Bindslev-Jensen C

Ebner C

Madsen C

Mäkinen-Kiljunen S

Peltre G

Poulsen LK

van Ree R

Viets S

 

 

 

Genetically modified foods and allergenicity

 

Position Paper

 

 European Academy for Allergology and Clinical Immunology

 

 

First Draft June 2000

 

 


 

Introduction

Introduction of genetically modified foods to the consumers carry a potential risk of introducing allergic reactions in susceptible individuals with food allergy (1). Although the risk and significance of the much quoted case, where allergenic proteins from Brazil Nut was introduced in soy for chicken feed (2) has been overestimated, new genetically modified plants with a real allergenic risk are now being developed. As an example,  the allergen β-casein from cow’s milk has been inserted into soy bean and glycinins from soy into rice (3,4).

 

Although these products are not (and probably never will reach) the market, the risk of contamination or spreading of genetic material always exist.

 

Therefore, the necessity of control procedures for ascertaining potential allergenicity in GMO-foods is obvious. Legislation in Europe (and in US) demands such proce­dures, but no mutual agreement upon the practical aspects has been reached so far.

A Decision Tree has been launched by FCBS/ILSI (5), aiming at enabling an evaluation for labelling of a GMO-foods based on available data on the insert protein, but a flow chart for risk evaluation does not exist at present.

 

This position Paper aims at reviewing the present knowledge within the field of GMO’s and allergenicity and at presenting a suggestion for ascertainment of allerge­nicity in genetically modified foods.

 

References to this chapter:

1.Bindslev‑Jensen C: Allergy risks of genetically engineered foods. Allergy 1998;53:58‑­61

2.Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK. Identification of a Brazil-nut allergen in transgenic soybeans. New Engl J Med 1996;334:688-92.

3 Katsube T, Kurisaka N, Ogawa M, Maryama N, Ohtsuka R, Utsumi S, Takaiwa F: Accumaulation of soy bean clycinin and its assembly with the glutenins in rice. Plant Physiology 1999:120, 1063-1073.

4. Maughan PJ, Philip R, Cho MJ, Widholm JM, Vodkin LO: Biolistic transformation, expression, and inheritance of bovine beta-casein in soy bean (glycine max). In vitro Cell Develop Biology Plant 1999:35; 344-349

5.Metcalfe DD, Astwood JD, Townsend R, Sampson HA, Taylor SL, Fuchs RL:  Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit Rev Food Sci Nutr 1996;36; Suppl:S165‑­86





Legislation

 

The following is an attempt to give an overview of the EC regulation concerning the safety assessment and labelling of genetically modified organisms (GMO’s) with special emphasis on allergenicity. The description is restricted to EC regulation not because it is not recognised that there are countries in Europe not members of the EU but because the Eu constitutes the largest group of countries in Europe and is likely to expand in the future.

 

Novel foods


Rules concerning GMO’s are covered in the regulation dealing with novel foods (NF) (1). Novel foods are defined as foods and food ingredients, which have not hitherto been used for human consumption to a significant degree within the Community and which fall under the following categories:

 

(a)           foods and food ingredients containing or consisting of genetically modifi­ed organisms;

(b)           foods and food ingredients produced from, but not containing, genetical­ly modified organisms;

(c)           foods and food ingredients with a new or intentionally modified primary molecular structure;

(d)           foods and food ingredients consisting of or isolated from micro-orga­nisms, fungi or algae;

(e)           foods and food ingredients consisting of or isolated from plants and food ingredients isolated from animals, except for foods and food ingredients obtained by traditional propagating or breeding practices and having a history of safe use;

(f)           foods and food ingredients to which has been applied a production process not currently used, where that process gives rise to significant changes in the composition or structure of the foods or food ingredients which affect their nutritional value, metabolism or level of undesirable substances.

 

From the above it will be clear that the regulation is designed to cover a variety of different situations and that GMO’s are only a subset of novel foods.

 

It is stated that foods and food ingredients falling within the scope of the regulation must not:

-   present a danger for the consumer,

-   mislead the consumer,

-   differ from foods or food ingredients which they are intended to replace to such an extent that their normal consumption would be nutritionally disadvantageous for the consumer.

 

Before marketing a novel food

The manufacturer wanting to place a GMO on the Community market shall submit a request to the Member State in which the product is to be placed on the market for the first time. At the same time, he shall forward a copy of the request to the Com­mission. The request shall contain the necessary information, including a copy of the studies carried out and any other material which is available to demonstrate that the food or food ingredient do not present a danger to the consumer, mislead the consumer, etc. The request shall be accompanied by a summary of the dossier. This summary shall be sent to the other Member States without delay.

 

The Member State concerned shall notify the Commission of the competent food assessment body responsible for preparing the initial assessment report. This report must be finished within three months from receipt of a request. The report shall be send to the other Member States. After that and within 60 days a Member State or the Commission may make comments or present a reasoned objection to the marketing of the food or food ingredient concerned.


Notification procedure

Food or food products that are substantially equivalent to existing foods do not have to go through the above procedure. Here it is just necessary to notify the Commis­sion together with the relevant details, including the confirmed statement that the products are, in fact, substantially equivalent. This statement should be made by an official food assessment body in one of the member states. Products of this nature may be marketed straight away, i.e. immediately after being notified to the Commis­sion. A GMO can not be substantially equivalent, but a product made from a GMO can, e.g. soybean oil and maize flour or starch.

 

Information necessary for safety assessment

The EU Scientific Committee for Food (SCF) has developed recommendations concerning the scientific aspects of the information necessary to support an applica­tion, how such information must be presented and how the initial report must be prepared (2).

 

SCF has suggested structured schemes to identify the types of information that are likely to be required to establish the safety of particular classes of novel foods. It is underlined that the schemes only can be used for guidance. It must be decided on a case by case basis what precise information is needed.

 

The information requested is:

I0     Specification of the novel food (NF)

II0    Effect of the production process applied to the NF

III0   History of the organism used as the source of the NF

IV0   Effects of the genetic modification on the properties of the host organism including,

-   characterisation of the parent food organism,

-   characterisation at the molecular level of the nature of the genetic modification including      insertional position, copy number and biochemical expression level,

-   establishment, as far as possible, of substantial equivalence between the parent food organism and its new derivative through chemical and phenotypic analysis,

The concept of ’substantial equivalence’ embodies the idea that existing organisms used as foods can serve as basis for comparison when assessing the safety of human consumption of a food that has been modified or is new. If a new food is found to be substantially equivalent to an existing food, it can be treated in the same manner with respect to safety, keeping in mind that establishment of substantial equivalence is not a safety or nutritional assessment in itself, but an approach to compare a potential new food with its conventional counterpart.

 

-   if substantial equivalence cannot be established, conventional safety studies on specific chemicals occurring in the food due to the phenotypic change involving either the new product of the new gene or the safety of inherent natural toxins now present in altered amounts. The potential allergenicity of the new components also needs to be addressed.


 

IX.    Genetic stability of the GMO used as NF source

X.     Specificity of expression of novel genetic material

XI.    Transfer of genetic material from GMO

XII.   Ability of GMMicroorganisms to survive in and colonise the human gut

XIII.  Anticipated intake/extent of use of the NF

XIV.  Information on previous human exposure to the NF or its source

XV.   Nutritional information on the NF

XVI.  Microbiological information on the NF

XVII. Toxicological information on the NF

If substantial equivalence to a traditional counterpart cannot be established the safety assessment based on a case-by-case evaluation must consider the following elements:

-   consideration of the possible toxicity of the analytically identified individual chemical components,

-   toxicity studies in vitro and in vivo including mutagenicity studies, reproduction and teratogenicity studies as well as long term feeding studies, following a tiered approach on a case-by-case basis,

-   studies on potential allergenicity.

 

 

The allergenicity of a novel food from a GM source should include consideration of the allergenic potential of the donor and of the recipient organism. It is suggested to do in vitro and in vivo tests in individuals allergic to the traditional food counterpart, although it is recognised that it raises ethical problems. If the novel protein comes from a source that is known to be a food allergen specific immunological test with sera from allergic individuals are suggested. If these tests are negative, in vivo skin prick tests and oral challenges may be performed. It is recommended to include factors such as sequence epitope homology with known allergens, heat stability, sensitivity to pH, digestibility by gastrointestinal proteases, detectable amounts in plasma, and molecular weight as possible indicators of allergenicity. Additional evidence might emerge from pre-marketing human results and reports of workers’ sensitisations. As with the other toxicological endpoints decision on which test it is possible and necessary to perform should be done on a case-by-case basis. It is stated that new approaches are needed to assess the potential allergenicity of NF in humans.

 

References to this chapter:

 

1 Regulation (EC) No 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and novel food ingredients.

 

2 Commission recommendation of July 1997 concerning the scientific aspects and the presentation of information necessary to support applications for the placing on the market of novel foods and novel food ingredients and the preparation of initial assessment reports under Regulation (EC) No 258/97 of the European Parliament and of the Council.

 







Methods used in the production of transgenic plants

 

Until roughly 15 years ago, conventional plant breeding was the only way to improve agricultural productivity and nutritional quality of food crops. By intercrossing varieties with different desired characteristics new improved varieties were (and are) created. The introduction of molecular biology in the field of plant research has resulted in the possibility to introduce selected genes from almost any life form into plants by genetic engineering. This development has increased the diversity of genes available for introduction and decreased the time needed for the development of new varieties. At the same time, complete genomes of living organisms are becoming available. The number of candidate genes for introduction in plants will, therefore, increase exponentially. Moreover, the elucidation of plant genomes will supply us with valuable new information on the regulation of expression of proteins in plants, facilitating new strategies of regulated protein expression. How are new genes introduced into plants? In principle there are two classes of plant transformation technologies: ‘natural’ and ‘non-natural’  QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\031;2\01\03\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00ÎPk\01Ö\00\01\00\00\01\0D\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00ÎPk\01Ö\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00ÎPk\01Ö\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03496\03496\00\03\00 1;2 QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\00\01\00\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03497\03497\00\03\00 . The ‘natural’ methodologies use natural pathogens of plants for introducing foreign genes, i.e. viruses and bacteria. The ‘non-natural’ approaches are based on physico-mechanical techniques.

