12007-03-29

f416 rTri a 19; Omega-5 Gliadinf416 rTri a 19; Omega-5 Gliadin

-- a risk marker for severe immediate or exercise-induced a risk marker for severe immediate or exercise-induced reactions in patients with suspected wheat allergyreactions in patients with suspected wheat allergy

22007-03-29

Classification of wheat proteinsClassification of wheat proteins

Wheatproteins

Wheatproteins

(Gluten 85%)

AlbuminAlbumin

GlobulinGlobulin

Gliadins(α-,β-,γ-,ω-1,2,3,4, ω-5)

Gliadins(α-,β-,γ-,ω-1,2,3,4, ω-5)

Glutenins(HMW*-, LMW-)

Glutenins(HMW*-, LMW-)

Water/salt insolubleWater/salt insoluble

Water/salt solubleWater/salt soluble

32007-03-29

What is on ImmunoCAPWhat is on ImmunoCAP® ®

f416 rTri a 19; Omega-5 Gliadin?f416 rTri a 19; Omega-5 Gliadin? Tri a 19; Omega-5 Gliadin from wheat (Triticum aestivum). Omega-5 gliadin is a storage protein and a major allergen

among water/salt–insoluble proteins, with limited presence on f4 Wheat ImmunoCAP.

Omega-5 gliadin can be found to some extent on f79 Gluten and to a higher extent on f98 Gliadin ImmunoCAP.

The immunodominant peptide epitopes of omega-5 gliadin have been identified.

A cDNA encoding a hydrophilic portion of Tri a 19, containing major epitopes for IgE antibodies, has been cloned and expressed in E. coli and the purified recombinant product coupled to ImmunoCAP.

42007-03-29

Possible clinical utility of ImmunoCAPPossible clinical utility of ImmunoCAP® ® f416 rTri a 19; Omega-5 Gliadin f416 rTri a 19; Omega-5 Gliadin

Identify patients at risk of developing severe immediate reactions to wheat.

Identify patient at risk of developing anaphylaxis to wheat in relation to exercise (WDEIA).

52007-03-29

Severe immediate reactions to wheatSevere immediate reactions to wheat

Are important to identify, especially in children. ~ 80% of patients with severe immediate reactions have

IgE antibodies to both omega-5 gliadin and wheat. The remaining 20% of patients have IgE antibodies to

other components in wheat and will thus be positive with f4 Wheat ImmunoCAP.

62007-03-29

Doctors diagnosis of wheat allergy and levels of IgE ab Doctors diagnosis of wheat allergy and levels of IgE ab to omega-5 gliadin in 88 children sensitized to wheatto omega-5 gliadin in 88 children sensitized to wheat

100

10

1

0.1No wheat allergy

n = 44Wheat allergy

n = 44

kU/L

A

Reference: Ito K et al. Poster presented at AAAAI in San Diego 2007

12/44 (27%)> 0.35 kUA/l

37/44 (84%)> 0.35 kUA/l

72007-03-29

Challenge outcome and levels of IgE ab to Challenge outcome and levels of IgE ab to omega-5 gliadin in children sensitized to wheatomega-5 gliadin in children sensitized to wheat

100

10

1

0.1Negative

Challenge/Historyn = 44

Mildn = 5

p<0.001

Moderaten = 8

Severen = 8

kU/L

A

Positive Challenge

Reference: Ito K et al. Poster presented at AAAAI in San Diego 2007

82007-03-29

Wheat-dependent, exercise-Wheat-dependent, exercise-induced anaphylaxis (WDEIA)induced anaphylaxis (WDEIA)

Wheat allergy induced by physical exercise. Symptoms are typically generalized urticaria and

severe allergic reactions such as anaphylactic shock or hypotension.

Skin tests and in vitro IgE ab tests for wheat are not always satisfactory.

Challenge test + intense physical exercise is the gold std for diagnosis, but is inconvenient and dangerous.

92007-03-29

Major allergens in WDEIAMajor allergens in WDEIA

~80% of patients with WDEIA have IgE ab to omega-5 gliadin.

~20% of patients with WDEIA have IgE ab to high molecular weight glutenin (HMW-glutinin).

30-50% of patient with WDEIA are, however, negative with f4 Wheat ImmunoCAP, but a majority are positive to omega-5 gliadin.

102007-03-29

IgE ab to different wheat IgE ab to different wheat proteins in patients with WDEIA proteins in patients with WDEIA

IgE ab to different wheat IgE ab to different wheat proteins in patients with WDEIA proteins in patients with WDEIA

Peptide A = Omega-5 gliadin

Peptide B = HMW-glutinin0.35

5

10

15

20

25

30

35

1

G luten Peptide A Peptide BW hea t

Spec

ific Ig

E (k

U/L

)A

W D E IA patients reac ting predom inan tly to pep tide A (n=23)W D E IA patients reac ting predom inan tly to pep tide B (n=6)W D E IA patients not reac ting to bo th pep tide A and B (n=1)A topic de rm atitis patients (n=25)N orm al con tro ls (n=25)

