Technology has been used for many years to improve our food supply since the
first cultivation of crops such as wheat and barley in Mesopotamia in 6000 bc
and the domestication of animals such as sheep and goats in southwestern Asia
over 10,000 years ago. More recently, improvement of our food supply through
genetic manipulation by breeding was accelerated in the development of hybrid
crop varieties in the 1960s and 1970s (a period referred to as the green revolution),
which more than doubled the crop production in developing countries. Breeding
and selection have been used for many domesticated animal species that are food
sources. A good example is chicken, one of the more expensive meats in the 1940s,
and now one of the least expensive sources of meat. Cultivation, domestication,
breeding, and selection of certain traits of plants and animals have created
environmental impacts and major changes to human societies. Although use of
technology in breeding plants and animals is not new, new methods of biotechnology
incorporate genetic engineering, also referred to as molecular breeding. Genetic
engineering facilitates the selection, identification, and transfer of genes
encoding for a specific protein into the genome of another organism. This process
can determine which proteins are introduced and where they are expressed; in
most cases, only minute amounts of a protein need to be expressed to obtain
the desired trait.
In assessing the public health aspects of genetically engineered foods, it
is the proteins that are expressed that are of interest. Three possible modes
of adverse health effects have been hypothesized: toxicity, impaired nutrition,
and food allergy. Modifications of expression of protein in foods occur with
all kinds of plant breeding, and these theoretical concerns are not unique to
genetically engineered foods. However, because genetic engineering is a more
powerful tool for making such changes, government authorities in the United
States, Europe, Japan, Canada, and elsewhere have taken actions to regulate
this class of foods.
In this article, we describe various clinical aspects of food allergy relevant
to the assessment of novel proteins in genetically modified foods. Food allergy
is among a spectrum of adverse reactions that can result when an individual
ingests food or a food additive. Evaluation of novel proteins for potential
allergenicity is based on a fundamental understanding of the clinical and biological
aspects of these responses. Among food allergy researchers, a number of definitions
of food allergy have been used. For the purpose of this article, allergy is
defined as hypersensitivity and implies an immunologic reaction to food; other
(nonimmunologic) adverse reactions to food will be referred to as "intolerance."
This reflects the standard definitions of terms in the United States. It is
important to note that the revised nomenclature for allergy proposed by the
European Academy of Allergy and Clinical Immunology uses the terms "nonallergic
food hypersensitivity" instead of "intolerance," and "food allergy" (IgE-mediated
or not) instead of "hypersensitivity." Defined in this manner, food allergy
affects only those individuals who have developed an abnormal immunologic response
to food. To understand the public health impacts of food allergy, we must appreciate
both the prevalence in the population and the clinical spectrum of food allergy.
Although there are a number of clinical manifestations of food allergy, most
of the focus for clinical evaluation of genetically modified foods has been
on IgE-mediated anaphylaxis. Various expert committees, including a committee
of the International Life Sciences Institute, an advisory committee to the U.S.
Environmental Protection Agency (U.S. EPA), and committees convened by the Food
and Agriculture Organization (FAO) and the World Health Organization (WHO),
have developed decision trees for identifying whether novel proteins cause such
anaphylactic reactions. These decision trees are largely based on the clinical
and laboratory methods used to assess the potential allergenicity of foods.
These methods were developed for clinical purposes but now are being used to
predict allergenicity and for postmarket surveillance. The StarLink corn episode
provided an opportunity for researchers to assess the utility of the decision
tree as well as the ability to conduct postmarket surveillance for allergic
reactions in foods in commerce. None of the reported allergic reactions were
confirmed, and it is possible that this episode resulted from the media coverage
and public perceptions about the risks. This episode demonstrated the limitations
of using clinical tools for assessment of food allergy in postmarket assessment.
Another clinical issue is the potential for development of inhalation allergy
from novel proteins in foods.
Clinical Spectrum of Food Allergy
H. A. Sampson presented the clinical spectrum of adverse reactions to food,
including food allergies. Estimates of the prevalence of adverse food reactions
vary widely depending on whether they are defined using the "gold standard"--DBPCFC
(double-blind placebo-controlled food challenge)--or as reported reports by
patients and their families. In one prospective study of children, 6% of children
had food allergy or intolerance confirmed by DBPCFC, which is lower than the
28% among children whose parents reported adverse food reactions (Bock 1987).
A number of prospective studies indicate that allergy to cow's milk is prevalent
among 2.5% of children under the age of 2 years (Bock 1987; Hide and Guyer 1983;
Host et al. 1988; Schrander et al. 1993), with an overall prevalence rate at
some time during childhood of 6%. Some children are at greater risk; food allergy
occurs in 30% of children who have atopic dermatitis (Burks et al. 1988). Food
allergy is less common in adults, affecting between 1.5 and 2% of adults in
the United States (Jansen et al. 1994; Young et al. 1994).
The
development of food allergy most often occurs in early childhood before the
age of 3 years. It involves mechanisms related to the nature of the food allergen,
the gastrointestinal tract, and the immune system (Table 1) (Sampson 1993).