 

Plant transformation technologies

Agrobacterium tumefaciens-mediated gene transfer

Agrobacterium tumefaciens is a natural pathogen for dicotyledonous plants QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\031;2\01\03\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00TPk\01ê\00\01\00\00\01\0D\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00TPk\01ê\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00TPk\01ê\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03496\03496\00\03\00 1;2 QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\00\01\00\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03497\03497\00\03\00 . It invades wounds in plant tissue, where it introduces small segments of its own DNA, the so-called Ti-DNA (tumor-inducing DNA). This results in local production of phytohormones that induce tumor growth, the so-called crown galls that function as sites of refuge for the bacterium. For the introduction of heterologous genes in plants, A. tumefaciens was made non-pathogenic by removing genes involved in tumor induction. The present A. tumefaciens plant transformation vectors are also suitable for replication in E. coli, allowing convenient manipulations. Usually the vector contains two resistance genes, one for selection in bacteria (e.g. spectinomy­cin) and one for selection in plants (e.g. kanamycin or a herbicide). The gene of interest is inserted behind a plant promotor sequence like the cauliflower mosaic virus 35S promotor (CaMV 35S).  A. tumefaciens can be used for transforming plant cells as well as plants. After transfer, the part of the DNA is integrated into the genome of the plant and the desired protein is expressed. The fragment that is inserted, T-DNA (transferred DNA), is flanked specific regions called ‘border sequen­ces’. Recognition of these sequences is essential for injection of DNA into the plant cell. The expression of heterologous genes has been shown to be stable for at least 5 generations. The integration into the plant genome can occasionally also be a disadvantage, because plant genes could be disturbed. Finally, most monocotelydo­nous plants are no natural host for A. tumefaciens. This implies that this method for plant transformation is not suitable for a number of very important food crops, i.e. cereals like wheat, rice and corn.

 

Physico-mechanical transformation methods


For transformation of monocotelydonous plants, originally protoplasts (plant cell from which the cell wall has been removed) were used QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\031;2\01\03\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00\01\00\00\01\0D\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03496\03496\00\03\00 1;2 QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\00\01\00\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03497\03497\00\03\00 . Several techniques have been applied to protoplasts including electroporation, microinjection, polyethylene glycol treatment, and calcium phosphate treatment. Unfortunately, it is hard to regenerate plants from protoplasts. This can, however, efficiently be achieved from immature embryos. To transform embryonic cells from cereals the ‘particle gun’ method or high-velocity microprojectile bombardment was introduced QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\031‑3\01\03\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00\01\00\00\01\09\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03496\03496\00\03\00 1-3 QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\00\01\00\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03497\03497\00\03\00  QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\00\01\00\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03498\03498\00\03\00 . This is now the most common technique used for cereals. Microscopic gold or tungsten particles coated with DNA are accelerated by explosive charge, high pressure helium, or by electric discharge, towards the embryonic cells. This enables the particles to pass the rigid cell wall and enter the cytoplasm. In this method, the transformed genes are also integrated into the plant genome, resulting in very stable transformation. As with A. tumefaciens selection of transformed cells is achieved by co-transformation of resistance genes.

 

In order to alter agronomic characteristics of food crops it will be necessary to manipulate complex metabolic pathways that are regulated by multiple genes. This requires the integration of multiple transgenes into the plant genome. Although possible with A. tumefaciens, it is very complex and demanding. Microprojectile bombardment has now been show to be an efficient method to introduce multiple genes. By mixing different genes and a sinlge selectable marker, up to 13 genes were simultaneously transformed to rice by so called cobombardment QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\014\01\01\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03499\03499\00\03\00 4.

 

Antisense approach: towards hypo-allergenic foods?

The methods described so far aim at introducing new genes into plants. Sometimes it may be desirable to suppress a gene. A method that has been successfully used in achieving this, is the antisense technique QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\012\01\01\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\19Pk\01â\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03497\03497\00\03\00 2. The method is based on the fact that DNA transcription is a unidirectional process, from the 5’ to the 3’ end along the so-called sense strand. By transforming plant cells with an inverted gene, the transcri­bed sequence will be identical to the antisense strand. The cell will contain both the normal gene and an inverted version. As a result, both sense and antisense mRNA will be produced. These molecules will hybridize because their sequences are exactly complementary. Translation of mRNA is prevented by the interference of the antisense mRNA. This technique has for example been used to slow down the ripening of fruits like tomato and melon by knocking out the gene for ACC oxidase QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\035;6\01\03\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\5CPk\01n\00\01\00\00\01\0D\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\5CPk\01n\00\01\00\00\01\00\00\00\00&\00\00\000år\00\00Pk\01â\01s\00\03\00\00\00.4S\00\5CPk\01n\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03500\03500\00\03\00 5;6 QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\00\01\00\00!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03501\03501\00\03\00 . This enzyme catalyzes the last step of ethylene biosynthesis. Ethylene is thought to trigger several processes observed during ripening. The antisense approach might in the future prove useful to suppress genes for allergens.

 

 

References to this chapter

1. Gasser CS, Fraley RT. Genetically engineering plants for crop improvement.  Science 1989;244:1299

2. Day PR. Genetic modification of proteins in foods.  Crit Rev Food Scie Nutr 1996;36(S):49-67.

3. Christou P. Rice transformation: bombardment.  Plant Mol Biol 1997;35:197-203.


4. Chen L, Marmey P, Taylor NJ, Brizard J-P, Espinoza C, D'Cruz P, Huet H, Zhang S, De Kochko A, Beachy RN, Fauquet CM. Expression and inheritance of multiple transgenes in rice plants.  Nat Biotechnol 1998;16:1060-1064.

5. Ayub R, Guis M, Amor MB, Gillot L, Roustan J-P, Latché A, Bouzayen M, Pach J-C. Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits.  Nat Biotechnol 1996;14:862-866.

6. Hamilton AJ, Lycett GW, Grierson D. Antisense gene that inhibits ripening of cantelou­pe melon fruits.  Nature 1990;346:284-287.



 

 

Present situation of the use of genetically modified foods in the food chain

 

Microbial Products and Microorganisms

Independent of genetic engineering, microorganisms are used for biotechnological production of food additives for many years. Examples for food additives produced by bacteria, yeasts or moulds are amino acids, flavourings , organic acids, hydrocol­loids, preservatives, and vitamins [1-3]. Current approaches of genetic engineering are targeted towards (i) increased production (ii) replacement of chemical synthesis by cheaper fermentation processes (iii) modifying the characteristics of microorga­nisms and (iv) design of new products such as enzymes with improved properties. Most of these products are still under development. However, many enzymes derived from genetically modified microorganisms are being used by the food industry (Table 1). These enzymes do not differ from their natural homologues as regards structure and function. Bacteria, yeasts and moulds that are recognised as safe are used as host organisms [2,4]. Compared to conventional production of these enzymes, use of GMO-derived products saves energy and other resources.

 

Table 1: Enzymes derived from genetically modified organisms.

 

 

Enzyme

 

Produced by

 

Use

_-Acetolactate decarboxylase

Bacillus brevis AAC gene in B.subtilis

Brewery

_-Amylase

several Bacillus species

Bakery, brewery, starch technology

Chymosin

calf prochymosin B gene in Aspergillus niger var. awamori

Cheese production

ß-Glucanase

Bacillus amyloliquefaciens

Brewery

_-Glucano transferase

Thermomyces anaerobacter gene in B.licheniformis

Starch modification

Glucose isomerase

N N

Starch technology

Glucoseoxidase

N N

Bakery, egg processing, mayonnaise production

Hemicellulase

Aspergillus niger, Bacillus subtilis

Bakery

Invertase

N N

Invertation of saccharose

Catalase

N N

mayonnaise production

Lipase

Candida antarctica gene or Thermomyces lanuginosus gene in Aspergillus oryzae

Processing of fats and oils, bakery

Maltogenic amylase

Bacillus stearothermophilus gene in B.subtilis

Bakery, starch technology, malt sirup

Pektinase

NN

Prduction of fruit juces

Phytase

Aspergillus niger

Animal feeds, starch technology

Protease

Bacillus amyloliquefaciens

Bakery, brewery, distilling, diary, starch technology, processing of meat, fish and vegetables

Pullulanase

NN

Brewery, starch technology

Xylanase

Thermomyces lanuginosus gene in Aspergillus oryzae, self cloned Aspergillus awamori, self cloned Bacillus subtilis

Bakery, starch technology

 

 


Moreover, microorganisms are widely used as starter cultures for fermented foods and beverages. In 1990, a genetically modified yeast was approved in the UK [5]. This yeast presented enhanced maltase and maltose permease activity, resulting in improved fermentation of maltose and improved dough-leavening characteristics.  However, this organism is not used on a commercial scale.

 

In 1994, another genetically modified yeast obtained approval from the UK authoriti­es for use in the production of pasteurised beer [6]. The yeast contains a glucoamy­lase gene from a different yeast species, resulting in increased degradation of dextrins, reduced carbohydrate, and increased alcohol content. By pasteurisation, viable yeast cells are deactivated in the final product.

 

Plants and Plant Products

Genetically modified herbicide tolerant soybeans developed by Monsanto obtained approval for use in food products within the European Union in April 1996. This decision was based on directive 90/220/E­EC [7] which is a part of the general legislation regulation the application of gene technology. Insect-resistant maize developed by Ciba-Geigy (now Novartis) was released on the basis of the same directive in January 1997. Since then, food products containing these transgenic foods can be marketed in the EU. Moreover, in 1996, national approval was obtained in the UK for tomato puree produced from delayed ripening tomatoes and products from transgenic maize.

 

In May 1997 a European Community Regulation was established applying to novel foods including those derived from genetically modified organisms [8]. Foods and food ingredients containing or consisting of GMO undergo an authorisation proce­dure. By contrast, notification is required for those foods and food ingredients that are substantially equivalent to existing foods as regards their composition, nutritional value, metabolism, intended use and the level of undesirable substances contained therein. According to this regulation rapeseed oil derived from transgenic crops and additional foods and food ingredients produced from transgenic maize have been notified and can thus be marketed within the EU (Table 2).

 

In addition, the foods listed in Table 3 (tomatoes, radicchio, chicoree, and again soybeans and maize) have been submitted to the authorisation procedure.

 

Outside the EU, genetically modified food plants have been approved in USA, Canada, Mexico, Argentina, China, Japan, South Africa, Australia, Ukraine, and Romania [4] [9]. In 1999, transgenic crops were grown world-wide on an area of 39.9 million ha, with 74 % in the USA, 6.7 % in Argentina, and 4.0 % in Canada. 71 % of the plants were herbicide-tolerant, 22 % insect-tolerant, 7 % were resistant to both herbicides and insects, and less than 1 % were virus-resistant [9].

 

Table 2: Authorisations/notifications for the placing on the market of foods derived from genetically modified organisms.

