1. Atsuo Urisu Department of Pediatrics Fujita Health University Nagoya Japan

2. Food Allergy and Atopic Dermatitis in young children

3. Guidelines for the Diagnosis and Management of Food Allergy in the United States: Report of the NIAID-Sponsored Expert Panel J Allergy Clin Immunol 2010;126:S1-S58.

4. In summary: AD and FA are highly associated. When tolerance develops to a food, the reintroduction of the food in the diet will not result in recurrence or worsening of the AD. Up to 37% of children under 5 years of age with moderate to severe AD will have IgE-mediated FA. Whether FA can exacerbate AD is still controversial, in part because the signs and symptoms of food allergen exposure are so pleomorphic and because well-designed relevant food allergen avoidance trials have rarely been done in patients with AD. Food allergy ＆ Atopic dermatitis J Allergy Clin Immunol 2010;126:S1-S58.

5. Cutaneous reactions to foods Cutaneous reactions to foods are some of the most common presentations of FA and include (1) IgE-mediated (urticaria, angioedema, flushing, pruritus), (2) cell-mediated (contact dermatitis, dermatitis Herpetiformis) (3) mixed IgE- and cell-mediated reactions. (atopic dermatitis) J Allergy Clin Immunol 2010;126:S1-S58.

6. Food Allergy ＆ Atopic Dermatitis The prevalence of FA in individuals with moderate to severe AD is 30% to 40%, and these patients have clinically significant IgE- mediated FA (as assessed by some combination of convincing symptoms, SPTs, sIgE levels, or oral food challenges) 47 or a definite history of immediate reactions to food. 48 47. Eigenmann PA, et al, Prevalence of IgE-mediated food allergy among children with atopic dermatitis. Pediatrics 1998 ;101:E8. 48. *Thompson MM, et al, Patterns of care and referral in children with atopic dermatitis and concern for food allergy. Dermatol Ther 2006 ;19:91. J Allergy Clin Immunol 2010;126:S1-S58.

7. 0 20 40 60 80 100 Immediate Non-immediate Deterioration of AD Score 0 Score 1Score 2Score 3Score 4 Positive Rate Deterioration of Atopic Dermatitis by Oral Food Challenge with Egg White Antigens 1/207/24 0/205/24 2/10 1/10 3/10 0/12 7/1212/2020/245/8 4/8 3/8 Figure ； Positive No/Total No

8. (PRU/ml) Case Age IgE for EggWhite Non-immediate R.T ♀ 1y7m 17.50 Urtcaria, Wheezing Deterioration of AD T.M. ♂ 4y10m 17.50 Abd Pain Deterioration of AD K.I. ♂ 4y2m 17.50 Rush, Itching Deterioration of AD A.K. ♀ 3y0m 4.80 Rush, Urticaria Deterioration of AD S.Y. ♂ 1y6m 10.30 Itching,Wheezing,Vomiting Deterioration of AD M.O. ♂ 9y8m 5.00 Rush, Pharingial irrtaiton Deterioration of AD A.W. ♀ 1y2m 4.40 Rush, Urticaria, Rhinorrhea Deterioration of AD Y.Y. ♂ 1y10m 4.30 Rush, Itching Deterioration of AD T.S. ♀ 1y2m 0.34 Diarrhea Deterioration of AD Deterioration of Atopic Dermatitis is followed by Immediate Reactions after egg white challenge Immediate Deterioration of AD was observed after immediate-type reactions by oral food challenge with egg white antigens.

9. Food-specific IgE Immediate-type Food Allergy Cell-mediated immunity Deterioration of Atopic Dermatitis Causative Food AD: mixed IgE- and cell- mediated reactions. Th2 cytcains from activated mast cell T cell activation by antigen presentation by FcεR I+Langerhans cell 30- 40% ？%？% Atopic Dermatitis Associated with Food Allergy The prevalence of FA in individuals with moderate to severe AD is 30% to 40%,

10. Diagnosis of Egg Allergy

11. Allergenic Activity of Hen’s Egg White Proteins Ovalbumin54.045++ Unstable Ovotransferrin12.076,6 Unstable Ovomucoid11.028+++ Stable Lysozyme3.414,3++ Unstable ProteinConstitute (%) MW )kDa) Allergenic Activity Heat Digestive enzyme-treated + IgE-binding activity

12. PlaceboFreeze-dried or heated (90 ℃, 60 min) egg white Oral Challenge Test by Freeze-dried or heated (90 ℃, 60 min) egg white

