Apple Allergy

Allergen Data Collection - Update: Apple (Malus domestica)
Internet Symposium on Food Allergens 2(Suppl.4): 1-23 (2000) [http://www.food-allergens.de]

1 Prevalence of Apple Allergy

Prevalence data are based on different diagnostic procedures. While the prevalence of sensitization (sensitivity) can be estimated by SPT, RAST, and immunoblot, a clinical relevant sensitization (allergy) is evaluated by convincing history (anamnesis) or food challenge tests (ideally by DBPCFC).

Country / Subjects Sensitivity / Allergy to References

Denmark, Hellerup
101 birch and/or hazelnut allergic patients apple extract 8%
apple peel, fresh 28%
(SPT) Andersen & Lowenstein 1978

France, Paris
a) 24 patients with latex and pollen allergy
b) 20 patients with latex allergy (no pollen allergy)
c) 25 patients with pollen allergy (no latex allergy)

clinical symptoms SPT

a) apple in 4% and 29%

b) apple in 0% and 16%

c) apple in 48% and 56%

Levy et al. 2000

France, Pierre Benite
580 patients with adverse reactions to food (study period 1984-92) apple 15% (RAST) Andre et al. 1994

Finland, Helsinki
73 birch pollen allergic patients apple 63 % (case history)
apple pulp 43 % and apple seed 59% (SPT) Lahti et al. 1980

Finland, Oulu
children with atopic dermatits apple 0% and 13% in patients 1-3 years and 3-15 years of age (n=14 and 32) (SPT) Hannuksela 1987

France, Toulouse
142 food allergic children apple 0.7 % (labial food challenge) Rance & Dutau 1997

Germany, Berlin
167 pollen and food sensitive patients apple 93 % and 84 %
(SPT and case history) Jankiewicz et al. 1996

Germany, Frankfurt
80 patients with pollen associated food allergy apple 41% (SPT, RAST) Boehncke et al. 1998

Germany, Hamburg
30 patients with pollen associated food allergy apple 87 % (EAST) Möller et al. 1997

Italy, Genoa
132 pollen and food sensitive patients apple 38% (self-reported) Troise et al. 1992

Italy, Milan
262 fruit and/or vegetable allergic patients apple 53 % (clinical history) Ortolani et al. 1988

Italy, Milan
100 fruit and/or vegetable allergic patients apple 45 % (clinical history) Ortolani et al. 1989

Japan
171 birch pollen allergic patients apple 13 % (RAST) Yamamoto et al. 1995

Netherlands
131 cases of food- induced anaphylaxis
(from 1993-1997) apple 1.5% (survey, reported to the TNO Nutrition and Food Research Institute) European Commission 1998

Netherlands, Rotterdam
79 tree-pollen allergic patients apple 65%, 68%, and 44%
(SPT, RAST, and case history) de Groot et al. 1996

Spain, Madrid
29 plant-derived food allergic patients apple 24% (SPT) Diez-Gomez et al. 1999

Spain, Salamanca
84 mugwort sensitive patients without other pollen sensitizations apple 1.2% (RAST) Garcia-Ortiz et al. 1996

Sweden, Halmstad / Malmö
a) 380 birch pollen allergic patients
b) 103 patients without birch pollen allergy a) apple 47%
b) apple 6%
(questionnaire) Eriksson et al. 1982

Sweden, Uppsala
128 pollen allergic patients
a) birch pollen b) grass / mugwort pollen a) apple 90%
b) apple 46%
(SPT, controls = 17% positive) Dreborg & Foucard 1983

Switzerland, Vaudois
111 patients with pollen- associated food allergy apple 83 % (RAST) Bircher et al. 1994

Switzerland, Zurich
402 food allergic adults apple, kiwi 1.5 % (clinical history, diagnostic tests) Wüthrich 1993

Switzerland, Zurich
383 food allergic patients (study period 1990-94) apple 26% (clinical history, diagnostic tests) Etesamifar & Wüthrich 1998

UK, London
100 patients with food intolerance apple 1%
(repeated challenge) Lessof et al. 1980

UK, Manchester
90 patients expierenced anaphylactic reactions to foods (study period 1994-1996) apple 1% (suspected cause of patients' worst reaction) Pumphrey & Stanworth 1996

USA, Long Beach, CA
137 patients with latex allergy apple 2 % (convincing history of possible IgE mediated symptoms occurring within 60 minutes of ingestion) Kim & Hussain 1999

USA, Ohio
148 respiratory- allergic children with reproduced symptoms after food challenge apple 2 % (open challenge) Ogle et al. 1980

USA, Rockville, MD
34 patients allergic to tree pollens apple 71 % (SDS-PAGE immunoblot) Hsieh et al. 1995