Food allergens are proteins, usually glycoproteins, and they generally share
certain physical and chemical characteristics (Table 1). Only a few foods are
known to cause the vast majority of allergic reactions: in children--eggs, peanuts,
milk, soy, and wheat; in adults--shellfish, fish, nuts, and peanuts. The gastrointestinal
tract has a number of nonspecific barriers to the entry of foreign proteins,
and one specific barrier, secretory IgA, produced by the immune system (Table
1). Nonetheless, food proteins can be absorbed into the bloodstream and carried
to target organs. Mechanisms have evolved to ensure that the immune system does
not attack one's own proteins ("self") and proteins in food. The general process
by which the immune system is programmed not to attack such proteins is called
"tolerance." Tolerance is therefore a barrier to the development of food allergy,
and when people become sensitized to food, this is a breakdown of tolerance.
Table 2
 |
Clinical manifestations. Food allergies are mediated either by
IgE or by other immunologic mechanisms. IgE-mediated reactions are the most
common in the general population. Non-IgE-mediated food allergies account
for a significant proportion of food allergies in infants and young children.
As shown in Table 2, food allergy can cause a multitude of clinical manifestations.
IgE-mediated food allergies. IgE-mediated food allergies typically
develop in an individual within minutes to hours after ingesting a food allergen.
IgE-mediated food allergies can have a longer onset in the case of food that
has been ingested more frequently, thus blunting the immediate symptoms and
resulting in symptoms of a chronic inflammatory reaction that may last for several
days. A number of skin manifestations can occur. On an immediate basis, acute
urticaria/angioedema often occurs following the ingestion or contact with a
food. Chronic urticaria/angioedema related to food allergy is rare (Champion
et al. 1969; Volonakis et al. 1992). Children may develop atopic dermatitis.
During blinded food challenges, an itchy, red, morbilliform (measleslike) rash
develops within 10 to 90 min of allergen ingestion (Sampson and McCaskill 1985).
Repeated ingestion of the allergen may result in the development of an itchy,
eczematous rash (Sampson 1992).
Food allergy can cause both upper and lower respiratory symptoms (Bock 1992;
James et al. 1994). Recently, James et al. (1996) established that food allergy
can provoke increased airway hyperreactivity in food-allergic patients with
asthma, and two studies (Novembre et al. 1988; Oehling and Baena Cagnani 1980)
demonstrated food-induced wheezing in 6-8% of unselected asthmatic patients.
Symptoms involving the oropharynx and gastrointestinal tract may occur within
minutes of ingesting a food allergen. Itching and swelling of the lips, tongue,
and soft palate as well as nausea, abdominal pain, vomiting, and diarrhea have
all been demonstrated secondary to food allergy. The oral allergy syndrome consists
of symptoms confined exclusively to the oropharynx and is most commonly reported
in patients with seasonal allergic rhinitis after the ingestion of one of a
variety of fresh fruits and vegetables (Ortolani et al. 1988; Pastorello et
al. 1994). Gastrointestinal anaphylaxis frequently accompanies symptoms in the
skin or respiratory tract and presents as nausea, abdominal cramping, vomiting,
and diarrhea. Repeated ingestion of food allergens in young children may induce
partial desensitization, resulting in less-obvious symptoms, e.g., gastroesophageal
reflux (GER) instead of projectile vomiting (Iacono et al. 1996). A minority
of patients with infantile colic (inconsolable, agonized crying, drawing up
of the legs, abdominal distention, and excessive gas associated with feeding
during the first several months of life) have symptoms attributed to IgE-mediated
food hypersensitivity (Sampson 1989).
Food-induced systemic anaphylaxis has been reported to be the leading single
cause of anaphylaxis in emergency departments in the United States (Kemp et
al. 1995; Yocum and Khan 1994 ). In two reports of fatal anaphylactic reactions
(Sampson et al. 1992; Yunginger et al. 1988), the authors noted that all subjects
had asthma, had unknowingly ingested the responsible food allergen, and had
tended to minimize the symptoms initially, and that the initiation of emergency
medical management was delayed. Anaphylactic shock in association with exercise
2-4 hr after the ingestion of certain foods is being recognized increasingly,
especially in young women (Horan and Sheffer 1991; Romano et al. 1995).
Non-IgE-mediated allergic reactions. Non-IgE-mediated allergic
reactions are believed to take several hours to days to develop, and a variety
of disorders have been delineated. A variety of gastrointestinal disorders believed
to have an immunologic basis have been described. Food-induced enterocolitis
syndrome is seen most frequently in young infants ingesting cow's milk- or soy-based
formulas. It generally presents between 1 week and 3 months of age, with protracted
diarrhea and projectile vomiting often severe enough to produce dehydration
(Powell 1978). The syndrome also is seen in exclusively breast-fed infants (secondary
to the passage of food proteins in maternal milk) and occasionally in older
children (associated with ingestion of egg, wheat, rice, peanut, nuts, chicken,
turkey, and shellfish). Benign eosinophilic proctocolitis, also present in the
first few weeks to months of life, is often secondary to cow's milk or soy,
although about half the infants are being exclusively breast-fed (Machida et
al. 1994; Odze et al. 1995). Patients appear clinically well and present only
with bloody stools (gross or occult) or hematochezia. Lesions are confined to
the distal bowel and vary from mucosal edema to ulceration and linear erosions.
