Recent Patents on Inflammation & Allergy Drug Discovery 2008, 2, 47-57 47

1872-213X/08 $100.00+.00 © 2008 Bentham Science Publishers Ltd.

Allergy After Inhalation and Ingestion of Cereals Involve Different Allergens in Allergic and Celiac Disease

Alicia Armentia, Eduardo Arranz*, Nora Hernandez , Antonio Garrote*, Raphael Panzani *** and Alfredo Blanco*

From Allergy and Medicine Sections, Rio Hortega Hospital, Valladolid, Spain, *From IBM Department. Hospital

Clínico Universitario, Valladolid. Spain, ***Laboratoire de Recherches. Marseille. France

Received: December 14, 2007; Accepted: December 26, 2007; Revised: December 31, 2007

Abstract: Cereals are among the major foods in type I food hypersensitivity reactions. Hypoallergenic cereals and

recombinant immunotherapy have been recently patented. In celiac disease, limited information is available regarding

cereal allergens responsible for allergic reactions. The allergenic reactivity of ingested and inhaled cereal allergens in

allergic and celiac people are discussed in the manuscript. Allergic sensitisation IgE mediated to cereals may be observed

in celiac children. Inhalation and ingestion routes causing cereal allergy seem to involve similar allergens, but, in celiac

disease specific response to CM3 may be important.

Keywords: Celiac disease, food allergy, allergy to wheat, baker’s asthma.

INTRODUCTION

Food incompatibilities affect approximately 20% of the general population in Western countries. In about one quarter of the affected children and one tenth of affected adults, the incompatibility is based on an allergy sensitisation [1]. Celiac disease and cow’s milk protein allergy are key samp-les of chronic enteropathy. Enteropathy defines abnor-malities of the small intestinal mucosa, of various etiologies, that can be separated into acute versus chronic conditions [2].Celiac disease is a lifelong intolerance to the gluten found in wheat, barley and rye, genetically determined as in allergic diseases. Of the patients with celiac disease 95% are human leucocyte antigen (HLA-DQ2 or HLA-DQ8 positive). Characteristically, the jejunal mucosa becomes damaged by a T-cell-mediated autoimmune response that is thought to be initiated by a 33-mer peptide fragment in A2 gliadin, and patients with this disorder have raised levels of anti-endomysium and tissue transglutaminase antibodies in their blood. Kuendig has also recentkly patented use of vaccine for intralymphatic injection for desensitisation of individuals [3]. This disease is the major diagnosable food intolerance and, with the event of a simple blood test for case finding, prevalence rates are thought to be approximately 1:100 [3]. Kuendig has recently patented use of vaccine for intralym-phatic injection for desensitisation of individuals. [4]. Albrecht has also recently patented recombinant modified vaccinia virus Ankara (MVA) for the treatment of type I hypersensitivity resulting from allergens from the group of weed pollens, grass pollens, tree pollens, mites, animals, fungi, insects, rubber, worms, human autoallergens, and foods [5].

Early exposure to solid food in infancy has been associated with the development of allergy [6,7]. In a study, carried out on reviewed data on 16281 patients, the early

*Address correspondence to this author at the Hospital Rio Hortega. Sección de Alergia Cardenal Torquemada sn, 47010 Valladolid, Spain; Tel:

983-420400; Fax: 983-331566; E-mail: ALICIAARMENTIA@terra.es

introduction of cereals in the diet of children was found to be a risk-factor of grass pollen asthma [7]. By another hand association of asthma and allergic rhinitis with celiac disease have been reported [8,9], and also chronic urticaria have been associated with celiac disease in children [10,11].

During the past few years, we have purified and characterized several major IgE binding proteins from wheat, barley and rye, which are associated with flour allergy [12-17]. These allergens have molecular masses of 12-15 kDa, inhibitory activities against heterologous -amylases or trypsin and a potential role as a biological defence against the insect parasitation of the grain.