2 Symptoms of Apple Allergy

Symptoms & Case Reports References

systemic reactions
anaphylaxis (2, 8, 12), exercise-induced anaphylaxis (6)
cutaneous symptoms
angioedema (1, 5, 13), urticaria (1, 7, 8, 12, 13)
gastrointestinal symptoms
glottis edema (13), tongue edema (13), itching in mouth (1, 5, 9), itching in throat (1, 9), swelling of lips (9), tingling in mouth (1, 5), vomiting (13), oral allergy syndrome* (3, 4, 7, 8, 10, 11, 12), in general (7, 12)
respiratory symptoms
allergic rhinitis (1), asthma (5, 7, 13)
* symptoms, which could be involved in oral allergy syndrome: local symptoms as intra-oral and lip-irritation, angioedema and systemic symptoms as rhino-conjunctivitis, urticaria-angioedema, asthma, and anaphylaxis (4)
(1) Kremser & Lindemayr 1983
(2) Pigatto et al. 1983
(3) Pastorello et al. 1987
(4) Ortolani et al. 1988
(5) Ortolani et al. 1989
(6) Anibarro et al. 1994
(7) van Ree et al. 1995
(8) Fernandez-Rivas et al. 1997
(9) Möller et al. 1997
(10) Wüthrich 1997
(11) Wüthrich et al. 1997
(12) Fernandez-Rivas & Cuevas 1999
(13) Sánchez-Monge et al. 1999

Percentage of reactions
Oral allergy syndrome 82%, with additional systemic symptoms 17%, extra-oral symptoms 18% in 139 apple-allergic patients (1)
Onset of symptoms within 5 min after food ingestion in > 50% of patients with oral allergy syndrome and within 30 min in > 90% (1)
In 10 apple allergic patients without related pollinosis: 64% anaphylaxis, 27% oral allergy syndrome, and 18% urticaria (2)
(1) Ortolani et al. 1988
(2) Fernandez-Rivas et al. 1997

Threshold for Elicitation of Symptoms
Amounts of apple fruit inducing symptoms ranged from 4 g to 32 g (estimated protein content: 12 - 96 mg), lower doses not tested (open challenge, 37 apple allergic patients) (1) (1) Pastorello et al. 1999

Symptoms & Case Reports	References
systemic reactions anaphylaxis (2, 8, 12), exercise-induced anaphylaxis (6) cutaneous symptoms angioedema (1, 5, 13), urticaria (1, 7, 8, 12, 13) gastrointestinal symptoms glottis edema (13), tongue edema (13), itching in mouth (1, 5, 9), itching in throat (1, 9), swelling of lips (9), tingling in mouth (1, 5), vomiting (13), oral allergy syndrome* (3, 4, 7, 8, 10, 11, 12), in general (7, 12) respiratory symptoms allergic rhinitis (1), asthma (5, 7, 13) * symptoms, which could be involved in oral allergy syndrome: local symptoms as intra-oral and lip-irritation, angioedema and systemic symptoms as rhino-conjunctivitis, urticaria-angioedema, asthma, and anaphylaxis (4)	(1) Kremser & Lindemayr 1983 (2) Pigatto et al. 1983 (3) Pastorello et al. 1987 (4) Ortolani et al. 1988 (5) Ortolani et al. 1989 (6) Anibarro et al. 1994 (7) van Ree et al. 1995 (8) Fernandez-Rivas et al. 1997 (9) Möller et al. 1997 (10) Wüthrich 1997 (11) Wüthrich et al. 1997 (12) Fernandez-Rivas & Cuevas 1999 (13) Sánchez-Monge et al. 1999
*Percentage of reactions* Oral allergy syndrome 82%, with additional systemic symptoms 17%, extra-oral symptoms 18% in 139 apple-allergic patients (1) Onset of symptoms within 5 min after food ingestion in > 50% of patients with oral allergy syndrome and within 30 min in > 90% (1) In 10 apple allergic patients without related pollinosis: 64% anaphylaxis, 27% oral allergy syndrome, and 18% urticaria (2)	(1) Ortolani et al. 1988 (2) Fernandez-Rivas et al. 1997
*Threshold for Elicitation of Symptoms* Amounts of apple fruit inducing symptoms ranged from 4 g to 32 g (estimated protein content: 12 - 96 mg), lower doses not tested (open challenge, 37 apple allergic patients) (1)	(1) Pastorello et al. 1999

3 Diagnostic Features of Apple Allergy

Parameters / Subjects Outcome References

IgE
birch pollen allergic patients
a) 24 with apple allergy, b) 9 apple tolerant Apple specific serum IgE (RAST):
a) 1.76 +/- 3.39 PRU/mL
b) 0.76 +/- 0.44 PRU/mL
no significant differences Pastorello et al. 1987

IgE
birch pollen allergic patients:
a) responding and b) non-responding to DBPCFC with birch pollen related foods Apple specific serum IgE (RAST):
a) 6.1 kU/L
b) 4.0 kU/L
(mean values, no significant difference) Reekers et al. 1999

Histamine Release (HR)
40 birch pollen-allergic patients
a) with and b) without fruit allergy Dose-dependent HR in both groups:
apple peel = apple pulp > peach = cherry
(to significant higher extent of HR in b)
significant increase of basophil sensitivity to birch pollen in group b) Kleine-Tebbe et al. 1992

IgE and Clinical Relevance
patients with clinical apple allergy (a), birch pollen allergy (b), or other allergies (c) apple specific IgE > 0.35 kU/L in
a) 90% (in 85% >0.7 kU/L),
b) 44%, and
c) 5-10% of patients (RAST) Bjorksten et al. 1980