Both enterocolitis and proctocolitis show dramatic clinical resolution within
72 hr of allergen elimination.
Food protein-induced enteropathy includes a spectrum of malabsorption
disorders that generally present with protracted diarrhea, vomiting in up to
two-thirds of patients, failure to thrive, and carbohydrate malabsorption. Increased
fecal fat and abnormal D-xylose absorption generally are present. Cow's milk
sensitivity is the most frequent cause of this syndrome, but it also has been
associated with soy, egg, wheat, rice, chicken, and fish hypersensitivity. Patchy
villous atrophy with cellular infiltrate on biopsy is characteristic of this
disorder (Kuitunen et al. 1975; Nagata et al. 1995). A more extensive enteropathy,
with total villous atrophy and extensive cellular infiltrate (celiac disease),
is associated with sensitivity to gliadin, a component of gluten. These patients
often present with diarrhea or frank steatorrhea, abdominal distention and flatulence,
weight loss, and occasionally nausea and vomiting.
Dermatitis herpetiformis is a highly itchy skin rash (sometimes mistaken for
atopic dermatitis) associated with gluten-sensitive enteropathy (Hall 1987).
Biopsy of the skin rash reveals an infiltration of polymorphonuclear leukocytes
and deposits of IgA at the dermal-epidermal junction. Administration of dapsone
or other sulfones often relieves the skin itching within 24 hr. Like celiac
disease, elimination of all gluten for 3-4 months may be required to normalize
intestinal biopsy findings.
Allergic eosinophilic gastroenteritis (AEG) may involve food allergy (Min
1991), as may infantile colic. AEG often presents as postprandial nausea with
vomiting, abdominal pain, diarrhea, occasionally steatorrhea, and weight loss
in the adult, or failure to thrive in the infant (Lee et al. 1993). In the mucosal
form, patients often have atopic disease, elevated serum IgE levels, positive
immediate skin tests to a variety of foods and aeroallergens, peripheral eosinophilia,
iron deficiency anemia, and hypoalbuminemia. Protein-
losing enteropathy or pyloric obstruction may be the main feature in some infants
with AEG (Snyder et al. 1987; Waldman et al. 1967).
A recent study of 10 patients with AEG and severe GER found that non-IgE-mediated
food allergy may be a much more common cause of AEG than previously appreciated
(Kelly et al. 1995). Food hypersensitivity is a frequent cause of GER in young
infants. Milk allergy was the cause of GER in 85 of 204 (42%) of infants less
than 1 year of age (Iacono et al. 1996). Removal of the suspect allergen for
up to 12 weeks may be required to bring about resolution of symptoms and intestinal
histologic changes.
Natural history of food allergy. Experience and follow-up challenge
studies on food-allergic individuals indicate that food allergies are not necessarily
lifelong. Studies have demonstrated the loss of food allergy in up to one-third
of children (Sampson and Scanlon 1989) in 1-3 years of age, even though
results of skin tests and radioallergosorbent assays (RASTs) may not change.
Evidence suggests that the likelihood of losing a food allergy is dependent
upon the food provoking the symptoms and the degree to which the patient maintains
the allergen elimination diet. Allergy to peanut, tree nuts, fish, and other
seafood appears to be more long-lasting (Sampson and Scanlon 1989).
Diagnosis. Many subjective complaints have been ascribed to
adverse food reactions, including neurologic (dizziness, weakness, headaches,
numbness, loss of concentration, depression), gastrointestinal (generalized
bloating, abdominal distention, constipation), musculoskeletal (muscle cramps,
myalgia, arthralgia, vasculitis), and miscellaneous complaints (sweating, chest
pain, fatigue, itchy earlobes) (National Research Council 2000b). Such symptoms
can rarely, if ever, be confirmed with blinded food challenges.
Evaluation of a patient for suspected adverse food reactions involves a thorough
history, physical examination, and laboratory tests. With the history, an attempt
is made to establish whether the patient is suffering from an intolerance or
hypersensitivity reaction, and if the latter, whether a non-IgE- or IgE-mediated
mechanism is involved. If an IgE-mediated food hypersensitivity is suspected,
the clinical impression may be reinforced by performing prick skin tests or
in vitro diagnostic tests.
Skin testing with food extracts by the prick or puncture method may be helpful.
Routine intradermal skin tests with food extracts are too sensitive and nonspecific
(leading to excessive false-positive tests), carry a higher risk of provoking
systemic reactions, and are not indicated. A positive skin test denotes the
presence of allergen-specific IgE antibodies bound to cutaneous mast cells;
it does not mean the patient will develop symptoms when ingesting the specific
food. In fact, the positive predictive values of most prick skin tests are less
than 50%. In contrast, the negative predictive accuracies are excellent, and
IgE-mediated allergic reactions are extremely rare in the face of negative skin
tests.