 


 

Applicant/

Notifier

 

GMO and derived Products

 

Authorisation (A)/

Notification (N)

 

Legislation/

Competent Authority

 

Monsanto

 

Foods and food ingredients from herbicide to­lerant soybean

 

A: April 1996

 

Directive 90/220/EEC/

United Kingdom

 

Novartis

(Ciba-Geigy)

 

Foods and food ingredients from insect tolerant maize Bt 176

 

A: February 1997

 

Directive 90/220/EEC/

France

 

AgrEvo

 

Refined oil from herbicide tolerant rape-seed

TOPAS 19/2

 

N: June 1997

 

Regulation (EC) No 258/97/

UK ACNFP

 

PGS

 

Refined oil from male sterile and herbicide tolerant rape-seed MS1XR­F1, MS1XRF2

 

N: June 1997

 

Regulation (EC) No 258/97/

UK ACNFP

 

Monsanto

 

Refined oil from herbicide tolerant rape-seed GT 73

 

N: November 1997

 

Regulation (EC) No 258/97/

UK ACNFP

 

Monsanto

 

Foods and food ingredients from insect tolerant maize MON 810

 

N: December 1997

 

Regulation (EC) No 258/97/

UK ACNFP

 

AgrEvo

 

Foods and food ingredients from herbicide tolerant maize T 25

 

N: January 1998

 

Regulation (EC) No 258/97/

UK ACNFP

 

Novartis

 

Foods and food ingredients from insect and herbicide tolerant maize Bt 11

 

N: January 1998

 

Regulation (EC) No 258/97/

UK ACNFP

 

Pioneer

 

Foods and food ingredients from insect and herbicide tolerant maize MON 809

 

N: October 1998

 

Regulation (EC) No 258/97/

UK ACNFP

 

AgrEvo

 

Refined oil from herbicide tolerant rape-seed

Liberator L 62

 

N: October 1999

 

Regulation (EC) No 258/97/

BgVV, Germany

 

AgrEvo

 

Refined oil from herbicide tolerant rape-seed

FALCON GS 40/90

 

N: October 1999

 

Regulation (EC) No 258/97/

BgVV, Germany

 

Plant Genetic Sy­stems

 

Refined oil from herbicide tolerant hybrid rape-seed MS8XR­F3

 

N: October 1999

 

Regulation (EC) No 258/97/

BgVV, Germany

ACNFP = Advisory Committee on Novel Foods and Processes, UK

BgVV = Federal Institute for Health Protection of Consumers and Veterinary Medicine, Germany

 

Table 3: Applications for the placing on the market of genetically modified organisms and derived foods according to Regulation (EC) No. 258/97.

 

Applicant

 

Description of

Food or Food Ingredient

 

Application in

 

Authorisation

 

Zeneca

 

Genetically modified processing to­matoes

 

ES

 

pending

 

Bejo Zaden

 

Transgenic Radicchio rosso with ma­le sterility

 

NL

 

pending

 

Bejo Zaden

 

Transgenic green hearted chicoree with male sterility

 

NL

 

pending

 

DuPont

 

High oleic soybeans

 

NL

 

pending

 

Monsanto

 

Herbizide tolerant maize GA21

 

NL

 

pending

 

Plant Genetic Sy­stems

 

Herbizide tolerant soybeans

 

BE

 

pending

 

Novartis

 

Insect tolerant sweet maize Bt 11

 

NL

 

pending

 

 


In addition to the plants and foods approved by the EU members, e.g corn, rapeseed and soybean crops with resistance to other insects and herbicides have been released. Further examples are various tomatoes with delayed softening, delayed ripening, or virus resistance, insect resistant potatoes, and virus resistant squash and papaya. The first product with modified physiological properties was a high laureate rapeseed that was approved 1995 in the USA and 1996 in Canada [4]. In contrast to the first generation of transgenic crops which almost exclusively had improved agronomic properties, there is an ongoing agronomic trend towards introducing benefits for the consumer by improving the nutritional value. High oleic soybean with the potential to reduce the „bad“ blood cholesterol, which has been released in the United States is another example for such a food [9]. There is a rapid development in this area. A popular example is the introduction of genes facilitating the synthesis of beta-carotene, the precurser of vitamin A, into rice. Keeping in mind that Vitamin A deficiency causes the death of 2 Million children in developing countries every year, this could be a substantial health benefit in countries with a rice-based diet [9].

 

Animals

The most important efforts to generate genetically modified animals have been made with fish [4, 10].for herbicide-tolerant soybeans. Eleven of the produtcs were found to contain roundup ready soybean, but only one product was labelled as containing genetically modified material [12]. In a Norwegian study, 300 food samples were taken. Out of 150 samples of soy-containing products, DNA could be isolated from 117 samples. Genetically modified soybean was detected in 59 of these 117 samples (about 50 %). Six samples from ordinary grocery stores, all belonging to one type of product, contained only traces of GMO soybean. By contrast 52 positive samples were from health food stores. 22 of these samples representing eight different types of products contained about 2 % of GMO soy , and 24 samples with seven different categories of products contained more than 2 % GMO-derived material [13]. Another 150 samples were taken from maize-containing foods. DNA could be successfuly isolated from 137 of these samples. Genetically modified maize at levels below 2 % was detected in 23 of the 137 samples (17 %); 13 of the samples were from ordinary grocery stores and 10 were from health food stores [13].

 

In a German study three out of 25 seed corn m The attempts are targeted towards e.g. improvement of growth rates, cold tolerance, disease resistance, or optimised composition. However, genetically modified animals have not been released to the market either inside or outside the EU.

 

Declaration

GMO derived foods and food ingredients are subject to labelling in order to inform the consumer of the genetic modification if the recombinant DNA or resulting new proteins are present in a proportion higher than one percent of the food or food ingredient [11].

 

Detection of GMO derived foods

The analysis of transgenic material in food samples is generally based on PCR techniques. Analyses to detect the presence of GMO foods in commercially available food products are underway. The first data have recently been published.

 

In a Swedish study 45 food samples,all labelled as containing soybean protein, soya lecithin, or soybean oil were analysed aize samples have been shown to contain between 0.1 % and 0.5 % insect-tolerant maize of the Monsanto variety MON 810 [14]. Moreover, it has been shown that Canadian rapeseed honey contains pollen of glufosinate-resistant rapeseed [15].


 

Conclusions

It is difficult to estimate to which extend the released products prepared using GMO are already present in the food chain. However the studies from Sweden and Norway have clearly shown that  many foods containing soybeans and maize already contain genetically modified material [12, 13]. The data also show that the major GMO foods, transgenic soybean and maize, are widely used. The results obtained with Canadian rapeseed honey may indicate that transgenic material can occur in a wider range of foods than anticipated by the consumer.

 

References to this chapter

1. Heller, K J Potential of the application of genetically engineered microorganisms in food production: an overview. in: Schreiber G A, Bögl K W (eds.) Foods produced by means of genetic engineering, 2nd status report, BgVV-Hefte 01/1997, Federal Institute for Health Protection of Consumers and Veterinary Medicine, P. 14-21.

2. Roller, S. Harlander, S. (Eds.), Genetic Modification in the Food Industry - A Strategy for Food Quality Improvement, Blackie Academic & Professional, London, 1998

3. Deutsche Gesellschaft für Ernährung e.V. (Hrsg.), Ernährungsbericht 1996, DGE, Frankfurt a.M., 1997

4 Braunschweiger G, Conzelmann C. Food industry supplies through genetic modification: what’s already on the market? in: Schreiber G A, Bögl K W (eds.) Foods produced by means of genetic engineering, 2nd status report, BgVV-Hefte 01/1997, Federal Institute for Health Protection of Consumers and Veterinary Medicine, P. 1-13.

5 Advisory Committee on Novel Foods and Processes (ACNFP) Annual Reports 1990, MAFF Ministry of Agriculture, Fisheries and Food Publications, London, UK

6. Advisory Committee on Novel Foods and Processes (ACNFP) Annual Reports 1993, MAFF Ministry of Agriculture, Fisheries and Food Publications, London, UK

7. Council Directive of 23 April 1990 on the deliberate release into the environment of genetically modified organisms (90/220/EEC), Official Journal of the European Communities L 117, 8.5.1990, p. 15

8. Regulation (EC) No 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and novel food ingredients, Official Journal of the European Communities L 43, 14.2.1997, p. 1

9. James C. Transgenic foods plant: traits, transformants, and deployment. ISAA (International Service for the Acquisition of Agri-Biotech

10. Applications) Brief No. 12-1999

11. Rehbein H. Development and production of transgenic fish for human consumption in: Schreiber G A, Bögl K W (eds.) Foods produced by means of genetic engineering, 2nd status report, BgVV-Hefte 01/1997, Federal Institute for Health Protection of Consumers and Veterinary Medicine, P. 40-47.

12. Commission Regulation (EC) No 49/Ö2000 of 10 January 2000 amending Council Regulation (EC) No 1139/98 concerning the compulsory indication on the labelling of certain foodstuffs produced from genetically modified organisms of particulars other than those provided for in Directive 79/112/EEC, Official Journal of the European Communities No L 6/13, 11.1.2000

13. Lundberg L, Malmheden Yman I, Genmodifierad soja i elva av 45 produkter.Vår Föda 6/1999 10-12.

14. Prosject: Genmodifisert soya og mais i utvalgte naeringsmidler på det norske markedet. Veterinaerinstittutet. Rapport february 1999.

15. Waiblinger H U, Pietsch K, Ungermann A, Kroh R A. Gentechnisch veränderter Mais in Saatgut - Aktuelle Befunde und deren Interpretation. Deutsche Lebensmittel-Rundschau 2000; 96: 1-3

16. Waiblinger H U, Wurz A, Freyer R, Pietsch K. Spezifischer Nachweis von gentechnisch verändertem Raps in Honig. Deutsche Lebensmittel-Rundschau 1999; 95: 192-195






 

Potential food allergens and transgenic food

 


Molecular biology and biochemistry have significantly increased the knowledge of the nature of  allergens. However, only limited information about specific properties of food allergens is presently available. At present, it is useful to classify plant food allergens according to their biological functions into several families (Table 1). The majority of known plant food allergens belong to seed storage proteins (1-16), protease and amylase-inhibitors (17-22), profilins (23-27) or pathogenesis-related (PR) proteins (28-58). Other important protein families that include plant food allergens are the cereal peroxidases, thiol proteases and lectins (for review see 59). Less variety is found among allergenic proteins derived from animal sources. However, also in this case, cross-reactive allergen families can be subdivided (60-62). These proteins will not be discussed here as they found no use for the produc­tion of GMOs until now.