SPT, IgE and Clinical Relevance
apple-allergic patients positive reactions in 84% and 3.6% of patients with clinical apple allergy tested with fresh apples and commercial extracts, respectively (SPT)
apple specific IgE in 70% of patients with clinical apple allergy (RAST) Ortolani et al. 1988

a) RAST and Clinical Relevance
b) SPT and Clinical Relevance
44 patients with clinical history of apple allergy a) RAST (specific IgE > 0.7 kU/L):
positive results in 71%
positive preditive value 79%
negative preditive value 62%
b) SPT with commercial extracts and fresh food:
positive results in 2.3 % and 81%
positive preditive value 100% and 78%
negative preditive value 40% and 71% Ortolani et al. 1989

SPT, Extracts
72 patients with birch pollen associated apple allergy Positivity in SPT:
a) Self-prepared extracts (low temperature method)
Granny Smith 91% (n=67)
Golden Delicious 87% (n=71)
b) 4 commercial allergen extracts: negative in all patients Vieths et al. 1995a

SPT, Commercial Extracts, Stable Allergens
298 patients with OAS after eating of fruits (Rosaceae) and /or nuts No positive reaction to commercial apple extract (SPT), 135 positive reactions to fresh apple in Prick-to-Prick test; patients sensitive to stable allergens may be detected with commercial plum or walnut extracts Asero 1999

Birch Pollen spec. IgE
103 birch pollen-hypersensitive patients free of oral allergy syndrome (at begin of the followed-up study) Birch pollen specific serum IgE- levels in patients:
a) who developed Apiaceae sensitivity 15.5 AU/mL
b) who developed apple/hazelnut allergy only 8.5 AU/mL
c) who remained free of oral allergy syndrome 5 AU/mL
(median values, P < 0.05) Asero 1997

HLA Class II Genes
42 birch pollen allergic patients with and without food allergy HLA class II DR4 and/or DR7 alleles were present in 42.6% of patients and in 2.4% of controls, no significant differences in the frequency of DPB1 alleles;
HLA-DR7 significantly involved in the presentation of apple and pollen allergens Sénéchal et al. 1999

Parameters / Subjects	Outcome	References
*IgE* birch pollen allergic patients a) 24 with apple allergy, b) 9 apple tolerant	Apple specific serum IgE (RAST): a) 1.76 +/- 3.39 PRU/mL b) 0.76 +/- 0.44 PRU/mL no significant differences	Pastorello et al. 1987
*IgE* birch pollen allergic patients: a) responding and b) non-responding to DBPCFC with birch pollen related foods	Apple specific serum IgE (RAST): a) 6.1 kU/L b) 4.0 kU/L (mean values, no significant difference)	Reekers et al. 1999
*Histamine Release (HR)* 40 birch pollen-allergic patients a) with and b) without fruit allergy	Dose-dependent HR in both groups: apple peel = apple pulp > peach = cherry (to significant higher extent of HR in b) significant increase of basophil sensitivity to birch pollen in group b)	Kleine-Tebbe et al. 1992
*IgE and Clinical Relevance* patients with clinical apple allergy (a), birch pollen allergy (b), or other allergies (c)	apple specific IgE > 0.35 kU/L in a) 90% (in 85% >0.7 kU/L), b) 44%, and c) 5-10% of patients (RAST)	Bjorksten et al. 1980
*SPT, IgE and Clinical Relevance* apple-allergic patients	positive reactions in 84% and 3.6% of patients with clinical apple allergy tested with fresh apples and commercial extracts, respectively (SPT) apple specific IgE in 70% of patients with clinical apple allergy (RAST)	Ortolani et al. 1988
a) RAST and Clinical Relevance b) SPT and Clinical Relevance 44 patients with clinical history of apple allergy	a) RAST (specific IgE > 0.7 kU/L): positive results in 71% positive preditive value 79% negative preditive value 62% b) SPT with commercial extracts and fresh food: positive results in 2.3 % and 81% positive preditive value 100% and 78% negative preditive value 40% and 71%	Ortolani et al. 1989
*SPT, Extracts* 72 patients with birch pollen associated apple allergy	Positivity in SPT: a) Self-prepared extracts (low temperature method) Granny Smith 91% (n=67) Golden Delicious 87% (n=71) b) 4 commercial allergen extracts: negative in all patients	Vieths et al. 1995a
*SPT, Commercial Extracts, Stable Allergens* 298 patients with OAS after eating of fruits (Rosaceae) and /or nuts	No positive reaction to commercial apple extract (SPT), 135 positive reactions to fresh apple in Prick-to-Prick test; patients sensitive to stable allergens may be detected with commercial plum or walnut extracts	Asero 1999
*Birch Pollen spec. IgE* 103 birch pollen-hypersensitive patients free of oral allergy syndrome (at begin of the followed-up study)	Birch pollen specific serum IgE- levels in patients: a) who developed Apiaceae sensitivity 15.5 AU/mL b) who developed apple/hazelnut allergy only 8.5 AU/mL c) who remained free of oral allergy syndrome 5 AU/mL (median values, P < 0.05)	Asero 1997
*HLA Class II Genes* 42 birch pollen allergic patients with and without food allergy	HLA class II DR4 and/or DR7 alleles were present in 42.6% of patients and in 2.4% of controls, no significant differences in the frequency of DPB1 alleles; HLA-DR7 significantly involved in the presentation of apple and pollen allergens	Sénéchal et al. 1999

