James D. Astwood

Assessment of the allergenic potential of foods derived from genetically engineered crop plants

This article provides a science-based, decision tree approach to assess the allergenic concerns associated with the introduction of gene products into new plant varieties. The assessment focuses on the source from which the transferred gene was derived. Sources fall into three general categories: common allergenic food proteins; less common allergenic foods or other known allergen sources; and sources with no history of allergenicity. Information concerning the amino acid sequence identity to known allergenic proteins, in vitro and/or in vivo immunologic assays, and assessment of key physiochemical properties are included in reaching a recommendation on whether food derived from the genetically modified plant variety should be labeled as to the source of the transferred gene. In the end, a balanced judgement of all the available data generated during allergenicity assessment will assure the safety of foods derived from genetically engineered crops. Using the approaches described here, new plant varieties generated by genetic modification should be introduced into the marketplace with the same confidence that new plant varieties developed by traditional breeding have been introduced for decades.

Bioinformatic Methods for Allergenicity Assessment Using a Comprehensive Allergen Database

Background: A principal aim of the safety assessment of genetically modified crops is to prevent the introduction of known or clinically cross-reactive allergens. Current bioinformatic tools and a database of allergens and gliadins were tested for the ability to identify potential allergens by analyzing 6 Bacillus thuringiensis insecticidal proteins, 3 common non-allergenic food proteins and 50 randomly selected corn (Zea mays) proteins. Methods: Protein sequences were compared to allergens using the FASTA algorithm and by searching for matches of 6, 7 or 8 contiguous identical amino acids. Results: No significant sequence similarities or matches of 8 contiguous amino acids were found with the B. thuringiensis or food proteins. Surprisingly, 41 of 50 corn proteins matched at least one allergen with 6 contiguous identical amino acids. Only 7 of 50 corn proteins matched an allergen with 8 contiguous identical amino acids. When assessed for overall structural similarity to allergens, these 7 plus 2 additional corn proteins shared ≧35% identity in an overlap of ≧80 amino acids, but only 6 of the 7 were similar across the length of the protein, or shared >50% identity to an allergen. Conclusions: An evaluation of a protein by the FASTA algorithm is the most predictive of a clinically relevant cross-reactive allergen. An additional search for matches of 8 amino acids may provide an added margin of safety when assessing the potential allergenicity of a protein, but a search with a 6-amino-acid window produces many random, irrelevant matches.

Evaluation of protein safety in the context of agricultural biotechnology

One component of the safety assessment of agricultural products produced through biotechnology is evaluation of the safety of newly expressed proteins. The ILSI International Food Biotechnology Committee has developed a scientifically based two-tiered, weight-of-evidence strategy to assess the safety of novel proteins used in the context of agricultural biotechnology. Recommendations draw upon knowledge of the biological and chemical characteristics of proteins and testing methods for evaluating potential intrinsic hazards of chemicals. Tier I (potential hazard identification) includes an assessment of the biological function or mode of action and intended application of the protein, history of safe use, comparison of the amino acid sequence of the protein to other proteins, as well as the biochemical and physico-chemical properties of the proteins. Studies outlined in Tier II (hazard characterization) are conducted when the results from Tier I are not sufficient to allow a determination of safety (reasonable certainty of no harm) on a case-by-case basis. These studies may include acute and repeated dose toxicology studies and hypothesis-based testing. The application of these guidelines is presented using examples of transgenic proteins applied for agricultural input and output traits in genetically modified crops along with recommendations for future research considerations related to protein safety assessment.

Digestive Stability in the Context of Assessing the Potential Allergenicity of Food Proteins

Assessment of the potential allergenicity of proteins introduced into food crops through biotechnology is required by international regulatory agencies that govern the release and production of genetically modified plants. Currently, one aspect of this assessment includes analysis of the protein in a simulated gastric fluid (SGF) assay that tests the digestibility of the target protein to pepsin. The logic behind this test was that proteins that are nutritionally desirable tend to be rapidly digested and have greater bioavailability of amino acids than stable proteins. In addition, proteins that are highly digestible would be expected to have less opportunity to exert adverse health effects when consumed. The assay was not meant to predict the fate of the protein of interest under in vivo conditions, but rather to evaluate the susceptibility of the protein to digestion under fixed conditions in vivo. The purpose is to provide information that, in conjunction with other evidence, would be useful in predicting whether a dietary protein may become a food allergen. Therefore, the relationship of the resistance to digestion by pepsin and the likelihood that a dietary protein is an allergen was identified as a means of aiding the assessment of proteins added to commodity crops through biotechnology. In this article, we discuss the predictive value of this assay and the practical and theoretical aspects of allergen resistance to pepsin digestion in the context of food safety.