 

In response to pathogens, plants synthesize and accumulate a variety of PRs which are part of a plants defence system. As plant protection against bacteria, fungi, viruses and insects is a major challenge to agriculture world-wide, overexpression of PRs in transgenic plants has been applied to increase the defense potential. Transgene-encoded, heterologous PRs are used in addition to, and not in place of, the inducible defences of the host plant. On the other hand, genes encoding proteins with a high content of essential amino acids, and therefore high nutritive

value, have also been considered for the transformation of crop plants.

 

Proteins with allergenic potential which are considered for use in the production of GMOs to increase the resistance to microbial and insectal attack

 

Anti-fungal proteins

Transformation of crop plants with either ß-1,3-glucanases or chitinases provides protection against fungal attack, as both enzymes hydrolyze cell walls of several plant-pathogenic fungi. A basic ß-1,3-glucanase found in Hevea brasiliensis latex has been identified as Hev b 2,  a relevant latex allergen (30-31). The coincidence of Hevea latex allergy and hypersensitivity to plant-foods, especially banana, avocado, and chestnut, has been is called latex-fruit syndrome. Indeed, ß-1,3-glucanases from Hevea brasiliensis reacted with specific IgE from food-allergic patients suffering from adverse reactions to banana and other fruits (32). Chitinases are proteins present in many seed-producing plants. In chestnut and avocado class I chitinases have been identified as allergens (33-34). An endochitinase designated as Pers a 1, a major allergen of avocado, was cloned and expressed in the yeast Pichia pastoris (35). It is believed that sensitization to Pers a 1 is a consequence of IgE-production against latex-hevein (36). Moreover, two banana allergens were identified as class I chitina­ses containing a hevein-like domain (37).


Thaumatin-like and osmotin-like proteins possess diverse functions including antifungal activity (63). Due to sequence similarities, the PR-group 5 proteins (28) and thaumatin (an intensely sweet tasting protein produced by the African shrub Thaumatococcus daniellii) are now designated "thaumatin-like proteins". Transgenic potato plants overexpressing a thaumatin-like protein showed increased resistance to fungal infection  (63). A major allergen of apple was the first thaumatin-like protein described as an allergen (38). The complete cDNA sequence has been decoded and the allergen received the designation Mal d 2 from the international nomenclature committee (Krebitz M., unpublished). A thaumatin-like protein representing a major allergen was cloned from sweet cherry, and designated Pru av 2 (39). Furthermore, the N-terminal sequence of an important bell pepper allergen was displayed a high degree of identity with a corresponding portion of the osmotin-like protein P23 from tomatoes (40).

Transfection of tomato plants with prohevein (Hev b 6.01) from Hevea brasiliensis led to a retardation of the growth of the fungus Trichoderma hamatum (64). Peptide sequences of a recently identified turnip allergen revealed 70% identity to prohevein and high similarities to wound-induced proteins from tomato and potato (41).

 

Insecticidal proteins

Besides endochitinases (see above), trypsin-inhibitors and patatins have been considered as transgene-candidates for the protection against insect pests. Aller­gens can be found in all three categories.

It has been shown that plant-derived protease-inhibitors are potent inhibitors of larval growth (65). Concerning the use of trypsin inhibitors in transgenic plants, proof of concept was established in non-edible plants (66). Feeding of transgenic peas expressing a bean proteinase inhibitor to rats caused no harmful effects. The authors concluded that such GMOs might be used in the diet of farm animals in the future (67). The Kunitz soybean trypsin inhibitor binds IgE of soy challenge positive patients, indicating that this protein is an allergen (17, 18). Inhibitors of proteases and alpha-amylases are also found in high concentrations in plant storage organs such as cereal seeds or tubers (19-22).

Patatin is the major storage protein of potato tubers and has been reported to inhibit the growth of insect pest larvae (68). Recently, patatin has been demonstrated to represent a major allergen of potato designated Sol t 1(9). Moreover, Hevea latex contains a patatin-like allergen with sequence homology to Sol t 1, designated Hev b 7 (10, 69).

 

Resistance to bacteria

LTPs can take part in plant defense, as some LTPs have potent antifungal and antibacterial activities (49-50). An enhanced tolerance to bacterial pathogens was achieved in tobacco transformed with a barley lipid transfer protein gene. The results obtained encourage the use of this strategy also in crop plants (70)  LTPs are important allergens of the Prunoideae, such as peach, plum and cherry (51). The LTP of peach was named Pru p 3, LTP of aplle Mal s 3, respectively, by the interna­tional allergen nomenclature committee (52-56). IgE-mediated reactions to beer can be caused by barley-LTP which is involved in beer foam formation (57).

 

Improvement of nutritive value

Mammalian organisms are incapable of synthesizing essential amino acids. There­fore, one of the major targets of genetically modifying crop plants is the improvement of their amino acid composition by introducing heterologous seed storage proteins, which are rich in essential amino acids, such as albumins. However, several exam­ples of albumins exist showing that these molecules also possess allergenic properti­es. The major allergens of  Sin a 1 (yellow mustard), and Bra j 1 (oriental mustard), can elicit allergic reactions (1-3). The 2S albumins are also found to be allergens, present abundantly in nuts, e.g. in the Brazil nut and the English walnut (5-8). Plans for commercial development of GMOs expressing the brazil nut 2S albumin were abandonned because of the proteins allergenicity (71). However, the expression of an amaranth albumin gene in transgenic potato was not associated with allergenic complications (72).


Conclusion

The classification of proteins into certain families according to their biological functions facilitates the unmasking of potential allergens encoded by genes used for the transfection of plants. Many PR proteins that represent very attractive candidates for the production of transgenic plants are allergens. Several of the seed storage proteins that are candiates for improving nutritive value of crop plants have also been identified as allergens.  It is suggested to apply detailed investigations whenever proteins with these characteristics are taken into consideration.

 

References to this chapter

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2. Monsalve RI, Gonzáles de la Peña MA, Menéndez-Arias L, López-Otín C, Villalba M, Rodríguez R. Characterization of a new oriental-mustard (Brassica juncea) allergen, Bra j 1E: detection of an allergenic epitope. Biochem J 1993;293:625-32.

 

3. Gonzáles de la Peña MA, Monsalve RI, Batanero E, Villalba M, Rodríguez R. Expression in Escherichia coli of Sin a 1, the major allergen from mustard. Eur J Biochem 1997;237: 827-32.

 

4. Monsalve RI, Gonzáles de la Peña, MA, López-Otín, C, Fiandor A, Fernández C, Villalba M, et al. Detection, isolation and complete amino acid sequence of an aeroallergenic protein from rapeseed flour. Clin Exp Allergy 1997;27:833-41.

 

5. Gander ES, Holmstroem KO, De Paiva GR, De Castro LAB, Carneiro M, Grossi de Sá MF. Isolation, characterization and expression of a gene coding for 2S albumin from Bertholletia excelsa (Brazil nut). Plant Mol Biol 1991;16:437-48.

 

6. Teuber SS, Dandekar AM, Peterson R, Sellers CL. Cloning and sequencing of a gene encoding a 2S albumin seed storage protein precursor from English walnut (Juglans regia). J Allergy Clin Immunol 1998;101:807-14.

 

7. Teuber SS, Jarvis KC, Dandekar AM, Peterson, WR, Ansari, AA. Identification and clonin of a complemen­tary DNA encoding a vicilin-like proprotein, Jug r 2, from English walnut kernel (Juglans regia), a major food allergen. J Allergy Clin Immunol 1999;104:1311-20.

 

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9. Seppälä U, Alenius H, Turjanmaa K, Reunala T, Palosuo T, Kalkkinen, N. Identification of patatin as a novel allergen for children with positive skin prick test responses to raw potato. J Allergy Clin Immunol 1999;103:165-71

 

10. Kostyal DA, Hickey VL, Noti JD, Sussman GL, Beezhold DH. Cloning and characterization of a latex allergen (Hev b 7): homology to patatin, a plant PLA2. Clin Exp immunol 1998;112:355-62. Kostyal DA, Hickey VL, Noti JD, Sussman GL, Beezhold DH. Cloning and characterization of a latex allergen (Hev b 7): homology to patatin, a plant PLA2. Clin Exp immunol 1998;112:355-62.

 

 

11. Burks AW, Williams LW, Connaughton C, Cockrell G, O'Brien T, Helm RM. Identification and characteri­zation of a second major peanut allergen, Ara h II, with use of sera of patients with atopic dermatitis and positive peanut challenge. J Allergy Clin Immunol 1992;90:962-9.

 

12. Stanley JS, King N, Burks AW, Huang SK, Sampson H, Cockrell G, et al. Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2. Arch Biochem Biophys 1997;342:244-53.


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17. Burks AW, Cockrell G, Connaughton C, Guin J, Allen W, and Helm RM. Identification of peanut agglutinin and soybean trypsin inhibitor as minor legume allergens. Int Arch Allergy Immunol 1994;105:143-9.

 

18. Moroz LA, Lang WH. Kunitz soybean trypsin inhibitor. A specific allergen in food anaphylaxis. New Engl J Med 1980;302:1126-28.

 

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23. Valenta R, Duchene M, Ebner C, Valent P, Silaber P, Deviller P, et al. Profilins constitute a novel family of functional plant pan-allergens. J Exp Med 1992;175:377-85.

 

24. Van Ree R, Voitenko V, van Leeuwen WA, Aalberse RC. Profilin is a cross-reactive allergen in pollen and vegetable foods. Int Arch Allergy Immunol 1992;98: 97-104.

 

25. Kleber-Janke T, Crameri R, Appenzeller U, Schlaak M, Becker WM. Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology. Int Arch Allergy Immunol 1999;119:265-74.

 

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34. Diaz-Perales A, Collada C, Blanco C, Sánchez-Monge R, Carrillo T, Aragoncillo C, et al. Class I chitinases with hevein-like domains, but not class II enzymes, are relevant chestnut and avocado allergens. J Allergy Clin Immunol 1998;102:127-33.

 

35. Sowka S, Hsieh L-S,  Krebitz M, Akasawa A, Martin B, Starrett D, et al. Identification and cloning of Prs a 1, a 32 kDa major allergen of avocado and its expression in Pichia pastoris. J. Biol. Chem. 1998;273:28091-7.

 

36. Chen Z, Posch A, Cremer R, Raulf-Heimsoth M, Baur X. Identification of hevein (Hev b 6.02) in Hevea latex as a major cross-reacting allergen with avocado fruit in patients with latex allergy. J Allergy Clin Immunol 1998;102:476-81.

 

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38. Hsieh LS, Moos M, Lin Y (1995). Characterization of apple 18 and 31 kd allergens by microsequencing and evaluation of their content during storage and ripening. J Allergy Clin Immunol 1995;96:960-70.