In vitro tests of food-specific IgE antibodies are often used in patients
with extensive skin disease, significant and prolonged dermatographism, or a
history of exquisite sensitivity (i.e., exposure to minute quantities of a specific
food resulted in a life-threatening reaction). A newer test, the CAP system
FEIA (fluorescent-enzyme immunoassay; Pharmacia Diagnostics, Uppsala, Sweden)
quantitates the amount of food-specific IgE antibodies, which correlates better
with clinical reactivity.
Elimination diets are used when the history and/or preliminary laboratory
studies suggest certain foods may be provoking a patient's symptoms. Foods (and
all hidden sources of that food) suspected of inducing symptoms are totally
eliminated from the patient's diet for 1-2 weeks. If symptoms appear to
improve, further characterization of the sensitivity should be pursued (e.g.,
endoscopy and biopsy, blinded challenge, and so forth). In several chronic disorders
(e.g., atopic dermatitis, asthma, or chronic diarrhea), factors in addition
to the food hypersensitivity may be triggering symptoms, so that failure to
see resolution of symptoms during the elimination period does not necessarily
rule out food hypersensitivity. In cases where food hypersensitivity or intolerance
are suspected but no specific foods can be incriminated, a brief trial (i.e.,
2-4 weeks) of an oligoantigenic or elemental diet may be helpful. If patient
symptoms persist unabated, it is very unlikely that food is a contributing factor.
In the practice setting, open or single-blind oral food challenges may be
used to screen for food allergic reactions. However, in cases where multiple
food allergies are diagnosed, positive responses should be confirmed by DBPCFCs.
DBPCFCs have been used successfully in both children and adults for examining
a variety of food-related complaints. The choice of foods used in DBPCFCs is
based on history, skin test RAST results, and/or foods suspected on the basis
of elimination diets.
The diagnosis of food allergy is a clinical exercise that requires a careful
history, selective skin tests or RASTs in cases of suspected IgE-mediated disorders,
appropriate exclusion diets, and blinded provocation challenges. Currently,
there is no evidence of diagnostic utility for the following assays: quantitation
of food-specific IgG or IgG4 antibodies or food antigen-antibody complexes,
evidence of lymphocyte activation (3H uptake, interleukin-2 production,
leukocyte inhibitory factor production, etc.), or sublingual or intracutaneous
provocation. In gastrointestinal disorders where pre- and postchallenge biopsy
studies are required for diagnosis (e.g., malabsorption syndromes, including
celiac disease), the challenge does not require blinding.
Assessment of Allergenicity to Genetically Modified Foods
and Novel Proteins
S. Lehrer recounted the efforts undertaken to assess the potential allergenicity
of genetically modified foods. Recently, the U.S. National Academy of Sciences
and other national science academies reviewed the issue of genetically modified
foods. The report cited good reasons for the development of the science of agricultural
biotechnology. The potential to alter the food supply suggests the possiblity
of developing less-expensive and healthier foods that could play a role in the
elimination of deficiency diseases and aid in feeding the growing world population.
There is also the potential to reduce chemical pesticide use and increase the
productivity of land, thus protecting habitat for other species (National Research
Council 2000b). Another panel of the National Academy of Sciences looked specifically
at plants genetically modified to include pesticidal properties. This panel
concluded that there was no evidence that the technology is unsafe. At the same
time, the committee conveyed concerns with ecologic risks of gene spread from
the genetically modified crop to wild relatives and the development of pesticide-resistant
superweeds. They also have cited a number of theoretical health concerns, including
changes in nutritional composition or availability of nutrients in food, production
of toxins, and potential of developing more allergenic foods or novel allergens
in new foods (National Research Council 2000a).
Figure 1. Decision-tree
approach to determining the potential allergenicity of novel food products. |
Allergenicity risk assessment for genetically modified foods. During
the last 5 years, a number of national groups, governmental agencies, and industry
organizations, as well as international organizations such as the FAO of the
United Nations and WHO, have become interested in issues of allergy and allergenicity
assessment of these new food products. These organizations have supported a
number of meetings and reports in which allergy risk assessment for genetically
modified foods was addressed. The potential alteration of allergens in foods
concerned changes in endogenous protein levels, expression of known allergens
in different foods, and the expression of novel proteins that may be allergenic.
The U.S. Food and Drug Administration (U.S. FDA) held one of the earliest meetings
(in 1994) to address these issues (Metcalfe et al. 1996). Following this meeting,
a series of meetings held by different organizations resulted in an evolving
decision-tree process that continues to be altered according to new information
about allergen structure and activity. The most recent version of such a decision
tree came out of a WHO workshop in 2001 and is shown in Figure 1 (FAO/WHO 2001).
At this time, decision processes for allergenicity assessment make the initial
choice of study on the basis of the source of the gene, that is, whether it
is from a known allergen source or an unknown allergen source (Figure 1). If
the gene is from a known allergen source, solid-phase immunoassays can be used
to determine whether a known allergen is expressed in the new product. This
information can be used to make a judgment about the potential for allergenicity
of the product and whether it should be produced. Conversely, if the protein
is from an unknown allergenic source or a source with little human exposure
information, the ability to assess the potential for allergenicity is more problematic.