Hypersensitivity reactions to ingested cereals proteins have been reported by several investigators performing blinded food challenges in children. Varjonen et al. found IgE binding components of wheat, rye, barley and oats recognized by immunoblotting analysis with sera from adult atopic dermatitis patients [18]. After analysis of serum samples collected from seven children with wheat allergy and an adult with baker's asthma, James et al. [19] showed that wheat -amylase inhibitor is a relevant sensitizing allergen after both ingestion and inhalation. Jones et al. [20] implicated 20 kDa and 47 kDa wheat proteins as potential allergens in children with confirmed wheat hypersensitivity and found immunologic cross-reactivity among cereal grains and grasses in children with food hypersensitivity. Sandiford et al. [21] showed that sensitized patients to soluble proteins from wheat and barley were able to produce IgE against insoluble proteins, and identified some of the major water/salt insoluble proteins involved in cereal sensitivity. Recently, we have described Ma wheat lipid transfer protein (Tri a 14) as a major allergen associated with baker’s asthma [22].

Although, there are some similarities in the possible adverse reactions to cereal (baker's asthma, IgE mediated food allergy, non-IgE mediated enteropathy, wheat-depen-dent exercise induced anaphylaxis) and celiac, the possibility of allergens involved in clinic responses have not been

48 Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 Armentia et al.

completely resolved. In celiac disease, very limited infor-mation is available regarding cereal allergens responsible for allergic reactions after ingestion of cereal proteins and both diseases are considered independent.

This prompted us to evaluate the allergenic reactivity of ingested and inhaled cereal allergens in different ages, in allergic and celiac people, in order to investigate the existence of some similarities and study if the response to different allergens would depend on the sensitisation route.

METHODS

Patients Recruitment and Collection of Serum Samples

Eighty-eight patients were included in the study and distributed in groups according to their clinical data. Group

1: 17 patients (13 adults, 4 children) with food allergy related to cereals in the diet in which celiac disease or other digestive disturbances had also been discarded. These patients with allergy after ingestion of cereal were selected from the reviewed data of a 20041 data-base of patients studied in our Allergy Unit during the last 15 years. Group

2: Fourteen patients previously diagnosed as having baker's asthma after specific bronchial challenge tests. These patients were chosen because they had not still began specific immunotherapy and had not symptoms related to the ingestion of cereal or another food that may interfere in the immunoblotting results.

Group 3: 57 children of ages from 3 to 6 months who suffered from growth retarded or digestive and skin symptoms (atopic dermatitis, diarrhea, vomiting) related to the introduction of cereal formulas in their diet. The children were selected from the Paediatric Department data base and were studied in the last years. We chose children in whom a clear relationship between the introduction of a new cereal in their diet and their symptoms was very probable: The symptoms disappeared when this cereal was retired from diet and came back when the mother tried to introduce them again. The possibility of celiac disease or another non-IgE mediated enteropathy have been diagnosed with specific analysis (anti-transglutaminase (ATG), anti-gliadin antibody (AGA) assays, anti-reticulin antibody (ARA) test and anti-endomysial antibody (AEA assays) and small intestinal biopsy if necessary. In addition to all these clinical data, the diagnosis of cereal allergy was based on the positivity of CAP (Uppsala, Sweden), skin prick tests (SPT) and double blind placebo-controlled food challenge (DBPCFC), except in the children where the last tests (pricks and DBPCFC) were not performed by ethical considerations. As control group, we selected 12 children and 10 adults in which

allergic sensitisation and celiac disease were discarded with the specific analysis described before.

Food allergy was defined as atopic dermatitis, urticaria/ angioedema, asthma or diarrhea/vomiting occurring on at least two occasions or anaphylaxis induced by the specific food concurrent with food-specific IgE [23].

Informed consent was obtained from each patient or patient's parents and ethical approval was obtained from the Ethical Committee of the Rio Hortega Hospital.

We summarized the inclusion criteria in Table 1.

Allergy Tests

Skin Prick Testing

We used the followed extracts:

Commercial extracts from a standard battery of allergens (pollens, mites, molds and different foods) were provided by ALK-ABELLO Laboratories, Madrid, Spain.

Cereal Extracts

Whole seeds of bread wheat (Triticum aestivum, cultivar Astral) were ground, deffated with cold acetone (2x1; 1:5 [wt/vol] for 1 hour at 4ºC), and, after drying, extracted with 0.1 mol/L Tris-HCl pH 7.5, 10mM EDTA (1:5 [wt/vol] for 1 hour at 4ºC). After centrifugation (9000g for 30 minutes at 4ºC), the supernatant was dyalized against H2O (cut-off point, 3.5kd) and freeze-dried. This crude extract was used for both immunoblotting and SPTs.

