harvested tomato fruits.

Mass spectrometry and pollen allergies.

Allergic disorders affect more than 25% of the population, especially in industrialized countries [1]. Allergy is defined as an adverse reaction of the immune system towards normally harmless substances, generally proteins. During the sensitization process in which one develops an allergy, a sequence of cellular activation mechanisms takes place, which eventually results in the production and release by B cells of allergen-specific IgE antibodies. IgE specifically binds to receptors on mast cells. Upon renewed allergen exposure, cross-linking by the allergen of two receptor-bound IgE molecules triggers an immediate response: the mast cells degranulate and release several inflammatory mediators, such as histamines and leukotrienes, which subsequently cause the allergic reaction. IgE synthesis is further regulated in a late-phase response that involves secretion of cytokines and interleukins by T lymphocytes.

Socio-Economic Issues Associated with Food Allergy

Food allergies affect a substantial proportion of the population, with prevalence estimates ranging from 1% to 11% of the population suffering from some complaints. Dietary exclusion of allergenic foods, ingredients and derived products, if necessary, represents the primary risk management strategy at the present time. Given the potentially profound consequences of experiencing an allergic reaction, food allergy has the potential to negatively affect the socio-economic functioning of those food allergic patients, as well as their families. The results of research focused on the socio-economic impact of food allergy suggest that, whilst food allergy has the potential to have a profound impact on the well-being of consumers, not all food allergy management activities are equally preferred by food allergy sufferers. The introduction of novel hypoallergenic products produced using potentially controversial food technologies is not universally accepted by food allergic consumers. There is considerable consumer demand for accurate allergen labeling of food products. Questions arise as to whether current regulatory frameworks (for example, the General Food Law in Europe) are adequate in terms of optimizing consumer protection. Potential future areas of research will be discussed, in particular those where international collaboration is needed to attain the research objectives.