112007-03-29

Investigation of risk for severe immediate or exercise-Investigation of risk for severe immediate or exercise-induced reactions in patients with suspected wheat allergyinduced reactions in patients with suspected wheat allergy

NoYes

Test resultsf4 neg / f416 pos

Test resultsf4 pos / f416 pos

Test resultsf4 pos / f416 neg

Test resultsf4 neg / f416 neg

Test with ImmunoCAP Allergen:™

f4 Wheat + f416 rTri a 19; Omega-5 Gliadin

Conclusion

No sensitization to wheat; symptoms are probably caused by another food or due to a different mechanism

Conclusion

High risk for severeimmediate or exercise-induced reactions due to wheat

Conclusion

High risk for severe immediate reactions due to wheat

Conclusion

High risk for exercise- induced reactions due to wheat

Conclusion

Low risk for severeimmediate or exercise-induced reactions due to wheat

Is there a history of exercise-induced symptoms?

122007-03-29

Recommended test procedure in Recommended test procedure in patients with suspected wheat allergypatients with suspected wheat allergy

f4 Wheat f4 Wheat ++ f415 rTri a 19; Omega-5 gliadin f415 rTri a 19; Omega-5 gliadin The clinical relevance of a positive f4 Wheat test are sometimes difficult to interpret. Omega-5 gliadin is found to be a risk marker for severe immediate reactions including

WDEIA (80% pos). 30-50% of patients with WDEIA have no IgE abs to wheat (f4). f4 neg & f416 neg tests: no sensitization to wheat is found; another food or mechanism

is probably the cause. f4 neg & f416 pos tests: high risk for severe immediate reactions including WDEIA due

to wheat. f4 pos & f416 pos tests: high risk for severe immediate reactions including WDEIA due

to wheat. f4 pos & f416 neg tests: low risk for severe immediate reactions including WDEIA due to

wheat. However, many of these patients will have immediate type reactions such as skin and gastrointestinal symptoms.

132007-03-29

A case reportA case report

Age: 57 Gender: Male Race group: White Country of birth: Zambia Living in: South Africa Occupation: Engineer, Project Manager (often very stressful) Activities: Camping, Cycling, Scuba diving, Hiking

142007-03-29

AnamnesisAnamnesis

Onset three years ago, June 2003. 15 adverse reactions between June -03 and Feb -06. Reaction one hour after eating with extreme itching from hairline

to armpits, buttocks and groin where hives break out. Steadily increasing hart rate (180 beats vs. 80 normally). Tight chest with labored breathing. Loss of conscious ones with cardiac arrest. Mild reactions on a few occasions that were stopped with 3

antihistamine tablets. Many hospital visits with intravenous adrenaline and cortisone.

152007-03-29

What is the offending allergen (s)? What is the offending allergen (s)? - ImmunoCAP- ImmunoCAP® ® tested in tested in

South AfricaSouth Africa

13 Nov 2003 Total IgE 1601 kU/l Phadiatop positive Cat 1.09 kUA/l Mixed Cockroach 0.56

kUA/l Negative test results*:

Dog, mites, trees, weeds, timothy grass, mould, egg, milk, soy, peanut, wheat, fish and shrimp

31 March 2004 Total IgE 1818 kU/l Phadiatop positive Milk 0.58 kUA/l Negative test results*:

soy, peanut, wheat, fish, shrimp, cheese

* Negative < 0.35 kUA/l

162007-03-29

What is the offending allergen (s)? What is the offending allergen (s)? - - ImmunoCAPImmunoCAP® ® tested in South Africatested in South Africa

11 March 2006 Total IgE 2315 kU/l Mixed cockroach 0.81 kUA/l (tropomyosin) Avocado & pear negative* (Chitinase) Pineapple negative* (Profilin) Sunflower seed negative* (2S albumin) Peach negative* (LTP) Strawberry negative* (Bet v 1) Salmon negative* (Calcium binding protein/Polcalcin) Yeast negative* (Enolase) Bell pepper/paprika negative* (Thaumatin protein)* Negative < 0.35 kUA/l

172007-03-29

What is the offending allergen (s)? What is the offending allergen (s)? - ImmunoCAP- ImmunoCAP® ® tested at Phadia in Swedentested at Phadia in Sweden

Fall 2006 Phadiatop 0.47 PAU/l Pork 0.81 kUA/l Mushroom 0.1 kUA/l Sesame 0.19 kUA/l Black pepper 0.13 kUA/l Olive 0.15 kUA/l Casein 0.19 kUA/l Bovine lactoferrin 0.64 kUA/l Beef 1 kUA/l Anisakis 0.15 kUA/l Echinococcus 0.91 kUA/l

Wheat 3.3 kUA/l Gluten 4.4 kUA/l Gliadin 30.5 kUA/l Omega-5 Gliadin 30.5 kUA/l

Negative test results*: tomato, Gly m 4, Ara h 8, onion, grape, mustard, alfa-lactalbumin, beta-lactoglobuline, CCD; MUXF3

* Negative < 0.1 kUA/l

182007-03-29

Summary case reportSummary case report

Omega-5 gliadin from wheat was finally found to be the offending allergen.