Additionally, grains were milled in a Laboratory Mill CD1 (Chopin Technologies, Villeneuve-la-Garenne Cedex, France) to separate flour and bran (final yield 52.0% and 33.6%, respectively), which were independently extracted by the method described above. Protein concentration in all extracts was quantified according to Bradford [24].

Isolation and Characterization of Tri a 14

The wheat bran extract was fractionated by cation exchange chromatography on an Acell Plus CM Waters

TM

SepPakR cartridge (Waters, Milford, Mass., USA) using

10mmol/L ammonium acetate pH 6.8 as equilibration buffer. The retained material, enriched in Tri a 14, was then eluted with 50 mmol/L ammonium acetate pH 6.8, dyalized and freeze-dried. This Tri a 14-enriched fraction was further separated by reverse-phase HPLC on a Nucleosil 300-C4 column (8x250mm; particle size 5μm; Scharlau Science, Barcelona, Spain), eluting with a linear gradient of aceto-nitrile in 0.1% trifluoroacetic acid (10% to100% in 150min.; 1ml/min.). Chromatographic fractions containing wheat LTP

Table 1. General Inclusion Criteria

Group (n) Age at the time of the diagnosis Allergy symptoms after eating

cereal

Allergy symptoms after inhalation

of cereal

1 (17, 13 adults, 4 children) 1.5-37 years old Yes No

2 (14 adults) 17-30 years old No Yes

3 (57 celiac children) < 1 years old Yes No

Allergic Sensitisation to Cereals in Celiac Children Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 49

Tri a 14 were located by means of immunodetection after SDS-PAGE with both anti-peach LTP Pru p 3 antibodies and a serum pool from baker’s asthma patients. These fractions were then pooled and freeze-dried.

Peach Pru p 3 allergen was purified as previously [25]. The isolated allergens were quantified by using a com-mercial bicinchoninic acid test (Pierce, Cheshire, United Kingdom).

SDS-PAGE was performed on Bio-Rad Miniprotean III System gels (15% polyacrylamide), according to Laemmli [26]. Bands corresponding to Tri a 14 in bran and flour extracts were quantified by means of the GelDoc 2000 System and the Quantity One software (BioRad, Richmond, Calif, USA).

The prick-tests were carried out using standard methods. A positive SPT response was defined when the mean diameter (= half of the sum of the largest diameter and its perpendicular) of the wheal was at least 3 mm higher than the negative control. Pricks were not performed in the group of children because generalized reactions after pricks with food in children under 6 months of age have been reported [27].

Food Challenges

Patients were challenged with specific cereal protein extracts as indicated by history and SPT results, following the method reported by Jones et al. [20] and the recom-mendations for DBPCFC by Bock et al. [28].

Bronchial Challenge Tests

Specific bronchial reactivity to cereal flour extract was evaluated in asthmatic patients using the technique of Chai with minor modifications, as described previously [29].

Cereal Exclusion Diet

In the case of allergic children, we eliminated cereal formula until the age of 10 months. We introduced after-wards gradually cereals beginning with rice and corn. In the case of adults, we eliminated from diet all source of cereals: bread, pastas, cakes, pastry in general, beer, cocoa drinks, cacao powder, soluble coffee, soups, syrups, other food with possibility of hidden cereals as sausages and other butcher's shop goods and sweets.

in vitro Tests

Specific IgE

Determination of specific IgE to wheat, barley and rye flours and a battery of food allergens: whole milk, alpha-lactoalbumin, beta-lactoglobuline, caseine, egg ( white and yolk), legumes, nuts and fish was performed with the Pharmacia CAP System FEIA, (Uppsala, Sweden). We considered as positive a specific IgE level to wheat greater than 0.35 kU/L (mean: 16.3 kU/L; range: 0.51- >100 kU/L).