4 Therapy of Apple Allergy

Treatment * Outcome References

Tree Pollen Immunotherapy
72 children with birch pollinosis (age of 6-16 years), prevalence of adverse reactions to apple before immunotherapy 78%
I. subcutaneous immunotherapy for 3 years with a) birch pollen preparation or b) a mixture of birch, alder, and hazel pollen
II. oral immunotherapy for 10 months with c) birch pollen preparation or d) placebo capsules Assessment of food allergy after treatment (self-reported):

improved unchanged worse

a) (n=19) 37% 42% 21%

b) (n=20) 55% 30% 15%

c) (n=14) 21% 64% 14%

d) (n=14) 14% 86% 0%

no significant more decrease in birch pollen immunotherapies as compared to placebo oral immunotherapy Möller 1989

Birch Pollen Immunotherapy
20 birch pollen allergic patients 2 or 3 courses of immunotherapy in successive years:
fruit allergy improved in 9 patients
no improvement in 4 patients
fruit allergy developed in 3 patients
At the end of 3 years 16 patients were allergic to fruit, 13 of them to apple
IgE during immunotherapy:
birch and apple specific IgE increased temporary
IgG and IgG4 during immunotherapy:
increase of birch specific Ab only
histamine release:
sensitivity to birch pollen extract decreased significantly during immunotherapy, no significant changes to apple extract Herrmann et al. 1995

Birch Pollen Immunotherapy
49 birch pollen allergic patients with apple- induced oral allergy syndrome 12, 24, or 36 months of pollen- specific immunotherapy:
84 % improved (reported symptoms of OAS)
88 % improved (fresh apple, SPT)
IgE during immunotherapy:
significant decrease in birch pollen- specific IgE levels, apple- specific IgE reduction in 21%, no change in 43%, increase in 38% (RAST) Asero 1998

Birch Pollen Immunotherapy
15 patients with birch pollen allergy and associated apple allergy two courses of pre-seasonal birch pollen immunotherapy:
in 60% beneficial effects on apple allergy
in 87% improvement of pollen allergy
(immunoblot inhibition corroborated importance of homology from Bet v 1 and Mal d 1) Henzgen et al. 1999

Oral Desensitization
1 apple allergic patient
(effectiveness not confirmed by DBPCFC) A diluted food extract followed by increased pure food was administered following a standardized protocol, at the beginning pretreatment with oral sodium cromoglycate, length of therapy 3.5 months, after therapy apple was tolerated (maintenance dose: 1 apple twice a week) Patriarca et al. 1998

* Studies may be experimental, unproved, or controversial. Please notice the disclaimer !

5 Composition of Apple

6 Allergens of Apple

Proteins / Glycoproteins Allergen Nomenclature References

Bet v 1 - homologous Protein [18 kDa] Mal d 1 Ebner et al. 1991, Vieths et al. 1994, Vanek-Krebitz et al. 1995

Thaumatin-homologous Protein [31 kDa] Mal d 2 Hsieh et al. 1995

Lipid-transfer Protein [9 kDa] Mal d 3 Pastorello et al. 1999, Sánchez-Monge et al. 1999

Apple Profilin [14-15 kDa] Mal d 4* Vallier et al. 1992, van Ree et al. 1992, Ebner et al. 1995, van Ree et al. 1995

Art v 1 cross-reactive Allergen: 60 kDa Heiss et al. 1996, Grote et al. 1998

Allergen: 34-35 kDa Wellhausen et al. 1996

Allergens: >30 kDa (carbohydrate epitopes) Vieths et al. 1994b

Allergens: 8-16, 18, 35, 50 kDa Vieths et al. 1992

Allergens: 13, 14, 16, 18, 31 kDa Hsieh et al. 1995

Allergens: 18 and 67-kDa Möller et al. 1997

* proposed name not yet listed in WHO/IUIS Allergen Nomenclature

6.1 Sensitization to Apple Allergens

Country / Subjects Sensitivity to References

Austria, Vienna
83 birch pollen allergic patients double band at 17 and 18 kDa in 97% (SDS-PAGE immunoblot) Ebner et al. 1991

Austria, Vienna
20 birch pollen allergic patients double band at 17 and 18 kDa in 95%
profilin-homologue 14 kDa allergen in 20 %
(SDS-PAGE immunoblot) Ebner et al. 1995

Germany, Berlin
23 (22) birch pollen and apple allergic patients Allergens from Golden Delicious:

Allergens mature semi-mature

> 50 kDa in 9% 27%

50 kDa in 13% 77%

40-50 kDa in 39% 68%

35 kDa in 4% 23%

18 kDa (Mal d 1) in 65% 9%

16 kDa in 26% 5%

10-15 kDa in 30% 36%

(SDS-PAGE / immunoblot) Vieths et al. 1993

Germany, Berlin
12 apple and celery allergic patients Carbohydrate epitopes on allergens > 30 kDa
(periodate treatment, immunoblot, EAST inhibition) Vieths et al. 1994b

Germany, Berlin
33 birch pollen and apple allergic patients Mal d 1 in 73 % and 76 % (purified allergen from Golden Delicious and Granny Smith) (SPT) Vieths et al. 1995b

Italy, Milan
apple allergic patients
a) with birch pollen allergy
b) without birch pollen allergy