 

39. Inschlag C, Hoffmann-Sommergruber K, O'Riordain G, Ahorn H, Ebner C, Scheiner O, et al. Biochemical characterization of Pru a 2, a 23-kD thaumatin-like protein representing a potential major allergen in cherry (Prunus avium). Int Arch Allergy Immunol 1198;116:22-8.

 

40. Jensen-Jarolim, E., Santner, B., Leitner, A., Grimm, R., Scheiner, O., Ebner, C., et al. Bell peppers (Capsicum annuum) express allergens (profilin, pathogenesis-related protein P23 and Bet v 1) depending on the horticultural strain. Int Arch Allergy Immunol 1998;116:103-9.

 

41. Hänninen AR, Mikkola JH, Kalkkinen N, Turjanmaa K, Ylitalo L, Reunala T, et al. Increased allergen production in turnip (Brassica rapa) by treatments activating defense mechanisms. J Allergy Clin Immunol 1999;104:194-201.

 

42. Vanek-Krebitz M, Hoffmann-Sommergruber K, Laimer da Camara Machado M, Susani M, Ebner C, Kraft D, et al. Cloning and sequencing of Mal d 1, the major allergen from apple (Malus domestica), and its immunological relationship to Bet v 1, the major birch pollen allergen. Biochem Biophys Res Comm 1995;214:538-51.

 

43. Scheurer S, Metzner K, Haustein D, Vieths S. Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen. Mol Immunol 1997;34:619-29.

 

44. Breiteneder H, Hoffmann-Sommergruber K, O'Riordain G, Susani M, Ahorn H, Ebner C, et al. Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens) and its immunological and structural relationships to a group of 17-kDa tree pollen allergens. Eur J Biochem 1995;233:484-9.

 


45. Hoffmann-Sommergruber K, O'Riordain G, Ahorn H, Ebner C, Laimer da Camara Machado M, et al. Molecular characterization of Dau c 1, the Bet v 1 homologous protein from carrot and its cross-reactivity with Bet v 1 and Api g 1. Clin Exp Allergy 1999;29:840-7.

 

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63. Liu D, Ragothama KG, Hasegawa PM, Bressan RA. Osmotin overexpression in potato delays development of disease symptoms. Proc Natl Acad Sci USA 1994;91:1888.

 

64. Lee HI, Raikhel NV. Prohevein is poorly processed but shows enhanced resistance to a chitin-binding fungus in transgenic tomato plants. Braz J Med Biol Res 1995;28:743-50.

 

65. Harsulkar AM, Giri AP, Patankar AG, Gupta VS, Sainani MN, Ranjekar PK, Deshpande VV. Successive use of non-host plant proteinase inhibitors required for effective inhibition of Helicoverpa armigera gut proteinases and larval growth. Plant Physiol 1999;121:497-506.

 


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67. Pusztai A, Bardocz GG, Alonso R, Chrispeels MJ, Schroeder HE, Tabe LM, Higgins TJ. Expression of the insecticidal bean alpha-amylase inhibitor transgene has minimal detrimental effect on the nutritional value of peas fed to rats at 30 % of the diet. J Nutr 1999;129:1597-1603.

 

68. Strickland JA, Orr GL, Walsh TA. Inhibition of Diabrotica larval growth by patatin, the lipid acyl hydrolase from potato tubers. Plant Physiol 109;667-74:1995.

 

69. Sowka S, Wagner S, Krebitz M, Arija-Mad-Arif S, Yusof F, Kinacyian T, Brehler R, Scheiner O, Breiteneder H. cDNA cloning of the 43-kDa latex allergen Hev b 7 with sequence similarity to patatins and its expression in the yeast Pichi pastoris. Eur j Biochem 1998;255:213-19.

 

70. Molina A, Garcia-Ulmedo F. Enhanced tolerance to bacterial pathogens caused by the rtansgenic expression of barley lipid transfer protein LTP2. Plant J 1997;12:669-675.)

 

71. Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK. Identification of a Brazil-nut allergen in transgenic soybeans. New Engl J Med 1996;334:688-92.

 

72. Chakraborty S, Chakraborty N, Datta S. Increased nutritive value of transgenic potato expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus Proc. Natl Acad Sci USA 2000, in press.






The implications of IgE against carbohydrate determinants for GMO’s

 

It is known for approximately two decades now, that IgE antibodies of pollen allergic patients can be directed to complex N-linked glycans on pollen glycoproteins QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\011\01\01\00\01\00\00\01\00\00\00¿\00ðWÅ\00\00\00\00ì\05\5CÅ\00\00\00\00\10ß\5CÅ\10ß\5CÅ\0Cß\5CÅ;®!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03102\03102\00\03\00 1. These IgE antibodies are extremely crossreactive between different pollen, vegetable foods and even foods of invertebrate animal origin. The structural basis for this high degree of crossreactivity is largely elucidated QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\012\01\01\00\01\00\00\01\00\00\00¿\00ðWÅ\00\00\00\00ì\05\5CÅ\00\00\00\00\10ß\5CÅ\10ß\5CÅ\0Cß\5CÅ;®!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03494\03494\00\03\00 2. Essentially, plant complex N-glycans have two typical monosaccharide substitutions that are not found in mammals: an a(1,3)-fucose linked to the proximal N-acetylglucosamine and a b(1,2)-xylose linked to the core mannose. These monosaccharides are pivotal in immunogenicity and allergeni­city of plant N-glycans. There is a dispute concerning the clinical relevance of N-glycan specific IgE antibodies. Some groups consider these IgE responses clinically irrelevant QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\013\01\01\00\01\00\00\01\00\00\00¿\00ðWÅ\00\00\00\00ì\05\5CÅ\00\00\00\00\10ß\5CÅ\10ß\5CÅ\0Cß\5CÅ;®!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03122\03122\00\03\00 3. Patients with carbohydrate-specific IgE antibodies frequently demonstrate a positive in vitro test results for vegetable foods, but they do not have clinical food allergy. This implies that these antibody responses would preferably not be picked up in in vitro food allergy diagnostics QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\014\01\01\00\01\00\00\01\00\00\00¿\00ðWÅ\00\00\00\00ì\05\5CÅ\00\00\00\00\10ß\5CÅ\10ß\5CÅ\0Cß\5CÅ;®!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03495\03495\00\03\00 4. There are however also groups that have reported clear biological activity of carbohydrate-specific IgE antibodies in basophil histamine release assays QUOTE{ ADDIN REFMAN #\11\05ê\19\01\00\00\00\015\01\01\00\01\00\00\01\00\00\00¿\00ðWÅ\00\00\00\00ì\05\5CÅ\00\00\00\00\10ß\5CÅ\10ß\5CÅ\0Cß\5CÅ;®!C:\5CAPPS\5CWinRM8N\5CProgram\5Crefmanrvr\03\00\03124\03124\00\03\00 5. Whether this biological activity is linked to clinical food allergy was left in the dark in these reports. 


What is the relevance of N-glycan reactive IgE antibodies for GMO’s? If proteins with putative N-glycosylation sites are expressed in transgenic plants, they will be glycosyated with plant N-glycans. These structures will thus be transformed into IgE-binding structures. This does not imply an extra allergenic risk for GMO foods, because they are full of glycoproteins carrying IgE-binding N-glycans anyway,with or without an additional transgene for a glycoprotein. If the lack of biological activity of anti-carbohydrate IgE is confirmed, this has repercussions for the selection of sera to be used for judging whether a transgene codes for an allergen or not. Sera with anti-carbohydrate IgE will recognize virtually any plant- or  invertebrate animal-derived glycoprotein. The candidate transgene will then unjustly be designated as a potential allergenic risk on the basis of recognition by anti-carbohydrate IgE. Because sera from clinically well-defined food allergic partients are rare, it might sometimes be tempting to use these highly crossreactive sera. This should preferably be avoided.

 

References for this chapter

 

1.       Aalberse RC, Koshte V, Clemens JGJ. Immunoglobulin E antibodies that crossreact with vegetable foods, pollen and Hymenoptera venom.  J Allergy Clin Immunol 1981;68:356-364.

 

2.       van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, Villalba M, Koppelman S, Aalberse R, Rodriguez R, Faye L, Lerouge P. beta(1,2)-Xylose and alpha(1,3)-Fucose Residues Have a Strong Contribution in IgE Binding to Plant Glycoallergens.  J Biol Chem 2000;275:11451-11458.

 

3.       van der Veen MJ, van Ree R, Aalberse RC, Akkerdaas JH, Koppelman SJ, Jansen HM, van der Zee JS. Poor biologic activity of cross-reactive IgE directed to carbohydrate determinants of glycoproteins.  J Allergy Clin Immunol 1997;100:327-334.

 

4.       van Ree R, Akkerdaas J, van Leeuwen A, Fernandez-Rivas M, Asero R, Knul-Brettlova V, Knulst A, Aalberse R. New perspectives for the diagnosis of food allergy.  Allergy Clin Immunol Intern 1999;12:7-12.

 

5.       Batanero E, Crespo JF, Monsalve R, Martïn-Esteban M, Villalba M, Rodríguez R. IgE-binding and histamine-release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e 1.  J Allergy Clin Immunol 1999;103:147-153.

 





Detection methods for allergenic transgene products

 

There are several reasons why it is important that the content of potential allergens can be precisely and sensitively determined in GMO foods. Since it is the ultimate goal to secure safe food consumption for the individual patient, the allergic popula­tion, and the society in total, both consumers, producers, health care personal, and legislators should be able to have information on the concentration of ingredients in a given food. It is important to emphasize, that this information not only pertains to the actually products of modified genes, but to all potential allergens, since the amount of gene products that are native to the organism, may very well be changed by the introduction of a new or changed gene in the organism.

 

Determination of potential allergens by biological methods

The biological activity of food allergens or mixtures thereof may be determined by various in vivo and in vitro methods that may quantitatively or semi-quantitatively express the combined effects of individual allergenic molecules in a mixture. Even in the rare case of testing an individual food allergen molecule, a response will emerge that is only declared relatively to other allergenic substances or mixtures. Thus, an important feature of testing the biological activity of mixtures is the lack a response which can be directly linked to individual molecular entities, and this put special emphasis on the definition of both the test systems and the mixtures that are tested.

 

The biological activity of a substance in relation to food allergy may be understood in various ways. In the context of this review only food allergic diseases believed to be mediated by immunoglobulin E (IgE) will be considered even though adverse reactions to foods include other disease entities and even the term food allergy may comprise diseases elicited by several other mechanisms (1).