IgE Immunodetection

We selected for IgE immunodetection four serum pools corresponding to groups 1 (mixture of 17 sera of patients with wheat allergy), 2 (baker's asthma adults; mixture of 14 sera) , 3 (mixture of 53 sera from celiac children without specific IgE to wheat) and 4 (mixture of four sera from celiac

children with positive IgE to wheat. Extracts from cereal flours (15 μg of protein wheat extracts and 3 g of purified allergens) were separated by SDS-PAGE [22] on Bio-Rad Miniprotean II system gels, and then electrotransferred to polyvinylidene difluoride (PVDF) membranes.

After blocking, membranes were incubated overnight with a pool of 17 sera from patients with baker’s asthma (1:5 dilution), washed and then treated with mouse anti-human IgE mAb HE-2 ascitic fluid (1:3000 dilution for 1 hour) [24]. After washing, a rabbit anti-mouse IgG peroxidase-conju-gated antibody (1:5000 dilution for 1 hour; DAKO, Glostrup, Denmark) was added. Detection of IgE-binding components was achieved by enhanced chemiluminiscence, following the manufacturer’s instructions (Amersham Biosciences, Little Chalfont, United Kingdom). A pool of 3 sera (1:5 dilution) from bakers non-allergic to wheat (negative IgE and SPT and no symptoms) were tested as negative control.

Alternatively, replica gels were immunodetected with rabbit polyclonal antibodies to the peach LTP Pru p 3 (1:1000 dilution) and then treated with goat anti-rabbit IgG peroxidase-conjugated antibody (1:100000 dilution, Sigma, Steinheim, Germany) and revealed.

RESULTS

Description of the Results of the Diagnostic Tests

Tables 2, 3 and 4 summarizes the clinical characteristics of the patients groups and the results of the diagnostics tests. In the group of 57 children suffered from celiac disease (Table 4) only four children had positive IgE to wheat and not to other tested allergen. The clinical symptoms of these children were not different that the others included in this group. Fourteen adults patients with cereal allergy (Table 2) had positive DBPCFC response against the suspected cereal. These tests were not performed in the four children suffered from anaphylaxis. The most important allergen was wheat, followed by barley, rye flour and other seeds. The most important source of cereals allergens in the adults diets were soluble cacao powder (it contains malt extract) and beer. All the patients presented positive SPT to LTP from wheat Tri a 14 and to al alpha-amylase inhibitors including CM3. Table 3 shows the results of the tests performed in 14 patients suffered from baker's asthma. Eleven of the 14 patients were also sensitised to LTP of wheat Tri a 14. Seven patients with baker's asthma presented sensitisation to seed and fruits. The allergen that cause more positive results in bronchial challenge tests was wheat flour. All these patients tolerated cereal foods, seeds and fruits without problems. In the other groups of patients (children and adults with cereal food allergy), cereal exclusion diet was indicated. Whereas significant improvement could be seen in the group of children (their symptoms disappeared after exclusion of cereals formulas), light changes were observed in the adult group. Only the four children and three adult patients suffered from anaphylaxis and angioedema (35.29%) resolved their symptoms, possibly because of the severity of their symptoms they may have followed the exclusion diet more strictly than other patients with only light symptom relief.

50 Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 Armentia et al.

Table 2. Group 1: Patients Suffered from Asthma and Anaphylaxis Due to Ingestion of Wheat Flour