On the basis of their physical and chemical properties, it is possible to identify
novel proteins with little resemblance to known allergens and to judge that
such proteins would have a lower likelihood of allergenicity. However, the predictive
value of such an assessment is unknown. As mentioned in a previous paper in
this mini-monograph (Metcalfe 2003), amino acid sequence comparison with known
allergens, particularly known allergenic epitopes, has also been considered.
This is in the original decision tree presented by Metcalfe et al. in 1996 (Metcalfe
et al. 1996).
The practice of allergy risk assessment for genetically modified foods and
novel proteins has generally worked well but should be improved as our knowledge
of food allergy increases. Although stability in processing and enzymatic digestion
is useful, assessments are not well standardized and validated. Amino acid sequence
similarity, as mentioned earlier, is being used more frequently, particularly
as more allergens are sequenced. However, major questions concerning the comparison
remain. Should it be the whole molecule or parts of the molecule? If parts of
the molecule, should it be epitopes? What percent identity is important? All
of these issues need to be resolved, and obtaining more information on allergen
protein structure in particular will help make this process more precise.
A validated animal model would aid greatly in the assessment process. Such
a model should respond to known food allergens in a manner similar to the human.
One would expect that allergenic foods would have much more significant IgE
reactivity than nonallergenic foods in such an animal model; allergenic proteins
within the allergenic foods would have the same reactivity; and a pattern of
reactivity to epitopes similar to that seen in man would be demonstrated in
the animal.
The amount of food required for allergic sensitization or provocation in man
is not known. This could provide important information, particularly if there
is a level below that at which an allergen is not a risk. Several groups are
addressing this process and should yield important information. Finally, more
importance needs to be placed on the structure and sequence comparison, not
only with allergens but also with nonallergens. If a novel protein with no background
of allergenicity is very similar in structure and function to a nonallergen,
should this not be an important lesson?
StarLink corn, produced by Aventis Corporation (Bridgewater Crossing, NJ)
was approved by the U.S. EPA as a pesticide when produced for sale as an animal
feed. In September 2000, it was reported and confirmed that StarLink had contaminated
the human food supply. The basis for the approval process of StarLink only for
animals was that it contained Cry9c, a protein from Bacillus thuringiensis
(Bt), that appeared to be heat stable when compared with other Bt proteins and
thus could not be excluded as an allergen (because allergenicity could not be
ruled out), using an earlier version of the decision tree (Bucchini and Goldman
2002).
Because of the importance of this exposure, the U.S. FDA, the U.S. EPA, and
the U.S. Department of Agriculture Food Safety and Inspection Service asked
the National Food Processors Association (NFPA) to provide assistance in obtaining
member company information related to potential adverse events that could conceivably
be related to StarLink corn in processed foods. Eleven food companies submitted
data to the NFPA on consumer contacts associated with processed foods containing
yellow corn and possibly StarLink. Additional information estimating production
units, total consumer contacts, allergy health contacts, and allergy-related
product recalls was also provided. The U.S. Centers for Disease Control and
Prevention (CDC) selected four time periods for review: 1998, when 10,000 acres
of StarLink corn were planted; 1999, when 250,000 acres of StarLink corn were
planted; and the year 2000 up to 17 September, when 350,000 acres of StarLink
corn were planted. The fourth period was from 18 September through 11 November
2004, a 2-month period in which numerous product recalls ensued related to StarLink
and thousands of media reports were issued.
The NFPA studies found no correlation between the amount of exposure to StarLink
corn and the allergy/health contacts or allergy complaints to corn products.
There appears to be a positive association between the number of allergy/health
contacts for processed food containing yellow corn and the intense media coverage
of StarLink-related product recalls.
Clinical and Laboratory Methods in the Assessment of the
Allergenicity of Foods
R.G. Hamilton reviewed the methods for assessing food allergy. Any decision
tree is only as good as the clinical and laboratory methods used in the assessment
of the allergenicity of foods. The objective of this presentation was to discuss
each component of the International Food Biotechnology Consortium (IFBC)/Allergy
and Immunology Institute - International Life Sciences Institute (ILSI)/FAO/WHO
Decision Tree (FAO/WHO 2001), with an emphasis on laboratory and clinical methods
useful for investigating potential allergenicity of proteins in genetically
modified foods.
Allergenicity has been defined as the ability to induce IgE antibodies that
react with a protein (immunogenicity) or that cross-react with structurally
similar epitopes. Allergenicity is determined by the antigenicity or foreignness
of a molecule (size and stability), the extent of allergen exposure (allergen
concentration), and the genetic predisposition of a host for inducing an allergic
response (Hamilton 2001).
The IFBC/ILSI report estimates that approximately 160 foods and food-related
substances are associated with allergic reactions (Metcalfe 1996). As of December
2001, 98 foods specificities have been U.S. FDA-cleared for IgE antibody measurement,
and an additional 95 food specificities are in an allergen-specific reagent
status for human IgE antibody testing with the Pharmacia CAP System.