Preventing food allergy : Emerging technologies

Food allergy represents a largely avoidable threat. Technologies are presently available or are under development that better define the molecules (allergens) responsible for food allergy, better detect the presence of allergens, and can reduce or even eliminate specific allergens from the food supply. The incidence of food allergy and fatal anaphylaxis triggered by food allergens is expected to decline as a result of the application of a combination of key technologies such as biotechnology, bioinformatics and immunodiagnostics, and of the Internet, as well as following the development of well-defined science-based public health policies.

Bioinformatic Methods for Identifying Known or Potential Allergens in the Safety Assessment of Genetically Modified Crops

Agricultural crops have been genetically improved through centuries of breeding to select phenotypes that are controlled by combinations of genes, typically with undefined mutations, which produce the desired traits. Changes in the plant characteristics (e.g., disease resistance, insect resistance, food quality) typically have been slow, except when partial or full genomes have been combined. This combination as is hypothesized to have occurred thousands of years ago in the generation of modern hexaploid wheat as a hybrid cross of tetraploid and diploid progenitors (1). Such crosses result in the combination of hundreds to thousands of different proteins in a single food. In contrast, modern tools of biotechnology have allowed the introduction of one or a few new genes and resulting proteins into crop varieties that are carefully selected and studied for safety and performance before being allowed into commercial production (2, 3). As methods to introduce desired traits in plants have improved, so too has the public

Effect of substitution of low linolenic acid soybean oil for hydrogenated soybean oil on fatty acid intake.

Low linolenic acid soybean oil (LLSO) has been developed as a substitute for hydrogenated soybean oil to reduce intake of trans FA while improving stability and functionality in processed foods. We assessed the dietary impact of substitution of LLSO for hydrogenated soybean oil (HSBO) used in several food categories. All substitutions were done using an assumption of 100% market penetration. The impact of this substitution on the intake of five FA and trans FA was assessed. Substitution of LLSO for current versions of HSBO resulted in a 45% decrease in intake of trans FA. Impacts on other FA intakes were within the realm of typical dietary intakes. No decrease in intake of α-linolenic acid was associated with the use of LLSO in place of HSBO because LLSO substitutes for HSBO that are already low in α-linolenic acid.

Molecular Biology of Male Gamete Development in Plants—An Overview

This chapter describes the biology of pollen development emphasizing (where possible) biochemical, physiological, and genetic events as they are known to occur in cereals generally and barley (Hordeum vulgare) specifically. Inasmuch as it is ultimately impossible to separate these phenomena, attention to their discrete consequences will be instructive. Pollen mother cells undergo reductional divisions and further divide twice to become mature tricellular haploid gametes in a process that can last several weeks and even years in some gymnosperms. Throughout this program, enzyme activity, translation, transcription, and DNA replication all proceed in a differential but scheduled manner. That schedule is fatally tied to environmental conditions, hormonal conditions, and expression of specific genes in the reproductive phase plant.

Molecular Characterization Of Hor v 9

We have cloned, sequenced and expressed a recombinant group IX pollen allergen from barley (Hordeum vulgare). Hor v 9 is a polypeptide of 313 amino acids. The Hor v 9 cDNA clone was engineered into the E. coli protein expression vector pMAL and expressed as a fusion of maltose binding protein and truncated Hor v 9. Polyclonal antibodies to the fusion protein were raised in mice. Cross-reactive proteins, RNA and DNA homologues were found in many agricultural species including wheat, rye, triticale, oats, maize, sunflower and flax. The presence of group IX-like proteins in a variety of agricultural crops may represent a previously uncharacterized aeroallergenic occupational hazard. Sequence comparisons of the barley allergen, Hor v 9, with Poa p 9 and other cloned group IX pollen allergens revealed putative structural domains common to all. These include a signal peptide, two conserved immunoglobulin-like motifs, a 150 amino acid highly conserved carboxyterminal domain and a carboxyterminal transmembrane helix. This structural arrangement is also found in cell adhesion molecules. The highly conserved T-cell epitope previously characterized and mapped in group IX allergens (and present in Hor v 9) was found in several human cell adhesion molecule sequences (VCAM, NCAM and CD2). This T-cell epitope corresponded to the most highly conserved amino acid residues common to all group IX homologues sequenced to date. CD2 and VCAM are known to play a role in allergic inflammation: VCAM is involved in the recruitment of lymphocytes to sites of inflammation, and cross-linking CD2 leads to T-cell activation. .We anticipate that the similar structural arrangement of group IX allergens and human cell adhesion molecules, as well as the presence of a T-cell epitope common to group IX pollen allergens and cell adhesion molecules, will have important consequences in the natural history of the atopic immune response.