The term allergenic may be understood both as the capacity to sensitize, i.e. induce an IgE immune response, and as the capacity to elicit an allergic reaction in an individual already sensitized. In this chapter only the latter meaning will be discussed corresponding to the left branches of the decision trees. The induction of an allergic reaction in an already sensitized individual, has been much more successfully investigated and as described below numerous models exist for determination of the biological potency (Table 1), which lists the various test procedures in the opposite direction of what is seen in the decision trees .

Table 1.

 

TBL 1

 

Target

 

 

Species

 

Test system

 

Examples

 

In vivo

 

Entire orga­nism

 

Human

 

Challenge of allergic patients

 

DBPCFC, open challenges

 

Experimental animals

 

Peroral challenge of animals

 

Anaphylactic response

 

Skin

 

Human

 

Skin testing of allergic pati­ents

 

Skin prick tests, intrademal tests

 

Experimental animals

 

Actively or passively sensiti­zed animals

 

PCA

 

In vitro

 

Ba­sophils

 

Human

 

Actively or pas­sively sensiti­zed basophils + allergen

 

Basophil hista­mine release, cord blood basophil histamine release

 

Basophil or mast cells

 

Humanized, i.e. trans-fected with a human IgE receptor

 

Passively sensitized cells

 

Mediator release

 

Mast cells

 

Rodents

 

Peritoneal mast cells

 

Histamine or other mediator release

 

Modified from In vivo and in vitro techniques to determine the biological activity of food allergens (Review);  Poulsen LK Submitted.

 

The Table illustrates the hierarchy that exist among these test systems as challenge of human patients are considered as closest to the relevant biological response, i.e. elicitation of an actual allergic response, albeit under controlled and safe circumstan­ces. The next level in the hierarchy is to use the skin as a restricted and localized area for challenge. This system obviously involves the skin mast cells, which must be sensitized by IgE in order to respond to the offending allergen. Leaving the in vivo systems, the next step is to use the sensitized basophil granulo­cyte as a model for the sensitized mast cell present in the relevant organ of the patient. Moving further away from the actual patient, basophil from a non-allergic donor such as cord blood may even be used as an reagent which are then sensitized by IgE derived from an actual patient.

 

The above-mentioned human model systems all have their animal experimental counterparts which will not be further discussed since many of the parameters of the human systems discussed below, will also apply to animal models. The basic problem with experimental animals is to actually make them allergic. Although several immunization schemes - often parenteral - are available, which will readily produce an IgE response, it is still not known whether the mere presence of IgE specific to a food allergen gives a good prediction of allergenicity.

 

 

The human challenge model: Double-blinded, placebo-controlled food challenges


The ultimate determination of the biological activity of a food allergen or a mixture of these is the effect on a sensitized food allergic patient. The first report on double-blinded placebo controlled food challenges (DBPCFC) is probably a study of May, where asthmatic children were challenged with freeze-dried foods in capsules (2). By 1991 Bock and Atkins reviewed about 500 challenges performed at their pediatric centre, and a pattern emerged with relatively few placebo reactions and a high degree of safety (3). Thus DBPCFC has been said to be the gold standard of food hypersensitivity diagnosis (4), and is recommended by the European Academy for Allergy and Clinical Immunology as the only conclusive evidence of a food allergy, provided it is performed properly (1).

 

For both practical and ethical reasons it is obvious, that patients cannot be routinely challenged to potential allergenic preparations. Thus the main reason for conducting controlled food challenges in patients is to verify or rule out a suspicion of food allergy, establishing a clinical diagnosis for the benefit of the patient. It is possible however to perform clinical trials on allergic patients in order to obtain knowledge about the allergenicity of GMO foods, provided that all other toxicologal and safety issues have been satisfactory solved..

 

Skin tests in humans


For the standardization of inhalation allergens the skin test has been the most important tool, and it is the recommended method for biological standardization of allergen extracts (5; 6). For this reason skin tests have also been used extensively for diagnosis of food allergy (1), and it seems well justified to use it for biological activity measurements of food allergens including GMO-foods. The rationale behind skin testing is that by introducing a small volume of allergen in the skin - either intradermally or via a small puncture of the stratum corneum as in the skin prick test - mast cells sensitized with specific IgE are activated via allergen cross-linking of this IgE. The activation of mast cells results in release of mediators - primarily histamine - which induces a wheal and flare reaction of the skin. Within a certain concentration range there is a dynamic response, i.e. a wheal and flare with a larger area develop after application of a higher concentration of allergen. The biological response is measured by planimetry as the area of the wheal or the flare (7; 8) and the result may be quantified by end-point-titration, i.e. the highest concentration in a titration which produces a negative response, or by comparison with a standard, typically histamine in a concentration of 10 mg/ml. The patient must - besides being well-defined as a patient - fulfill certain conditions such as an intact skin, lack of dermo­graphism, and abstinence from drugs such as antihistamines which will dramatically inhibit the skin reaction (9). For ethical and safety reasons the test substance must be assured to be without infectious or toxicological potential besides its allergenic properties. A detailed outline of the technique is given in the guidelines, and will not be discussed here, but some points of special relevance for food allergens and GMO foods should be mentioned, however. The guidelines recommend the use of 20 patients with symptoms of moderate severity, but with the number of available patients that have undergone a DBPCFC-procedure this may present a problem in many centres, especially if - for ethical reasons - only adults or adolescents are selected. Moreover, infants and small children have a good prognosis for outgrowing their food allergy (10) early in their life, which makes them less suited for participation in safety studies. Due to the paucity of DBPCFC+ patients and the individual respon­ses which may be quite varying, it can be difficult to perform a sufficient number of skin tests to ensure safety of the GMO.

 

Effector cells in vitro: Experimental systems for mast cell or basophil activation

As an alternative to the skin test the basophil granulocytes which are believed to be sensitized analogously to skin mast cells have been used extensively for many immunological studies of the allergic response (11,12). Being an in vitro method this technique has obvious advantages compared to the skin tests, since less strict requirements are posed on the test substance regarding non-toxicity and non-infectivity, albeit it should not be cytotoxic in the applied concentrations. For studies of inhalation and food allergy histamine release tests are correlating well with other measures of  IgE sensitization, such as skin prick tests or determination of specific IgE in plasma (13-16), and based on these findings it has been suggested to use the technique for biological standardization of allergen extracts including food allergens.

 

The direct histamine release method uses blood from a sensitized patient, and this limits the practical possibilities for running the method to patient-near centres, since the whole blood must be used within 24 h after drawing of the blood. This obstacle can be overcome by combining basophils from a non-sensitized person with serum containing specific and relevant IgE-antibodies. In its original form, basophils from adult donors were stripped of their original IgE by a brief treatment with low pH, followed by a new incubation with the sensitizing serum (12; 17). More recently cord blood basophils have been used as recipient cells, which has the advantage of eliminating the low pH IgE dissociation step, which may interfere with the biological functions of the cells. It is conceivable that basophil cell lines, such as the KU812 (18) or animal cell lines transfected with the the human FcεRI, i.e. the IgE high affinity receptor (19), may also be used as recipient cells for this purpose.

 

Determination of potential allergens by biochemical methods

A number of systems for biochemical detection of allergens are listed in Table 2. A pure system related to the allergic patient can be obtained by immunochemical assays detecting IgE-allergen binding directly or indirectly by inhibition designs. More indirect methods involve the use of animal antibodies for immunochemical detection, or molecular biology methods for detection via the DNA or mRNA levels.

 

Table 2. Hierarchy of biochemical test systems for testing of allergenic potency of pure substances or mixtures.

 

 

Vivo/

vitro

 

Target

 

 

Species

 

Test system

 

Examples

 

In vitro

 

IgE

 

Human

 

(Inhibition of) IgE-allergen bin­ding in immunochemical as­says

 

RAST or RAST-inhibition

 

IgG/IgE

 

Experimental animal inclu­ding mono­clonal antibodi­es, chimeric antibodies etc.

 

Immunochemical assays

 

ELISAs, Dip­sticks etc.

 

Phage display antibodies

 

Measurement of protein content in allergenic sour­ces

 

Immunochemical assays

 

ELISA-type assays

 

In vitro

(indirect)

 

PCR

 

Direct measurement of DNA coding for the gene in question

 

Polymerase chain reaction (PCR)

 

PCR

 

RT-PCR

 

Direct measurement of mRNA encoding the pro­tein in question

 

Reverse transcriptase-PCR

 

RT-PCR

 

Modified from In vivo and in vitro techniques to determine the biological activity of food allergens (Review);  Poulsen LK Submitted.

 

In vitro studies of IgE-allergen binding: RAST and RAST-inhibition

Since the binding between the allergen and the IgE is central in eliciting of the biological function in the test systems described above, it is obvious to use a test system that measures this binding, and the RadioAllergoSorbentTest and modifica­tions of this play an important role in allergen standardization. The initial design of the RAST was based on the use of dextran-derived materials (20; 21) but later solid phases have comprised the widely used paper discs (22), aluminium hydroxide gel (23), polystyrene tubes (24), cellulose polymers (25; 26), and magnetic micropar­ticles (27). Reviews of the available technologies and a discussion of method evaluation have been given in (28; 29).

 

Other immunochemical assays

In the Table 2 is also mentioned the production of animal antibodies to individual allergens and the use of such antibodies in ELISAs etc, forms the border between the biological assays and molecular identification of individual allergens A large number of animal antibodies has been raised against known or suspected food allergens, and may be used for testing. Several commercial assays for well-known food allergens have been described (30-33) [More references to be added, please give me all your inputs!] (summarized in Table 3) and more are to follow. A word of caution should be issued: If gene products are only slightly modified, it is important to carefully check how an antibody raised to the native protein (allergen) will react to the modified protein. In some cases it may be necessary to raise new antibodies to the modified protein.

 

Molecular biology assays

The final line in the Table 2 mentions the possibility of using determination of mRNA or DNA as a surrogate marker of the presence of allergens. Since the DNA may be transcribed with varying efficiency in the plant and the correlates between mRNA levels and protein levels may also vary, these measures may only be sei-qualitatively related to the potential allergen level. On the other hand, the molecular biology are very sensitive and may be the only way to determine extremely low levels of (genes coding for) allergens in GMO foods. Moreover, since the sequence of the targetted genes is often known, these techniques may be able to differentiate between native and genetically modified versions of the same gene products, since small dissimilari­ties may evade detection by immunochemically based techniques.