Patient Age/gender Symptons

Years of duration

IgE to

wheat KU/L

SPT to

wheat mm

IgE to others

foods

SPT

Tri a 14

SPT

Pru p3

SPT

Wheat extract

8320 30/F Asthma/4 7.72 5x5 Barley 28.6

Rye 8.61

6x6 5x5 6x6

8321 45/F Asthma/20 >100 10x10 Barley>100

Rye>100

Rize 15

4x4 5x5 10x10

8333 20/F Asthma/20 27.9 5x5 Barley 55.7

Rize 3.29

Corn 7.37

Peach 2

4x4 5x5 6x6

8380 61/F Asthma/anaphylaxis/35 4.92 6x6 Rye 5.03

Orange 3.38

Barley 5.45

5x5 5x5 6x6

8338 28/M Asthma/anaphylaxis/2 11.4 6x7 Soy 1.92

Lentil 1.41

Rye11.4

Barley 8.22

6x6 2x2 7x7

8407 48/F Asthma/24 3.1 5x5 Rye 1.8

Barley 1.5

5x5x 3x3 6x6

8408 25/F Asthma/urticaria/5 2.28 5x5 Rye 2.3

Barley 0,78

6x6 2x2 6x6

3795 22/F Asthma/4 2.26 5x5 Rye 2.70

Barley 2.82

Lolium 4.2

6x6 3x3 7x6

969628 73/F Asthma/urticaria/36 4.4 5x5 Rye 2.3

Barley 3.3

5x5 3x3 6x6

7912 25/F Astma/urticaria/6 3.3 5x5 Rye 2.1

Corn 7.2

5x5 2x2 6x6

5351 27/M Asthma/anphylaxis/2 4.2 7x7 Crisantemun 6.5 6x6 6x6 6x7

7526 32/M Asthma/8 5.3 6x6 Egg 10.3

Barley 5.6

6x6 3x3 5x7

7972 25/F Asthma/10 4,8 6x6 Rye 4.8

Barley 17.8

6x6 5x5 7x7

4889 12/M Asthma/urticaria/2 3.2 6x6 Lentil 0.45 6x6 3x3 6x6

BAC7 9/M Anaphylaxis/9 5.2 6x6 Rye 4. 6x6 6x6 6x6

BAC13 2/M Anaphylaxis/6 1.14 X5 Barley 2.3 5x5 5x5 6x6

BA10 1.5./M Anaphylaxis/3 7.3 5x5 Barley 3.4 5x5 6x6 6x6

Allergic Sensitisation to Cereals in Celiac Children Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 51

Table 3. Group 2: Patients Suffered from Asthma Due to Inhalation of Wheat Flour

Patient Age/gender Symptons

Years of duration

IgE to wheat

KU/L

SPT to

wheat mm

IgE to others

foods

SPT

Tri a 14

SPT

Pru p3

SPT

Wheat extract

8291 27/M Asthma /5 37.1 5x5 Rye 29.4

Barley 27.9

Apple 10

Peach 11.3

6x4 5x5 10x12

8306 25/M Asthma/6 1.47 5x5 Rye 6.5

Barley 3.5

Apple 0

4x4 2x2 12x14

8310 52/M Asthma/32 1.76 4x5 Rye 4.8

Barley 2.3

Kiwi 0

Melón 0

3x3 4x4 8x8

8309 30/M Asthma/12 0.97 4x4 Rye 0.5

Barley 0.5

2x2 2x2 5x5

8317 31/F Urticaria, asthma/10 7.3 5x5 Barley 10.3

Peanut 9.7

Lentil 5.8

Hazelnut 1.16

1x1 1x1 8x8

8315 36/M Uticaria, asthma/13 6.03 5x4 Barley 4.04

Yolk 5.30

Peanut 2

6x6 5x5 6x6

8318 38/M Rhinoconjunc

Asthma/12

0.51 4x4 Corn 2.24

Rye 0.51

Barley 1.04

2x2 4x4 5x5

8319 17/M Asthma/3 7.35 4x4 Barley 0.7

Almond 0.6

Lentil 18.1

5x5 10x10 10x10

5479 51/M Asthma/18 2.3 5x4 Rye 1.2

Barley 1.9

5x5 3x3 5x5

4608 31/M Asthma/8 12 6x6 Rye 3.2

Barley 6.7

6x6 3x3 6x6

1000 36/M Asthma/15 2.05 6x6 Rye 3.08

Barley 3.05

6x6 2x2 8x8

1302 52/M Asthma/22 22.2 6x7 Rye 20

Barley 18.3

7x7 5x5 8x8

8110 41/M Asthma/14 14.6 7x7 Barley 7.9 7x7 3x3 8x8

7990 35/M Asthma/17 0.7 5x5 Corn 3.4 5x5 2x2 6x6

52 Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 Armentia et al.