Allergen a) n = 32 b) n = 11

60 kDa in 22% 0%

43, 51, 84 kDa in 34% 0%

28 kDa in 6% 0%

18 kDa in 91% 0%

15 kDa in 50% 0%

Mal d 3 in 28% 100%

(SDS-PAGE / immunoblot) Pastorello et al. 1999

Spain, Madrid
10 apple and peach allergic patients Mal d 3 in 90 % (SDS-PAGE / immunoblot) Sánchez-Monge et al. 1999

USA, Rockville, MD
24 tree pollen and apple allergic patients

Allergens n = 24

> 38 kDa in 58%

31 kDa in 75%

18 kDa (Mal d 1) in 38%

16 kDa in 4%

14 kDa in 25%

13 kDa in 17%

(SDS-PAGE / immunoblot) Hsieh et al. 1995

6.2 Properties of Bet v 1 - homologous Protein (Mal d 1)
6.3 Properties of Thaumatin - homologous Protein (Mal d 2)
6.4 Properties of Lipid-transfer Protein (Mal d 3)
6.5 Properties of Apple Profilin

7 Isolation & Preparation

Extract / Purified Allergens Methods References

Protein extract Extraction of apple allergens in an active form, inhibition of reactions with phenolic compounds present in apple, chelators and solid PVPP in extraction medium Bjorksten et al. 1980

Protein extract Low temperature extraction method:
fresh fruit homogenized in acetone (-40°C), precipitates washed, filtered, lyophylized and water extracted Vieths et al. 1994c

Protein extract
(in vivo and in vitro- test solutions) Preparation of active extracts: application of inhibitors of cytoplasmic enzymes (phenol oxidases, peroxidases, proteases) during extraction, precipitation in organic solvents (diacetone alcohol) at -20°C and resolubilization in potassium phosphate buffer;
Storage of extract solutions at 4°C was most effective with PBS/glycerol and cysteine/sodium citrate/glycerol stabilizing additives Rudeschko et al. 1995a, 1995b

Protein extract Peeled apple pulp or powdered whole frozen apple were homogenized with extraction buffer (containing sucrose, polyvinylpyrrolidone, EDTA, and diethyldithiocarbamic acid, pH 9.5-10), filtered, centrifugated and stored at -20°C Hsieh et al. 1995

Protein extract Enhanced protein content of apple extracts after anion- exchange chromatography Martinez et al. 1997

Protein extract (apple peel)
(in vivo and in vitro- test solutions) Fresh peel (Granny Smith) homogenized with potassium phosphate buffer (pH 7.0, containing PVPP, EDTA, diethyldithiocarbamate and sodium azide), centrifuged, dialyzed, lyophylized and resuspended in saline plus phenol Asero et al. 1999

18-kDa allergen (Mal d 1) Isolation after modified low-temperature extraction: IEC followed by RP-HPLC Fahlbusch et al. 1995

18-kDa allergen (Mal d 1) Isolation after low-temperature extraction method by micropreparative SDS-PAGE followed by electroelution Vieths et al. 1995b

9-kDa allergen (Mal d 3) Isolation and purification from raw extract (Bjorksten et al. 1980) by cation exchange chromatography (Resource S column) with sodium citrate buffer (pH 6) applying salt gradient, followed by SEC (Superdex 75 column) with sodium citrate / sodium chloride buffer (pH 6) Pastorello et al. 1999

Extract / Purified Allergens	Methods	References
Protein extract	Extraction of apple allergens in an active form, inhibition of reactions with phenolic compounds present in apple, chelators and solid PVPP in extraction medium	Bjorksten et al. 1980
Protein extract	Low temperature extraction method: fresh fruit homogenized in acetone (-40°C), precipitates washed, filtered, lyophylized and water extracted	Vieths et al. 1994c
Protein extract (in vivo and in vitro- test solutions)	Preparation of active extracts: application of inhibitors of cytoplasmic enzymes (phenol oxidases, peroxidases, proteases) during extraction, precipitation in organic solvents (diacetone alcohol) at -20°C and resolubilization in potassium phosphate buffer; Storage of extract solutions at 4°C was most effective with PBS/glycerol and cysteine/sodium citrate/glycerol stabilizing additives	Rudeschko et al. 1995a, 1995b
Protein extract	Peeled apple pulp or powdered whole frozen apple were homogenized with extraction buffer (containing sucrose, polyvinylpyrrolidone, EDTA, and diethyldithiocarbamic acid, pH 9.5-10), filtered, centrifugated and stored at -20°C	Hsieh et al. 1995
Protein extract	Enhanced protein content of apple extracts after anion- exchange chromatography	Martinez et al. 1997
Protein extract (apple peel) (in vivo and in vitro- test solutions)	Fresh peel (Granny Smith) homogenized with potassium phosphate buffer (pH 7.0, containing PVPP, EDTA, diethyldithiocarbamate and sodium azide), centrifuged, dialyzed, lyophylized and resuspended in saline plus phenol	Asero et al. 1999
18-kDa allergen (Mal d 1)	Isolation after modified low-temperature extraction: IEC followed by RP-HPLC	Fahlbusch et al. 1995
18-kDa allergen (Mal d 1)	Isolation after low-temperature extraction method by micropreparative SDS-PAGE followed by electroelution	Vieths et al. 1995b
9-kDa allergen (Mal d 3)	Isolation and purification from raw extract (Bjorksten et al. 1980) by cation exchange chromatography (Resource S column) with sodium citrate buffer (pH 6) applying salt gradient, followed by SEC (Superdex 75 column) with sodium citrate / sodium chloride buffer (pH 6)	Pastorello et al. 1999