The first level of the allergenicity assessment framework involves an examination
of sequence homology to known allergens. What do we know about the predictive
value of sequence homology? The primary structures of amino acid sequences deduced
from cDNA are usually not complete because of posttranslational modifications
(e.g., glycosylation of asparagines, serines, threonines; hydroxylation of prolines,
lysines). Is the database of allergen amino acid sequences comprehensive? Aalberse
and Stapel (2001) identified four structural families among 40 allergenic food
proteins via 3D protein fold representations in 2D proximity plots. They concluded
that allergens have no characteristic structural features other than they need
to reach (and stimulate) immune cells and mast cells. Within this constraint,
any antigen may be allergenic, particularly if it avoids activation of T-helper
(Th)-2 suppressive mechanisms (CD8, Th1 cells).
Establishing serological assays for the detection of allergen-specific IgE
antibody involves the use of a number of reagents (Hamilton 1997; Hamilton and
Sobotka 2000). The first is a source of human IgE antibody. Pools of sera from
well-characterized patients are needed, as IgE antibody levels are low (in the
nanograms per milliliter range). The process of preparing serum pools dilutes
out IgE antibodies with minor allergen specificities, so the process favors
major allergen specificities. Such dilution is important, as biotechnology or
other plant breeding methods theoretically can increase the expression of minor
allergens, which would not be detected in such serum pools.
The second group of reagents needed for serological assays is a validated
allergen source prepared by extracting the plant or food in a clearly defined
manner with regard to extraction buffer, time, temperature, agitation, filtration,
dialysis, and concentration (Hamilton and Sobotka 2000). This creates obvious
challenges. Plant/food extraction conditions may vary widely, and such variability
can increase the chance that cross-reactivity may occur. Cross-reactivity occurs
when a protein other than the allergen source binds to the allergen-specific
IgE. Cross-reactivity may or may not be of clinical significance. Pastorello
et al. (2000) reported an experiment in which crude extracts of corn showed
significant cross-reactivity with peach, which was not found with the purified
corn protein. Such cross-reactivity can confuse the assessment of allergenicity.
Clinically, several U.S. FDA-cleared immunoassays are available for detecting
allergen-specific IgE antibody in serum. These include the CAP System (Pharmacia),
AlaSTAT (Diagnostic Products Corp., Los Angeles, CA), and Hy-TECH (Hycor, Garden
Grove, CA). These clinical assays use a total serum IgE heterologous reference
curve traceable to WHO IgE standard and quantitative IgE antibody results reported
in kilo-international units per liter. There are also a number of research immunoassays
that are less well characterized, including RAST, ELISA, and the immunoenzymetric
assay (IEMA). Research IEMA assays that employ the native and recombinant Cry
proteins will be useful in detecting IgE antibodies specific for these proteins
(Hamilton 1997, 2001; Hamilton and Sobotka 2000).
The clinical utility of assays for measuring food-specific IgE antibodies
in serum is another important issue. A clinical diagnosis requires confirmation
of clinical reactivity in vivo. Using DBPCFC, Sampson (2001) conducted
a prospective study of children and adolescents and established 95% predictive
decision points for egg-, milk-, peanut-, and fish-specific IgE antibody, as
measured in the Pharmacia CAP System.
All of this is predicated on the notion that a food is a well-defined package
of proteins and other constituents that can be easily characterized. However,
we know that this is not the case. The experience with latex allergy in the
United States illustrates the complexity of allergens in plant products. After
universal precautions were instituted in 1986, there began an epidemic of allergic
reactions and deaths associated with sensitization to a number of proteins in
natural rubber products derived from latex of the Hevea brasiliensis
tree (Hev b 1-Hev b 13). Use and exposure to latex in medical gloves was one
major risk factor for latex allergen sensitization; sensitization was documented
among 7% of health care workers and up to 50% of children with spina bifida.
Cross-reactivity between latex proteins and certain food allergens was one of
the factors that helped to identify latex-allergic individuals. Some of the
common foods with defined cross-reactivity to latex are avocado, banana, chestnut,
kiwi, raw potato, tomato, stone fruits (e.g., peach, cherry), hazelnut, melons,
celery, carrot, apple, pear, papaya, and almond. Foods with less well-defined
cross-reactivity to latex are peanut, peppers, citrus fruits, coconut, pineapple,
mango, fig, passion fruit, ugli fruit, and grape (Salcedo et al. 2001).
From the latex experience, evidence has emerged for a family of proteinous
plant pan-allergens, proteins that include profilin, patatin, plant stress proteins
(WIN 1 and 2), and Bet v 1 and Bet v 2 (birch [Betula verrucosa] proteins).