Since wheat was tested negative already in South Africa it was not at once retested at Phadia.

To test for gluten (f79) was a last try to find the offending allergen.

The positive gluten result (4.4 kUA/L) led into testing with gliadin (AGf98) but also omega-5 gliadin (f416) that was under development.

When omega-5 gliadin was tested positive (30.5 kUA/L) the wheat test was repeated and now found positive (3.3 kUA/L).

Similar levels of IgE ab to gliadin (AGf98) and omega-5 gliadin (f416), indicate that the amount of omega-5 gliadin is fairly good represented on gliadin ImmunoCAP AGf98.

192007-03-29

Information material availableInformation material available

See more info in Marketing Info No. 6 & 7/2007

New Horizon 1/2007; article & clinical leaflet, product leaflet and test chart

202007-03-29

References1. Matsuo H, Kohno K, Morita E.

Molecular cloning, recombinant expression and IgE-binding epitope of -5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis.FEBS Journal 2005;272:4431-8

2. Morita E, Matsuo H, Mihara S, Morimoto K, Savage AW, Tatham AS.Fast -gliadin is a major allergen in wheat-dependent exercise-induced anaphylaxis.J Dermatol Sci 2003;33(2):99-104

3. Palosuo K, Alenius H, Varjonen E, Kalkkinen N, Reunala T.Rye -70 and -35 secalins and barley -3 hordein cross-react with -5 gliadin, a major allergen in wheat-dependent, exercise-induced

anaphylaxis.Clin Exp Allergy 2001;31(3):466-73

4. Matsuo H, Morita E, Tatham AS, Morimoto K, Horikawa T, Osuna H, Ikezawa Z, Kaneko S, Kohno K, Dekio S.Identification of the IgE-binding epitope in -5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis.J Biol Chem 2004;279(13):12135-40

5. Tanabe S, Arai S, Yanagihara Y, Mita H, Takahashi K, Watanabe M.A major wheat allergen has a Gln-Gln-Gln-Pro-Pro motif identified as an IgE-binding epitope.Biochem Biophys Res Commun 1996;219(2):290-3

6. Battais F, Pineau F, Popineau Y, Aparicio C, Kanny G, Guerin L, Moneret-Vautrin DA, Denery-Papini S.Food allergy to wheat: identification of immunogloglin E and

immunoglobulin G- binding proteins with sequential extracts and purified proteins from wheat flour.

Clin Exp Allergy 2003;33(7):962-70

7. Palosuo K, Alenius H, Varjonen E, Koivuluhta M, et al.A novel wheat gliadin as a cause of exercise-induced anaphylaxis.J Allergy Clin Immunol 1999;103(5 Pt 1):912-7

8. Maruyama N, Ichise K, Katsube T, Kishimoto T, Kawase S, Matsumura Y, Takeuchi Y, Sawada T, Utsumi S.Identification of major wheat allergens by means of the Escherichia coli expression system.Eur J Biochem 1998;255(3):739-45

9. Palosuo K, Varjonen E, Nurkkala J, Kalkkinen N, Harvima R, Reunala T, Alenius H.Transglutaminase-mediated cross-linking of a peptic fraction of -5 gliadin enhances IgE reactivity in wheat-dependent, exercise-induced anaphylaxis.J Allergy Clin Immunol 2003;111(6):1386-92

10. Varjonen E, Vainio E, Kalimo K.Life threatening anaphylaxis caused by allergy to gliadin and exercise.Clin Exp Allergy 1997;27:162-6

11. Lehto M, Palosuo K, Varjonen E, Majuri ML, Andersson U, Reunala T, Alenius H.

Humoral and cellular responses to gliadin in wheat-dependent, exercise-induced anaphylaxis.

Clin Exp Allergy 2003;33(1):90-5

12. Morita E, Kameyoshi Y, Mihara S, Hiragun T, Yamamoto S.-Gliadin: a presumptive allergen causing wheat-dependent exercise-induced anaphylaxis. [Letter]Br J Dermatol 2001;145(1):182-4

13. Morita E, Yamamura Y, Mihara S, Kameyoshi Y, Yamamoto S.Food-dependent exercise-induced anaphylaxis: a report of two cases and determination of wheat--gliadin as the presumptive allergen.Br J Dermatol 2000;143(5):1059-63

14. Gall H, Steinert M, Peter RU.Exercise-induced anaphylaxis to wheat flour.Allergy 2000;55(11):1096-7

15. Palosuo K, Varjonen E, Kekki OM, Klemola T, Kalkkinen N, Alenius H, Reunala T.

Wheat -5 gliadin is a major allergen in children with immediate allergy to ingested wheat.J Allergy Clin Immunol 2001;108(4):634-8

16. Daengsuwan T, Palosuo K, Phankingthongkum S, Visitsunthorn N, Jirapongsananuruk O, Alenius H, Vichyanond P, Reunala T.IgE antibodies to -5 gliadin in children with wheat-induced anaphylaxis.Allergy 2005;60:506-9