8 Cross-Reactivities


Cross-Reacting Allergens	Subjects / Methods	References
*Apple* birch pollen *	Correlation between birch pollen allergy and apple hypersensitivity (1129 adults with bronchial asthma and/or allergic rhinitis, questionaire)	Eriksson 1978
*Apple fruit* birch pollen	Apple allergic patients' serum pool: birch pollen inhibited IgE- binding to apple allergens (RAST-inhibition)	Bjorksten et al. 1980
*Apple fruit* significant associations: apple and cherry, apple and pear, apple and birch pollen *	262 fruit and/or vegetable allergic patients (clinical history, SPT, RAST)	Ortolani et al. 1988
*Apple allergen* (17 to 18 kDa) birch pollen	32 patients with birch pollen allergy (pooled serum) birch pollen inhibited IgE- binding to 17-18 kDa apple- allergen apple extract did not diminish binding to Bet v 1 (immunblot- inhibition)	Ebner et al. 1991
*Apple* (15 kDa) celery profilin (15 kDa)	Sera reactive to 15 kDa celery allergen: Inhibition of IgE and celery profilin specific rabbit-mAb binding to 15 kDa apple allergen by celery crude extract and by celery profilin, respectively (immunoblot inhibition)	Vallier et al. 1992
*Apple allergens* birch pollen	Apple and birch pollen allergic patients (immunoblot inhibition)	Vieths et al. 1992
*Apple* kiwi fruit	3 kiwi allergic patients (RAST inhibition)	Gall et al. 1994
*Apple* birch pollen (Bet v 1)	Depletion of birch-pollen extract for Bet v 1 removed approximately 95% of the IgE cross- reactivity between birch pollen and apple extract; Cross-reactive human IgE antibodies reacted with an epitope nonoverlapping with the epitope recognized by a mAb (1 patient, RAST inhibition, two-site RIA)	Akkerdaas et al. 1995
*Apple allergens* (17 kDa / Mal d 1) birch pollen (Bet v 1)	7 Bet v 1 and Bet v 2 sensitive patients (pooled serum): complete inhibition of IgE- binding by rBet v 1 to 17 kDa apple allergen, no inhibition of binding to 14 kDa allergen (immunoblot inhibition)	Ebner et al. 1995
*Apple allergens* (14 kDa) birch pollen (Bet v 2 profilin)	7 Bet v 1 and Bet v 2 sensitive patients (pooled serum): complete inhibition of IgE- binding by rBet v 2 to 14 kDa apple allergen, no inhibition of binding to 17 kDa allergen (immunoblot inhibition)	Ebner et al. 1995
*Apple allergens* (18 kDa / Mal d 1) birch pollen (Bet v 1)	3 birch pollen and apple allergic patients and pooled serum: Allergenic potencies: Bet v 1 > Mal d 1 (EAST inhibition)	Fahlbusch et al. 1995
*Apple* a) grass pollen (Lolium perenne) b) profilin (Lolium perenne) c) carbohydrate moieties (Lolium perenne)	a) Inhibition of IgE- binding to apple extract by grass pollen (Lolium perenne) in 8 of 9 patients (RAST inhibition) b) Decrease of IgE- binding to apple extract (41%) from anti-profilin- IgE-depleted serum (RAST) c) Inhibition of IgE- binding to apple extract by carbohydrate moieties in 1 patient (proteinase K digested grass pollen extract, RAST inhibition)	van Ree et al. 1995
*Apple allergens* (18 kDa / Mal d 1) birch pollen (Bet v 1)	2 birch pollen and apple allergic patients and pooled serum: Allergenic potencies: Bet v 1 > Mal d 1 (EAST inhibition, histamine release)	Vieths et al. 1995b
*Apple* 60 kDa mugwort allergen (Art v 1)	Patients with food and pollen allergy: inhibition of IgE-binding to 60 kDa apple allergen by 60 kDa mugwort allergen in 2 of 3 patients (SDS-PAGE inhibition), 4-36% reduction of IgE-binding to apple proteins by 60 kDa mugwort allergen in 3 patients (RAST inhibition)	Heiss et al. 1996
*Apple* tomato	Tomato allergic patients (EAST inhibition)	Petersen et al. 1996
*Apple* pear	Serum pool from 7 birch pollen allergic patients: inhibition of IgE- binding to pear allergens by apple extract from Golden Delicious (EAST inhibition)	Vieths et al. 1996
*Apple* (34-35 kDa) birch pollen allergen 35 kDa and Bet v 1	Sera from birch pollen allergic patients reactive to 35 kDa allergen: IgE binding to apple extract inhibited by 35 kDa allergen and Bet v 1 of birch pollen (EAST inhibition) 35 kDa allergen from birch pollen inhibited IgE binding to 34-35 kDa apple allergen (immunoblot inhibition)	Wellhausen et al. 1996
*Apple* a) peach, pear, mugwort pollen b) rye grass profilin *	a) Patients allergic to Rosaceae fruits Allergenic potencies: peach > apple > pear (RAST inhibition) b) Cross-reactivity in patients with apple and pollen allergy, no cross-reactivity to profilin in patients without pollinosis (RAST, histamine release)*	Fernandez-Rivas et al. 1997
*Apple* (18-kDa / Mal d 1) birch pollen, Bet v 1	7 apple / kiwi allergic patients (EAST inhibition, immunoblot inhibition)	Möller et al. 1997
*Apple* (67-kDa-Allergen) kiwi allergens (43, 67 kDa)	7 apple / kiwi allergic patients: max. inhibition of IgE-binding to apple extract: kiwi extract 27% (EAST inhibition, immunoblot inhibition)	Möller et al. 1997
*Apple Extract* allergens rPru a 1 (cherry), rApi g 1 (celery), and rBet v 1 (birch pollen)	0% inhibition of IgE-binding to apple extract by rApi g 1, 90% inhibition by rPru a 1, and 100% inhibition by rBet v 1 (immunoblot inhibition estimated according to band intensities, 4 birch pollen and cherry allergic patients) appr. 50% max. inhibition of IgE-binding to rPru a 1 by rMal d 1, max. inhibition to rApi g 1 by rMal d 1 <20% (EAST inhibition, cherry and/or celery allergic patients)	Scheurer et a. 1999
*Apple* (18-kDa / Mal d 1) birch pollen, Bet v 1	13 birch pollen and apple sensitive patients 79% of Mal d 1-specific T-cell clones cross-reacted with Bet v 1; 44% Bet v 1-specific T-cell clones cross-reacted with Mal d 1; 6 cross-reactive T-cell epitopes from Bet v 1 Stimulating potencies: Bet v 1 > Mal d 1 (T-Cell proliferation, recombinant allergens)	Fritsch et al. 1998
*Apple extract, rMal d1* birch pollen, rBet v 1, rBet v 2 profilin), timothy grass pollen extract (21 patients with clinical relevant allergy to pollen and plant-derived food)	Mixture of rBet v 1 and rBet v 2 inhibited IgE-binding to 10-14 kDa (profilin related) and 17-21 kDa (Bet v 1 related) apple allergens, timothy grass pollen inhibited IgE-binding to 10-14 kDa and 30-100 kDa allergens from apple; only weak inhibition of IgE-binding to Bet v 1 by rMal d 1 (immunoblot inhibition); 92% (4.7-100%) inhibition of IgE-binding to rMal d 1 by mixture of rBet v 1 and rBet v 2 and 96% (23-100%) by mixture of rBet v1, rBet v 2, and timothy pollen extract (52 sera) (RAST inhibition)	Kazemi-Shirazi et al. 2000
*Apple pollen* birch pollen	Patients with birch pollen allergy and oral allergy to apple fruit; IgE binding inhibitory potency to birch pollen by apple pollen extract 1000-fold lower than homologous inhibition with birch pollen (RAST-inhibition)	Berrens et al. 1990
*Apple seeds* birch pollen	3 birch pollen-sensitive patients (RAST inhibition)	Lahti et al. 1980
*Birch pollen* (Bet v 1, Bet v 2 profilin) *	28 patients with oral allergy syndrome after ingestion of apples: 57% sensitive to rBet v 1 20 polysensitized patients with oral allergy syndrome after ingestion of apples: 20% sensitive to rBet v 2 (RAST)*	Rossi et al. 1996