Table 4. Group 3 ·: Children Suffered from Celiac Disease

Childen EmA IgA Grade Marsh IgE Wheat

KU/L

IgE Barley

KU/L

8563 Neg IIIb 0.51 0.67

8564 aTGt 0 0

8565 + 0 0

8566 aTGt IIIb 0 0

8567 + 0 0

8568 aTGt 0 0

8569 + 0 0

8570 + IIIa 0 0

8571 + IIIa-b 0 0

8572 + IIIc 0 0

8573 ATgt 0 0

8574 + I 0 0

8575 + II-IIIa 0 0

8576 + IIIc 0 0

8577 + II-IIIa 0 0

8578 + IIIc 0.71 0

8579 + IIIa 0 0

8580 DQ2- IIIb 0 0

8581 + IIIc 0 0

8582 + IIIb 0 0

8583 + IIIb 0 0

8584 + II 0 0

8585 + IIIc 0 0

8586 + IIIb 0.90 1.03

8587 + II 0 0

8588 + IIIc 0 0

8589 + IIIb 0 0

9890 + IIIc 0 0

8591 + 0 0

8592 + 0 0

8593 IIIb 0 0

8594 IIIb 0 0

8595 IIIc 0 0

8596 IIIa 0 0

8597 IIIC 0 0

8598 IIIc 0.67 1.16

Allergic Sensitisation to Cereals in Celiac Children Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 53

(Table 4) contd….

Childen EmA IgA Grade Marsh IgEWheat

KU/L

IgE Barley

KU/L

8599 IIIb 0 0

8600 IIIc 0 0

8601 IIIc 0 0

8602 IIIc 0 0

8603 II 0 0

8604 IIIa 0 0

8605 IIIc 0 0

8606 IIIc 0 0

8607 IIIb 0 0

8608 DQ2- II 0 0

8609 0 0

8610 I 0 0

8611 + IIIc 0 0

8612 + IIIc 0 0

8613 + II-IIa 0 0

8614 IIIb 0 0

8615 + 0 0

8616 aTTt + 0 0

8617 aTGt + 0 0

8618 AAG/ATG + 0 0

8619 IIIb 0 0

in vitro Results

In order to evaluate whether or not similar IgE-binding proteins were involved in allergy reactions after ingestion and inhalation of cereal products, salt extracts from wheat and their purified allergens, barley and rye flour were obtained, fractionated by SDS-PAGE and IgE Immu-nodetected Fig. (1). Proteins bands with apparent molecular sizes between 7 and 90 kDa were shown by the cereal extracts. Interestingly, IgE immunodetection using serum pools from patients sensitised to cereals by inhalation (adults suffering from baker's asthma) or ingestion (adults and children) rendered very similar patterns of IgE-binding components in all three extracts. Protein bands of around 11-16 Kda, (allergenic members of the -amylase inhibitor family) were immunodetected in wheat, barley and rye. The most prominent differences in IgE-binding patterns using serum pools from patients sensitised by inhalation or ingestion of cereal products were shown by the 35-80 Kda rye components and that samples from patients suffered from baker’s asthma no presented response to CM3 inhibitor.

In the case of immunodetection with pools from celiac people, the major differences were the absence of response to the wheat inhibitor CM3 in celiac patients without specific IgE to wheat. Response to LTPs Tria 14 and Pru p3 seemed to be more important in these last patients that alpha-amylase inhibitors.

DISCUSSION

The aim of this study was to evaluate the allergenic reactivity of ingested and inhaled cereal allergens in different ages, in allergic and celiac people, in order to investigate if the response to different allergens would depend on the sensitisation route. We also compared the in vitro reactivity to different purified cereal allergens in three different group of patients including tests with cereal alpha-amylase inhi-bitors and the recently purified wheat LTP Tri a 14 [22]. The combination of anti-tTG and AGA II serology may become a powerful non-invasive pairing for serologic diagnosis of celiac disease. In contrast, little information is available regarding specific cereal allergens responsible for IgE mediated allergic reactions after ingestion of cereal proteins.

54 Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 Armentia et al.

Possibility of sensitisation to different allergens depending of the sensitisation route (ingestion or inhalation) has not been fully resolved.