 

References to this chapter

1.      Bruijnzeel Koomen, C.A., Ortolani, C., Aas, K., Bindslev-Jensen, C., Björk­sten, B., Moneret-Vautrin, D., & Wüthrich, B. (1995).  EAACI Position paper: Adverse reactions to food.  Allergy,  50, 623-635.

 

2.      May, C.D. (1976).  Objective clinical and laboratory studies of immediate hypersensitivity reactions to foods in asthmatic children.  J Allergy Clin Immunol,  58, 500-515.


3.      Bock, A.S., & Atkins, F.M. (1990).  Patterns of food hypersensitivity during sixteen years of double-blind, placebo-controlled food challenges.  J Pediatr,  117, 561-567.

 

4.      Bindslev-Jensen, C. (1998).  Food al­lergy.  BMJ.,  316, 1299-1302.

 

5.      European Academy of Allergology and Clinical Immunology. (1989).  Skin tests used in type I allergy testing. Position paper. Allergy 44: (Suppl. 10).  Copenhagen:  Munksgård.

 

6.      Nordic Council on Medicines. (1989­).  Registration of allergen preparations. Nordic Guidelines.  (2 ed.).  Uppsala:  NLN Publication No 23.

 

7.      Poulsen, L.K., Liisberg, C., Bindslev-Jen­sen, C., & Malling, H.-J. (1993).  Precise area determination of skin-prick tests: validation of a scanning device and software for a personal computer.  Clin Exp.Allergy,  23, 61-68.

 

8.      Poulsen, L.K., Bindslev-Jensen, C., & Rihoux, J.P. (1994).  Quantitative determination of skin reactivity by two semiautomatic devices for skin prick test area measurements.  A­gents and Actions,  41, C134-C135

 

9.      Petersen, L.J., Bindslev-Jensen, C., Poul­sen, L.K., & Malling, H.J. (1994).  Time of onset of action of acriva­stine in the skin of pollen- allergic subjects. A double-blind, randomized, placebo-controlled comparative study.  Allergy,  49, 27-30.

 

10.      Host, A., & Halken, S. (1990).  A prospective study of cow milk allergy in Danish infants during the first 3 years of life. Clinical course in relation to clinical and immunological type of hypersensitivity reaction.  Allergy,  45, 587-596.

 

11.      Lichtenstein, L.M., & Osler, A.G. (1966).  Studies on the mechanisms of hypersensitivity phenomena. XII. An in vitro study of the reaction be­tween ragweed pollenantigen, allergic human serum and ragweed sensitive human leucocytes.  J Im­munol,  96, 169-179.

 

12.      Stahl Skov, P., Permin, H., & Malling, H.-J. (1977).  Quantitative and qua­litative estimations of IgE bound to ba­sophil leukocytes from hay fever patients.  Scand.J Immunol.,  6, 1021-1028.

 

13.      Østergaard, P.A., Ebbesen, F., Nolte, H., & Skov, P.S. (1990).  Basophil hista­mine release in the diagnosis of house dust mite and dander allergy of asthmatic children. Comparison between prick test, RAST, basophil histamine release and bron­chial provo­cation.  Allergy,  45, 231-235.

 

14.     Nolte, H., Storm, K., & Schiøtz, O. (1990).  Diagnostic value of a glass fibre-based histamine analysis for allergy testing in children.  Allergy,  45, 1-11.

 

15.      Hansen, T.K., Bindslev-Jensen, C., Stahl Skov, P., & Poulsen, L.K. (1996).  Codfish allergy in adults. Specific tests for IgE and histamine release vs double-blind, placebo-controlled challenges.  Clin Exp.Allergy,  26, 1276-1285.

 

16.      Nørgaard, A., Skov, P.S., & Bindslev-Jensen, C. (1992).  Egg and milk allergy in adults: comparison between fresh foods and commercial allergen extracts in skin prick test and histamine release from baso­phils.  Clin Exp.Allergy,  22, 940-947.

 

17.      Pruzansky, J.J., Grammer, L.C., Patter­son, R., & Roberts, M. (1983).  Dissociation of IgE from receptors on human basophils. I. Enhanced passive sensitization for histamine release.  J Immunol,  131, 1949-1953.

 

18.     Hara, T., Yamada, K., & Tachibana, H. (1998).  Basophilic differentiation of the human leukemia cell line KU812 upon treatment with interleukin-4.  Biochem.Biophys.Res.Commun.,  247, 542-548.

 

19.     Lowe, J., Jardieu, P., VanGorp, K., & Fei, D.T. (1995).  Allergen-induced hista­mine release in rat mast cells transfec­ted with the alpha subunits of Fc epsilon RI.  J Immunol Methods,  184, 113-122.


20.      Ishizaka, K., Ishizaka, T., & Hornbrook, M.M. (1967).  Allergen-binding activity of gE, gG and gA antibodies in sera from atopic patients. In vitro measuremnts of reaginic antibody.  J Immunol,  98, 490-501.

 

21.      Wide, L., Bennich, H., & Johansson, S.G.O. (1967).  Diagnosis of allergy by an in vitro test for allergen antibodies.  Lancet,  ii, 1105-1107.

 

22.      Ceska, M., Eriksson, R., & Arga, J.M. (1972­).  Radio-immunosorbent assay of allergens.  J.Allergy,  49, 1-9.

 

23.      Poulsen, L.K., & Weeke, B. (1985).  Aluminium hydroxide adsorbed allergens used in modified RAST.  Allergy,  40, 405-416.

 

24.      Poulsen, L.K., Pedersen, M.F., Malling, H.-J., Søndergaard, I., & Weeke, B. (1989).  Maxisorp RAST. A sensitive method for detection of absolute quantities of antigen-specific IgE.  Allergy,  44, 178-189.

 

25.      Ewan, P.W., & Coote, D. (1990).  Evaluation of a capsulated hydrophilic carrier polymer (the Immuno­CAP) for measurement of specific IgE antibodies.  Allergy,  45, 22-29.

 

26.      Bousquet, J., Chanez, P., Chanal, I., & Michel, F.B. (1990).  Comparison between RAST and Pharmacia CAP system: A new automated specific IgE assay.  J Allergy Clin Immunol,  85, 1039-1043.

 

27.      Kleine-Tebbe, J., Eickholt, M., Gätjen, M., Brunnée, T., O'Connor, A., & Kunkel, G. (1992).  Compari­son between Magic Lite- and CAP-system: two automated specific antibody assays.  Clin Exp.Allergy,  22, 475-484.

 

28.      Matsson, P., Hamilton, R.G., Adkinson, N.F., Esch, R., Homburger, H.A., Maxim, P., & Williams, B. (1996).  NCCLS. Evaluation methods and anlytical performance characteristics of immunological assays form human immunoglobulin E (IgE) antibodies of defined allergen specifici­ties; Proposed guidelines (NCCLS document I/LA20-P).  (1 ed.).  Wayne, PA, USA:  NCCLS.

 

29.      Bindslev-Jensen, C., & Poulsen, L.K. (1997­).  In vitro diagnostic methods in the evaluation of food hypersensitivity. In D. D. Metcalfe, H. A. Sampson, & R. A. Simon (Eds.),  Food allergy: Adverse reactions to food and food additives. (pp. 137-150).  Cambridge, Massachusetts:  Blackwell Scientific Publications.

 

30.      Hlywka, J.J., Hefle, S.L., & Taylor, S.L. (2000).  A sandwich enzyme-linked immunosorbent assay for the detection of almonds in foods.  J Food Prot.2000.Feb.;63.(2.):252.-7.,  63, 252-257.

 

31.     Leduc, V., Demeulemester, C., Polack, B., Guizard, C., Le, G.L., & Peltre, G. (1999).  Immunochemical detection of egg-white antigens and allergens in meat products.  Allergy,  54, 464-472.

 

32.      Jeoung, B.J., Reese, G., Hauck, P., Oliver, J.B., Daul, C.B., & Lehrer, S.B. (1997).  Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich ELISA.  J Allergy Clin Immunol,  100, 229-234.

 

33.      Yeung, J.M., & Collins, P.G. (1996).  Enzyme immunoassay for determination of peanut proteins in food products.  J AOAC.Int,  79, 1411-1416.

 






Clinical considerations

 

Adverse reactions to foods may be toxic or non-toxic in nature (Bruijnzeel-Koomen 1995, Anderson 1996). Non-toxic adverse reactions are either non-immune-mediated (food intolerance) or immune-mediated (food allergy). Allergic reactions may be either IgE-mediated or non-IgE-mediated (Bousquet 1997). Only those proteins which cause an IgE response in human can be assessed for the allergenicity in skin testing and in in vitro IgE-binding tests.


Allergenic food sources cover certain plant- and animal-derived foods. World-wide, 8 foods are considered to be the most important food stuffs causing IgE-mediated allergy, e.g. egg, milk, fish, crustacean, peanut, soybean, wheat, and tree nut (Sampson 1998). However, regional differences may exist depending on dietary references and cross-reactivities with inhalant allergens. In addition to these most common allergenic foods, many other foods are known to be allergenic such as several fruits, vegetables, and spices.

 

Symptoms of the IgE-mediated allergy typically involve the skin, respiratory and/or gastrointestinal tract (Bruijnzeel-Koomen 1995, Sampson 1999). However, a number of gastrointestinal food allergies are not IgE-mediated, but may be a result of a lectin, irritant or other type of effect. Therefore, understanding and identification of the mechanisms involved in adverse reactions to foods is an important prerequisite in selecting patients for tests on allergenicity of a genetically engineered food.

 

Symptoms of immunologic food allergy are classic immediate hypersensitivity reactions, e.g. respiratory manifestations such as rhinitis and asthma, dermatologic manifestation (urticaria, atopic dermatitis), GI manifestations or even anaphylaxis (Bruijnzeel-Koomen 1995, Anderson 1996, Bousquet 1997). Symptoms may derive from local buccal exposure or be systemic, and include tingling, laryngeal edema, diarrhoea, nausea, vomiting, bronchospasm, choking, anaphylaxis or even death.

 

Often, the only symptom from food allergy may be oral allergy syndrome (OAS) typically caused by fresh fruits, vegetables and spices (Kazemi-Shirazi 1999). In about 30-40% cases OAS is associated with pollen allergy (Bircher 1994). In OAS food allergens are in direct contact with the oral mucosa.

 

Foods are the most common cause of IgE-mediated anaphylaxis (Pumphrey 1996). World-wide, in fatal reactions peanut is the most common causative food allergen (Sampson 1998). However, also other foods such as milk, egg, fish, crustaceans, tree nuts, soy, celery, kiwi, and wheat may cause anaphylaxis (Foucard 1999). In addition, the assessment of the allergenicity of foods causing severe allergic reac­tions (anaphylaxis) only in association with exercise (e.g. exersice-indused anaphy­laxis, EIA) may turn out difficult (Shadick 1999).