* multiple sensitization (not proven by inhibition-tests)

9 Stability of Apple Allergens


Treatment / Ripening	Effects	References
*Apple* pulp	Apple-pulp allergens are unstable (skin test)	Lahti et al. 1980
*Apple* (Ripening) mature and unripe fruits	(1) Stronger IgE binding to allergens of mature Golden Delicious apple (SDS-PAGE immunoblot) (2) Higher relative amounts of 18-kDa allergen (Mal d 1) in mature apples than in unripe apples (Golden Delicious > Boskoop) (SDS-PAGE immunoblot, densitometry, EAST inhibition)	(1) Vieths et al. 1992 (2) Vieths et al. 1993
*Apple* (Storage, Ripening) a) store-purchased b) storage at 4°C c) different ripening stages of stored apples (controlled atmospheric conditions) d) sprayed to prevent microbial infection	a) Higher relative amounts of 18-kDa allergen (Mal d 1) as compared to fresh apple (Golden Delicious, Granny Smith) b) Increasing amounts of 18-kDa allergen (Mal d 1) after 3 weeks c) No clear correlation between ripening stages and amounts of 31- and 18-kDa allergens, reduction of 16-kDa allergen related to ripening d) Only 16-kDa allergen detected during regulated ripening conditions (see c) (SDS-PAGE immunoblot)	Hsieh et al. 1995
*Apple slices* (Heat) heating at 175°C for 0.5 h, storage at RT	allergenic activity: heat labile and decreased during storage at RT	Dreborg & Foucard 1983
Apple Extracts (Heat) heat (30 min, 100°C)	Heating of the food reduced allergenic activity, while semipurified protein extracts from apple retained immunoreactivity of the major allergens	Vieths et al. 1998
*Apple* (Enzymic Hydrolysis) gastric digestion assay	Mal d 1 and Bet v 1 degraded within seconds under physiological conditions	Jensen-Jarolim et al. 1999
*Apple Prick Test Solutions* 4 commercial prick test solutions	No positive results in SPT with commercial extracts in 72 patients with apple allergy (positivity in SPT with self-prepared extracts up to 91%)	Vieths et al. 1995a
*Apple Test Solutions* 4 commercial extracts 1 self-prepared peel extract	a) SPT: No reactivity using 3 commercial extracts, 14% sensitivity for 1 commercial extract, 91% sensitivity for peel extract b) RAST: 55% and 9% false negativ results for 1 commercial and for the peel extract, respectively (a) 35 and b) 11 birch- pollen allergic patients with OAS to apple and positive SPT to fresh apple)	Asero et al. 1999
*Apple Prick Test Solutions* 5 commercial prick test solutions	Only 1 solution showed weak allergenic activity as compared to highly active self-prepared apple extract (EAST)	Hoffman et al. 1999
*Apple Extracts* (Xenobiotica) spiked with pesticides (chlorpropham, iprodione or thiram)	15 Apple allergic patients: a) SPT: in 40% - 73% inhibitory effect of pesticides (weaker skin reactions), in < 20% stronger skin reactions b) EAST / mediator release: dose- dependent decrease of spec. serum IgE and histamine release by chlorpropham spiked apple extracts, no influence on LTC4 release (mediator release from basophils, HPLC)	Vieluf et al. 1997