We found that inhalation and ingestion routes causing cereal allergy seem to involve similar allergens Fig. (1). This similarity in patterns may be due to the usage of pooled sera. Nevertheless these results are in agreement with those of previous studies. [19,30]. The most important difference among celiac patients and the other two groups (patients suffered from baker’s asthma and asthma or anaphylaxis after eating cereals) were the response to CM3 in celiac with positive IgE to wheat and a more positive response to LTP that to the other alpha amylase inhibitors.

Allergenic properties to cereal inhibitors may be related the enzymatic activity [26], as occur with some cereals proteins [15-17]. This is particularly enhanced by the immaturity of the infant's gastrointestinal tract in terms of mucosal permeability, enzymatic function, and immunologic

defence mechanism. Newborns generally are fed with either breast milk or infant formulas. Early exposure to solid foods in infancy has been associated with the development of allergy [7]. The age of introduction of cereals varies widely, but it delaying initial exposure to cereal grain until after 6 months seemed to increase the risk of developing wheat allergy [6].

Different investigations have confirmed the presence of specific IgE against albumins and globulins in serum of children with cereal food allergy but not in patients with celiac [4]. In celiac sprue, wheat and rye flour are made up of a harmless starch fraction and a protein fraction or gluten which is injurious in genetically susceptible persons. Gluten in turn is made up of two proteins, glutenin and gliadin, of which only the gliadin fraction exerts a deleterious effect. The availability of serologic testing tests have identified patients who appear to have the disease but have variable degrees of histophatologic changes and /or symptoms. Thus, several categories of celiac disease have emerged. Where

Fig. (1). Detection of cereal IgE-binding components. Salt extracts (15 μg of protein) from seed, flour, bran, CM16, CM3, Tri a 14 from

wheat barley (B) and rye (R) flour and peach LTP (Prup3), were separated by SDS-PAGE. Replica gels were stained with Coomassie Blue

(A) or electrotransferred to PVDF membranes and immunodetected with a serum pool from celiac patients without positive IgE to wheat (B),

from a serum pool from celiac patients with IgE to wheat (C) from a pool of samples from patients suffered from baker’s asthma (D) and

with samples from patients allergic to ingested cereals (E).

Allergic Sensitisation to Cereals in Celiac Children Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 55

these phenotypes have clinical usefulness remains to be determined [31,32]. In four of our celiac patients, the in vitro testing results (specific IgE and anti-tTG) belong to the two diseases, apparently independent. Association of celiac disease and other allergic symptoms have been studied [8-11] and some publications have postulated previously if celiac disease would be a kind of food allergy [33,34]

During the past few years, we have identified and purified a number of allergenic proteins from wheat, barley and rye flours associated with cereal allergy, which belong to a single family of seed-specific inhibitors of insect and mam-malian -amylases [12-17]. Our next step was to investigate the in vivo allergenic activities of purified members of the inhibitor family, which have very different IgE-binding capacities in vitro in asthmatic patients. Recently, we are included the wheat LTP [22]. LTPs are abundant proteins in cereal grains, but, only those of maize and beer have been identified as food or beverage allergens previously [35]. Wheat LTP Tri a 14 was identified as a major allergen linked to baker’s asthma caused by wheat flour, showing a limited cross-reactivity wit the model LP food allergen from peach fruit [22]. The clinical characteristics of baker’s asthma and the high in vitro and in vivo reactivity of Tri a 14, clearly suggest that members of the LTP family can sensitized by the respiratory route, being linked to systemic symptoms, such as asthma. Their potential role as inhalant allergens has been previously suggested in three patients with rhino-conjunctivitis and asthma caused by rice power inhalation [36], and more extensively, in subjects with allergy to mugwort and plane pollens [37]. However, sensitization to Tri a 14 via ingestion induced by cross-reactivity between the LTP wheat allergen and its homologous from fruits, vegetables or nuts can not be ruled out, at least in some patients. The studied cross-inhibition between Tri a 14 and peach Pru p 3 shown in same individual sera by ELISA-inhibition assays, points to this sensitization route [22]. Nevertheless sensitization due to cereal ingestion may be the only sensitization route in children suffered from celiac disease, although fruits are introduced in children’s diet before the age of 4 months in or country [7]. Other interes-ting finding was the lack of response to the wheat tetrameric alpha amylase inhibitor subunit CM3 in celiac patients without specific IgE to wheat and also in our pool inhalation samples (Fig. 1). The WAI-CM·, but not its associated subunits WTAI-CM2 and CM16, reacts with IgE from sera from Japanese subjects with wheat-related atopic dermatitis [38].