At present, thirteen allergenic Hevea proteins have been identified (cloned
and sequenced) from latex, and these serve a variety of functions in the rubber
tree. These proteins have quite different structures and molecular sizes. Breiteneder
and Ebner developed a list of protein types among known food allergens (Breiteneder
and Ebner 2000): a) pathogenesis-related proteins involved with defense
against pests; b) seed-storage proteins; c) alpha amylase/protease
inhibitors (wheat/barley/rye); d) Kunitz trypsin inhibitors (inhibit
growth of larvae); e) thiol proteases (e.g., papain-papaya, ficin-fig,
Gly m 1 soybean); f ) profilins (12-15 kd actin cytoskeleton-binding
protein, Ara h 5 peanut, Gly m 3 soybean, Hev b 8 latex; g) peroxidases
(induced by pathogens in plant defense); and h) lectins (agglutinins
that bind specific sugars on glycoproteins: 31 kDa peanut). It is evident that
there will be no easy way to identify novel food allergens based on functional
attributes of each protein. In addition, it is clear that some of the functional
attributes of food allergens are those that may be the most desirable to plant
breeders, for example, increasing the plant resistance to pests. Hanninen et
al. observed in one experiment on turnips that plant stress can increase the
concentrations of certain allergenic proteins (Hanninen et al. 1999). Can food
processing increase the concentrations of allergenic proteins? One observation
is that South American latex-allergic children with spina bifida routinely eat
bananas without any apparent allergy problems. In contrast, latex-sensitized
children (and adults) in North America often experience allergic reactions after
eating bananas. In the United States, food distribution centers treat unripe
bananas and other produce with ethylene to ripen; this is not commonly done
in South America. Does treatment of food with ethylene induce banana proteins
that cross-react with latex? Some research indicates that this can be the case
for certain foods (Sanchez-Monge et al. 2000). Although we currently do not
know the magnitude of this problem, it does illustrate another source of variability
in the allergenicity of foods.
Can Postmarketing Surveillance Provide Useful Information
about the Allergenicity of Genetically Modified Foods? The StarLink Corn Experience
C. Rubin outlined the situation with respect to postmarket surveillance for
allergy to genetically modified foods. When a person manifesting signs or symptoms
(e.g., hives, facial swelling, difficulty breathing) consistent with acute allergic
reaction to a food product seeks medical care, the treatment is usually symptomatic,
the episode is often isolated, and the potential allergen is seldom identified.
The number of such medical visits is not tabulated. Even repeat visits for allergic
reactions to well-known allergens such as peanuts or milk are not counted as
part of any established surveillance system. Thus, in October 2000, when StarLink
corn intended only for animal consumption was identified in products on grocery-store
shelves, there was no way to easily determine if consumption of this genetically
engineered protein was resulting in allergic reactions in humans (Bucchini and
Goldman 2002).
In a coordinated effort to determine if StarLink corn was indeed responsible
for adverse human health effects, the U.S. FDA and the CDC used existing postmarket
surveillance resources to follow up people who self-identified as experiencing
allergic reactions (CDC 2001). After media reports about StarLink, individuals
in 18 states and territories called the U.S. FDA to report health effects potentially
related to ingesting a corn product. Fifty-one people reported signs and symptoms
that varied from gastrointestinal illness to anaphylactic shock. These reports
were entered into the adverse events reporting system (AERS) that the U.S. FDA
has used since 1997 to passively collect information about adverse human health
effects related to the use of drugs marketed in the United States. Self- or
physician-reports to AERS regarding food-related illness generate a standardized
form that chronicles food consumption history, details the timing and manifestations
of the adverse health event, and describes any medical treatment. The U.S. FDA
aggregated all of the AERS reports received between 1 July and 30 November 2000
that mentioned consumption of a corn product. With all personal identifiers
removed, these reports were sent to CDC for review to determine the likelihood
that any of the reports were potentially related to StarLink.
CDC developed a case definition that included a) a suspected anaphylactic
reaction (e.g., dizziness, weakness, or loss of consciousness) that occurred
within 1 hr of product consumption; or b) any of the following dermatological
or oropharyngeal symptoms (hives, rash, pruritus, oropharyngeal tingling or
swelling) that occurred within 12 hr of product consumption; or c) any
of the following gastrointestinal symptoms (vomiting, diarrhea, abdominal cramping)
that occurred within 12 hr of product consumption and that involved only one
individual among meal companions. It was also necessary that these symptoms
were not explained by a preexisting medical condition.
Twenty-eight of the 51 reports were consistent with the case definition. The
U.S. FDA then contacted each potential case person and requested permission
for CDC to follow up the adverse health event. Each potential case was administered
a detailed questionnaire, gave consent to obtain medical records, and was also
asked to contribute a serum sample that would be stored until a test could be
developed to measure IgE antibodies to Cry9c protein. Although all 28 people
appeared to have clinically experienced an immediate hypersensitivity to an
allergen, our postmarketing surveillance could not demonstrate that the Cry9c
protein was actually in the product consumed prior to the adverse health event.
A serological test for antibodies specific to acute hypersensitivity was considered
to be the safest way to evaluate whether any of these people were indeed sensitive
to the genetically modified protein in StarLink. The U.S. FDA developed an ELISA
test that found no IgE antibody reactivity to Cry9c in any of the serum samples.
This test was limited because the Cry9c used in the ELISA was a recombinant
protein produced in bacteria rather than the protein expressed in plants. Concern
that this difference may alter the protein's allergenicity has encouraged researchers
to work on developing additional tests to further demonstrate the lack of reactivity
to the Cry9c protein. This work is in progress, and the results will be published
in the near future.