10 Allergen Sources


Reported Adverse Reactions	References
*Food / Food additives* After ingestion of fresh fruits (1)	(1) see 2 Symptoms of Apple Allergy
*Peel vs. Pulp* More frequently and more severe adverse reactions after ingestion of the whole fruit (peel and pulp) , 44% of patients tolerated ingestion of apple pulp (18 apple allergic patients)	Fernandez-Rivas & Cuevas 1999


Allergens in Apple Products	Results	References
*Apple Allergens* tree pollen and apple allergic patients	trace ammounts of active allergens in canned apples (SDS-PAGE immunoblot)	Hsieh et al. 1995
*Peel vs. Pulp* In vitro allergenicity in apple allergic patients	Peels induced higher SPT, histamine release and RAST results than pulps; higher IgE-binding potency of peel extract than pulp extract in RAST inhibition	Fernandez-Rivas & Cuevas 1999

11 Allergenicity of Different Apple Varieties


Varieties / Subjects	Differences	References
*16 Apple strains* (Mal d 1) patients allergic to birch pollen and apples	Relative amounts of 18-kDa allergen (Mal d 1): great variation in 16 apple strains Allergenicity of strains decreased in the following order: Golden Delicious > Boskoop > Jamba (corresponding to high, low, and very low 18-kDa allergen content) (SDS-PAGE / immunoblot / densitometry, EAST, histamine release, open challenge)	Vieths et al. 1994a
*7 Apple strains* (Mal d 1) patients allergic to birch pollen and apples	Expression levels of Mal d 1 in 100 g of fresh apple: 4.5 mg Golden Delicious, 1.6 mg Granny Smith, 0.7 mg Jona Gold, 0.8 mg Idared, 1.8 mg Gala, 0.5 mg Jamba, and 0.4 mg Gloster (SDS-PAGE coomassie staining)	Son et al. 1999
*4 Apple strains* (Mal d 1, Mal d 2) patients allergic to tree pollen and apples	Relative amounts of 31-kDa allergen (Mal d 2): Golden Delicious and Granny Smith > McIntosh and Red Delicious Relative amounts of 18-kDa allergen (Mal d 1): Golden Delicious > other strains (SDS-PAGE immunoblot)	Hsieh et al. 1995
*4 Apple strains* (SPT) 72 patients with birch pollen associated apple allergy	Positivity in SPT: Granny Smith 91%, Golden Delicious 87%, Boskoop 12%, and Jamba 8% (n=67-72)	Vieths et al. 1995a
*2 Apple strains* (SPT) patients allergic to tree pollen	Granny Smith more positive skin reactions and a better agreement with clinical history than Golden Delicious	de Groot et al. 1996

Proteins / Glycoproteins	Allergen Nomenclature	References
Bet v 1 - homologous Protein [18 kDa]	Mal d 1	Ebner et al. 1991, Vieths et al. 1994, Vanek-Krebitz et al. 1995
Thaumatin-homologous Protein [31 kDa]	Mal d 2	Hsieh et al. 1995
Lipid-transfer Protein [9 kDa]	Mal d 3	Pastorello et al. 1999, Sánchez-Monge et al. 1999
Apple Profilin [14-15 kDa]	Mal d 4*	Vallier et al. 1992, van Ree et al. 1992, Ebner et al. 1995, van Ree et al. 1995
Art v 1 cross-reactive Allergen: 60 kDa		Heiss et al. 1996, Grote et al. 1998
Allergen: 34-35 kDa		Wellhausen et al. 1996
Allergens: >30 kDa (carbohydrate epitopes)		Vieths et al. 1994b
Allergens: 8-16, 18, 35, 50 kDa		Vieths et al. 1992
Allergens: 13, 14, 16, 18, 31 kDa		Hsieh et al. 1995
Allergens: 18 and 67-kDa		Möller et al. 1997