 

The prevalence of food allergy has been investigated in a limited number of studies. The prevalence of food allergy among adult population is 1- 2 % (Sampson 1999). The figure is higher (up to 8%) among children who, however, often outgrow their food allergy before the 3rd birthday (Bock 1984). The prevalence figures are higher when OAS is taken into account. These phenomenena may hamper the selection of appropriate subjects for in vivo testing of a novel food.

 

Another major problem is the presence of cross-reactivity between various groups of foods, where detection of specific IgE antibodies towards a cross reacting food may or may not be reflectedcin the clinical situation. This create a need for evaluation also of cross reacting potential of new foods.

 


Foods contain several proteins, only a few of which are allergens. However, no fixed safety limit can be assessed under which no allergic reactions would occur. It is thus important to emphasize, that a safe lower level of allergenic foods (No Effect Level, where even the most sensitive patient does not react) probably does not exist.

 

Experience on protein quantities causing severe allergic reactions has mainly accumulated from inadverted allergen exposure and from allergen challenges. Usually, dosages eliciting allergic reactions are at milligram(s) level or less (Steinman 1996).

 

References for this chapter

 

Bruijnzeel-Koomen C, Ortolani C, Aa K, Bindslev-Jensen C, Björksten B, Moneret-Vautrin D, Wüthrich B. Adverse reactions to food. Position paper. Allergy 1995;50:623-35.

 

Anderson JA. Allergic reactions to foods. Crit Rev Food Science Nutr 1996;36 (Suppl.):S19-S38

 

Bousquet J, Metcalfe DD, Warner JO. Food allergy position paper of the Codex alimentarius. Allergy Clin Immunol Int 1997;9:10-21.

 

Sampson HA. Fatal food-induced anaphylaxis. Allergy 1998;53 Suppl 46:125-30.

 

Sampson HA. Food allergy. Part 1. Immunopathogenesis and disorders. J Allergy Clin Immunol 1999;103:717-28.

 

Kazemi-Shirazi L, Pauli g, Purohit A, Spitzauer S, Fröschl R, Hoffmann-Sommergruber K, Breiteneder H, Scheiner O, Kraft D, Valenta R. Quantitative IgE inhibition experiments with purified recombinant allergens indicate pollen-derived allergens as the sensitizing agents responsible for many forms of plant food allergy. J Allergy Clin Immunol 1999;105:116-25

 

Bircher AJ, van Melle G, Haller E, et al. IgE to food allergens are highly prevalent in patients allergic to pollens, with and without symptoms of food allergy. Clin Exp Allergy 1994;24:367-72.

 

Pumphrey RSH, Stanwoth SJ. The clinical spectrum of anaphylaxis in north-west England. Clin Exp Allergy 1996;26:1364-70.

 

Foucard T, Malmheden Yman I. A study on severe food reactions in Sweden -is soy protein an underestimated cause of food anaphylaxis? Allergy 1999;54:261-5.

 

Shadick NA, Liang MH, Partridge AJ, Bingham C, Wright E, Fossel AH; Sheffer AL. The natural history of exercise-induced anaphylaxis: survey results from a 10-year follow-up study. J Allergy Clin Immunol 1999;104:123-7.

 

Bock SA. Prospective appraisal of complaints of adverse reactions to foods in children during the first 3 years of life. Pediatrics 1987;79:683-8.

 

Steinman HA. “Hidden“ allergens in foods. J Allergy Clin Immunol 1996;98:241-50.

 





Risk Evaluation

 

At the FAO/WHO Consultations on Genetically modified foods in Rome, 1996, the following statements were launched (ref in[1]):

 

1        The transfer of genes from com­monly allergenic foods should be discouraged unless it can be documented that the gene transferred does not code for an allergen.


2        Foods found to contain an allergen transferred from the organism which provided the DNA should not be considered for marketing approval unless such products can be clearly identified in the marketplace and this identity will not be lost through distribu­tion and processing. Fur­ther, that labelling approaches may not be practical in these situations, and that particular problems exist for consumers who cannot read, or who may not be provided with labels.

3        Involved organizations should consider the appropriateness of, and/or actions to take, in respect to foods containing new protein(s) that are determined to have the charac­teristics of an allergen.

4        The identification of food allergens and the characteristics of these allergens that define their immunogenicity be encouraged.

 

The ILSI decision tree so far constitute the only guideline for assessment of potential change in allergenicity of genetically modified organisms[2]:

 

This decision tree divides GMO’s into foods where the sour­ce of gene stems from a known allergenic sour­ce or a source not known to be allergenic. Furthermore, the allergenic sour­ce is further subdivided into sources from the commonly allergenic foods (the big eight) and less commonly allergenic foods, namely the remaining foods, only accounting for approx. 10 per cent of the clinical reac­tions. This latter statement is however, limited to the classical type I allergic reactions, elicited by e.g. peanut, milk, egg, soy, fish, crusta­ceans but does not take into account the much more abundant reactions to to cross- or common reactivity between e.g. pollens and fruits/vegatab­les or Latex and fruits[3].

 

The „left side“ of the decision tree dealing with known allergenic proteins contains the test systems sufficient to rule out a potential risk to the allergic patient, provided the tests systems (both in vitro and in vivo) are used with material from high quality patients, fulfilling the EAACI guidelines[4] and using validated test proce­dures[5],[6]. The subdivision into common and less common foods is, however based on availability of test material rather than an actual risk assessment and should thus be left out - there are no data in literature supporting an increased risk for the actual patient to common food allergens than to less common food allergens.

 

­The „right side“ of the decision tree, fig 1, deals with inserted proteins, not known to be allergenic:

 

 This aproach is based on the follo­wing assumptions:

 

1      The optimal peptide length for bin­ding appears to be between 8 and 12 amino acids for T-cell epitopes and even longer for B-cell epito­pes[7]

2      All epitopes are sequential, and conformational epitopes are wit­hout significance

3      All relevant epitopes has already been sequenced.

4      The stability to di­gestion is a significant and valid parameter that distinguishes food aller­gens from non-allergens[8].


None of these statements has been proven  - there are examples of exceptions for all the above statements. Further­more, the test is also likely to identify conserved sequences that are unrelated to the allergenic potential of the proteins.Furthermore, harmless proteins might also be excluded form market based on these tests.

 

The EAACI risk evaluation procedure

It therefore suggested to add a screening procedure to be applied to gene modified foods, not previously known to be allergenic (the right side), fig 2:

 

The various subsequent steps in the evaluation procedure is commented in the following (The numbers refer to the flow chart).

 

- The „left side“ of the decision tree dealing with known allergenic proteins contains the test systems sufficient to rule out a potential risk to the allergic patient, provided the tests systems (both in vitro and in vivo) are used with material from high quality patients, fulfilling the EAACI guidelines and using validated test procedures.

 


                 After a negative outcome of  testing for sequence similarity to known aller­gens, the food is subjected to solid phase immunoassays screning for allerge­nicity using sera from patients with established allergy to major allergens, especially allergens, where cross-reactivity to foods are abundant (pollen allergics).

 

It is suggested to use as a minimum 3 * 10 patients allergic to birch, grass, artemisia respectivelyor to other relevant allergens.  There are major regional differences between reaction severity and plants in question within Europe. Allergens shou­ld therefore be included according to origin e.g. ragweed, Parietaria or other types of food or inhalant allergens according to type of GM-food in qu­estion.

 

Positive results in these tests trans­fer the evaluation of the food to the left side of the flow chart.

 

Sero­logical cross-reactions should also be dealt with by transferral to the left side of the decision tree (see -).

 

                 This step constitute considerations on the more uncertain aspects of a novel food. In this phase of evaluation, aspects like e.g. models for evaluation of a possible immunogenetic role as weel as considerations on a possible sensiti­zing potential­­, possiblestimulation of TH-2 system or creation of Neo-allergens­­. Also, the anticipated dose of intake and other aspects may be included.

 

Finally, the natural variability of allergens in wild type foods must be taken into account when assessing quantitative aspects of measurement of allergens in GMO’s.

 

At present, the methods for such evaluation procedures are not fully develo­ped. The upcome of animal models, which at present are not sufficiently developed for use may elucidate these aspects in the future.

 

                 After evaluation of the above parameters, it will be possible for the authorities to perform a proper risk assessment of the GMO in question.

 

The left side of the flow chart contains various levels of safety. A positive outcome in step  (DBPCFC) of course constitutes the highest possible risk, whereas previous experience with safe ingestion diminishes the absolute risk.

 

The in vivo and in vitro investigations (-) thus results in data concerning the absolute risk of introduction of the GMO in question to the market, whereas data from ‘ enables a calculation on the relative risk at a certain level (deter­mined in  -).

 

(her indsættes LKP-figuren).

 

References to this chapter.

 

 



[1].Bindslev‑Jensen C: Allergy risks of genetically engineered foods. Allergy 1998;53:58‑­61

 

[2]. Metcalfe DD, Astwood JD, Townsend R, Sampson HA, Taylor SL, Fuchs RL:  Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit Rev Food Sci Nutr 1996;36; Suppl:S165‑­86

[3]. Pastorello EA, Incorvaia C, Pravettoni V, Ortolani C:  Crossreactio­ns in food allergy  Clin Rev Allergy Immunol 1997;15(4):415‑27.

[4].Bruinjzeel-Koomen C, Ortolani C, Aas K, Bindslev-Jensen C, Björksten B, Moneret-Vautrin D, Wütrich B: Adverse reactions to food. Position Paper. Allergy 1995, 50: 623-635.

[5].Bindslev-Jensen C, Poulsen LK: In vitro diagnostic tests.  Chapter 7 In: Sampson HA, Simons E, Metcalfe DD: Food Allergy 2nd edition. Blackwell Scientific Publications, 1996, 137-150.

[6].C.Ortolani, C.Bruijnzeel-Koomen, U.Bengtson, C.Bindslev‑Jensen, B.Björksten,  A.Høst, M.Ispano, R.Jarish, C.Madsen, K.Nekam, R.Paganelli, LK.Poulsen &  B.Wütrich: Controversial aspects of adverse reactions to food. Allergy 1999, 54: 27-46.

[7].( Roth­bard Jb and Gefter ML: Inter­actions between immunogenic peptides and MHC proteins. Annu. Rev. Immunol. 9:527-xxx, 1991).

[8]. Astwood JD, Leach JN, Fuchs RL:  Stability of food allergens to digestion in vitro. Nat Biotechnol 1996;14:1269-1273