The StarLink experience demonstrates many of the limitations in using postmarket
surveillance for adverse reactions to food as a method for assessing allergenicity
to a protein that has been newly introduced into the food supply. Intensive
epidemiologic investigation and laboratory test development by federal investigators
was not sufficient to determine whether individual allergic reactions were associated
with the inadvertent release of a genetically modified protein into the human
food supply. It is also unlikely that postmarketing physicians or hospital-based
surveillance would have been able to detect any increase in allergic reactions
during the time that StarLink corn was available to consumers. The symptoms
described in the case definition used in this investigation are generic and
could have been attributed to a variety of etiologies. The StarLink example
demonstrates many of the problems with any surveillance system that tries to
capture rare and somewhat generic health events such as food allergy.
Is Susceptibility to Food Allergy in Workers Sensitized by
Inhalant Exposure to B. thuringiensis kurstaki a Relevant Model for Sensitivity
to Genetically Modified Foods?
L. Bernstein reviewed the occupational allergy experience with inhalation
of Bt proteins. Novel proteins in genetically modified food may be present in
the dust in workplaces where such foods are processed or handled. Novel proteins
in genetically modified food may be present in the dust in workplaces where
such foods are processed or handled. Thus, occupational populations may be exposed
via inhalation. The possibilities of prior exposure and allergic sensitization
to proteins that are potentially cross-reactive with novel proteins contained
in genetically modified foods should be considered relevant risk factors prior
to general introduction of such foods into the human food chain. By serendipity,
this hypothetical circumstance actually occurred before StarLink corn was inadvertently
discovered in food products generally available to human consumers.
In 1999, researchers discovered that migrant health workers developed positive
skin tests and elevated specific IgE and IgG antibody levels to B. thuringiensis
kurstaki (Btk) spore extracts containing Cry1Aa and Cry1Ab delta endotoxin
proteins after respiratory exposure to Btk crop spraying (Bernstein et al. 1999).
A number of positive skin tests, as well as increased levels of specific IgE
and IgG antibodies, were present in more highly exposed groups than in medium-
to low-exposure groups. Preexisting atopy was also a risk factor for workers
with positive skin and serologic tests in the medium- and low-exposure groups.
Although consumers frequently use Btk spray products for gardening purposes,
similar risk assessment studies in this population have not been investigated.
Because of the documented success of Btk delta endotoxin insecticides in the
control of corn borer larvae, investigators postulated that direct incorporation
of genes encoding these proteins into corn and maize seeds would further enhance
productivity of these commodities. Cry1Aa and Cry1Ab genes were
among the first Btk genes to be used for this purpose. Later, the Cry9C
gene, uniquely modified to enable persistence of the encoded protein in the
larval gut, was used in StarLink transgenic corn seed. Although immunologic
cross-reactivity between Cry1A and Cry9C proteins has not yet been investigated,
it is of particular interest that these proteins share proteomic homology in
75% of their conserved amino acid residues as well as identity of the chief
domains that determine tertiary structure of these proteins.
Table 3
 |
For the above reasons and in view of prior experience with respiratory occupational
exposure to Btk in migrant workers, researchers extensively reviewed the literature
concerning the occurrence of subsequent food allergy in workers previously sensitized
after respiratory exposure to a variety of food proteins (Cartier et al. 1984;
Leser et al. 2001; Lybarger et al. 1982; Smith et al. 1987). Unfortunately,
there were a limited number of cross-sectional studies of occupational asthma
(OA) induced by inhaled food proteins where the question of subsequent food
allergy was addressed. However, in plants processing foods such as eggs, snow
crabs, and condiments, 18 (35%) of 51 workers with confirmed OA developed food
allergy symptoms at varying intervals after the onset of OA (Table 3).
A future approach to the potential allergenicity of genetically modified foods
during postmarketing surveillance should include human susceptibility and risk
factors in atopic subset populations; prior occupational exposure and sensitization
to related proteins; and prior consumer exposure and sensitization to related
proteins.
Occupational exposure to novel proteins and potential sensitization is an
issue that has had little study, yet could be of public health significance.
Approaches are available for further study, and exposed cohorts are available.
Conclusions
The introduction of genetically modified foods into the marketplace has brought
to light the challenges inherent in identification of food allergens and individuals
who are sensitive to those allergens. Although theoretically any plant-breeding
technique can modify (increase or decrease) the allergenicity of foods, biotechnology
has a greater potential to introduce novel proteins into the food supply, and
thus has been subjected to closer scrutiny by regulators. Expert bodies have
developed a decision tree for assessment of food allergy risks from such foods.
All aspects of current food allergy assessments, both clinical and laboratory
tools, have technical challenges that must be addressed if such tools are brought
into a regulatory context. However, our challenge scientifically is how to assess
novel proteins that have little or no exposure in the general population and
thus no readily available tools for the prediction of exposures. The experiences
with StarLink corn and in occupational cohorts exposed to grain dusts suggest
that the development of methods to be used for postmarket consumer and occupational
health surveillance may be useful. Thus, the current FAO/WHO decision tree for
assessment of food allergy risks may require revision to include evaluation
of appropriate diagnostic tests in these susceptible population groups, and
to address technical challenges in assessing proteins newly introduced into
the diet. Later papers in this monograph suggest research strategies for development
of tools that may be useful for the prediction and/or postmarket surveillance
of allergy to novel proteins in foods.
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Last Updated: June 13, 2003