Sensitisation to food allergens occurs in the majority of cases during the first years of life and is usually transient. The mechanism that predisposes children with food allergy to atopic or celiac disease remains poorly understood. Studies of various dietary regimens during the first months or first years of life have demonstrated a decreased incidence of food allergies and atopic dermatitis in children who followed a prescribed dietary regimen. Unfortunately, long-term follow-up studies have shown that once an allergenic food is subsequently introduced, the prevalence of allergic symptoms reverts to the normal level [39-42]. The poor result of the exclusion diet in our adult patients group may be due to difficulties in the elimination other sources of hidden cereal allergens in the diet or to the severe atopy of the

patients or as a result of sensitisation to other food with antigens that showed cross-reactivity with cereal allergens (others seeds, nuts and fruits). Nevertheless, in the case of children during lactation period this measure seemed to be effective.

At present, we can not modify the genetic factors that contribute to the allergic or celiac diseases. It is, however possible to avoid the risk factors which exist in our living environment and in our dietary customs Measurements of gliadins with validated enzyme-linked immunosorbent assay methods provides an appropriated marker for assessing gluten and/or wheat protein contents in food. Available data suggest that maximum gluten content for gluten-free foods could be set, which protects both wheat allergy sufferers and celiac patients [43]. Hotta et al. has recently patented ultra fine powders of nonglutinous rice to avoid wheat allergies [44]. Mclean has patented identification of loss-of-function mutations in the filaggrin gene and their use in diagnosing susceptibility to allergies (including food allergy) [45].The gut microenvironment maintains homeostasis by condi-tioning dendritic cells to remain in a quiescent state. Inhi-bitory signalling by commensal bacteria possibly contributes to this process. Elucidating their nature and how they are influenced by external factors such as probiotics may help develop novel therapies for allergy and help understand more celiac disease [46]. Kuendig has recently patented specific molecules for the preparation of a medicament, in particular, for the preparation of a vaccine for intralymphatic injection for desensitisation from allergies [47].

Furthermore, the study of clinical response of these patients to the different cereal proteins would be use in the elaboration of new transgenic cereals with the help of biotechnology technical support.

In conclusion, allergic sensitisation IgE mediated to cereals may be observed in celiac children. The in vitro allergenic immune response to cereals seem to involve simi-lar allergens in celiac children and in adults independently of the route of sensitisation but in celiac disease specific response to CM3 and LTPs may be important.

CURRENT & FUTURE DEVELOPMENTS

Specific IgE response to cereal antigens could be important in patients suffering from celiac disease. We found that inhalation and ingestion routes causing cereal allergy seem to involve similar allergens, but with some differences. This suggests the possibility of an association between celiac and allergic diseases.

Efficacy of specific immunotherapy in allergic disease has been clearly demonstrated. Thus, we will try to identify and purify allergens proteins from wheat in celiac people in order to test if a specific immunotherapy could be useful in their disease.

ACKNOWLEDGMENTS

This study was supported by the Hospital Universitario Rio Hortega and Hospital Clinico Universitario. SACYL.

Authors thank Profs Gabriel Salcedo and Arantxa Palacín from ETSI Ingenieros Agrónomos de Madrid for all their

56 Recent Patents on Inflammation & Allergy Drug Discovery 2008, Vol. 2, No. 1 Armentia et al.

help in the implementation of the study and for carrying immunologic tests with their purified cereal allergens.

We thank Prof. Keith Doorly for his help and all mem-bers of Allergy Section of Rio Hortega Hospital for technical assistance.

ABBREVIATIONS

ATG = Anti-transglutaminase antibody

AGA = Anti-gliadin antibody

ARA = Antireticulin antibody

AEA = anti-endomisyal antibody

SDS-PAGE = Sodium dodecylsulfate-polyacrylamide gel electrophoresis.

PVDF = Polyvinylidene difluoride

LTP = Non-specific lipid transfer protein

CM3 = Wheat tetrameric alpha amylase inhibitor subunit

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