13. Egg White-specific IgE and Outcome of Oral Food Challenge (OFC) Test 0.35 0.7 3.5 17.5 50 100 0.35 0.7 3.5 17.5 50 100 Specifc IgE (log10 ） UA/ml Egg White P= 6.48 ｘ 10 -6 OFCFail Pass Egg White-specific IgE; Failed result of OFC test in more than ９５％ of Subjects

14. Structure of Ovomucoid (Kato et al 1987 ）

15. GroupABC Number 382941 IHR at provocation Heated egg white （ 90 ℃,60min ） +-- Raw egg white （ Freeze-dried ） ND +- Male/female, ratio27/1117/1223/18 Age (months; median with range)36 (16-137)30 (14-72)33 (18-156) Number of patients <2 years of age (%) 8 (21%)10 (34%)10 (24%) IHR, immediate hypersensitivity reaction; ND, not determined Significant difference from group A: *, P<0.01; **, P<0.001 Ando H et al; JACI 2008 Subject

16. Specific IgE and Oral Food Challenge Test with Freeze-dried Egg-White OCT; Positive (n=67), Negative (n=41) Egg White OA OM TotalIgE ROC Curve Ando H et al; JACI 2008

17. OFC; Positive (n=38) Negative (n=70) OA OM 総 IgE Specific IgE and Oral Food Challenge with heated Egg-White Egg White ROC Curve Ando H et al; JACI 2008

18. Challenge food Raw egg whiteHeated egg white EWOVAOVMEWOVAOVM Assay cut-offs (0.35 kU A /l) 0.35 (97%, 29%) 0.35 (97%, 32%) 0.35 (87%, 41%) 0.35 (100%, 20%) 0.35 (100%, 21%) 0.35 (97%, 36%) Optimal cut-offs (shortest distance to ROC) 2.82 (81%, 85%) 3.88 (76%, 83%) 2.26 (73%, 83%) 7.38 (66%, 79%) 6.33 (74%, 73%) 4.40 (76%, 81%) Positive decision points based on 95% clinical specificity 7.38 (57%, 95%) 9.84 (58%, 95%) 5.21 (52%, 95%) 30.70 (42%, 96%) 29.30 (47%, 96%) 10.80 (55%, 96%) Negative decision points based on 95% clinical sensitivity 0.60 (96%, 39%) 0.79 (96%, 46%) ND0.85 (97%, 36%) 1.17 (97%, 36%) 1.16 (97%, 53%) Positive & negative decision points and optimal cut-offs using specific IgE antibody at challenge with raw or heated egg white. (sensitivity, specificity) ND, not determined (it could not be calculated due to the assay measuring range)

20. Bernhisel-Broadbent J et al; Allergenicity and antigenicity of chicken egg ovomucoid (Gal d III) compared with ovalbumin (Gal d I) in children with egg allergy and in mice. JACI. 1994 93:1047. IgE specific for ovomucoid is higher in outgrowen subjects than in intolerant subjects. IgE IgG OVM First OVM Second Intolerant Outgrow

21. Urisu A, et al ； Clinical significance of IgE-binding activity to enzymatic digests of ovomucoid in diagnosis and prediction of outgrow of egg white hypersensitivity, Int Arch Allergy Appl Immunol,120,192,1999

22. Electrophoresis of OVM which digested with pepsin, chymotrypsin and trypsin. Pepsin - - + Chymotrypsin - - + Trypsin - - +

23. Outgrow Intolerant IgE-binding activity to enzymatic digests of ovomucoid Antigen concentration of enzyme-digested ovomucoid requiring for 50% RAST inhibition NS P=0.033 NS PepsinChymoptrypsinTrypsin NS

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25. Protocol of Oral Immunotherapy with Heated & Ovomucoid-Reduced Whole Egg Heated & OM- reduced Whole Egg Fail; Heated EW Pass; Heated & OM- reduced Whole Egg Challenge Test (Pre) Challenge Test (Post) Oral Immunotherapy (4w) Placebo Heated & OM- reduced Whole Egg Heated EW Challenge Test (Post) Oral Immunotherapy (4w) Heated EW Heated & OM- reduced Whole Egg Oral Immunotherapy (4w) Heated & OM- reduced Whole Egg Continued Heated & OM- reduced Whole Egg

26. 26 Results of Oral Challenge Test after Oral Immunotherapy with Heated- & OM-reduced Whole Egg Placebo １ M Active 1M Active 2M Active 3M 1/30 (3.3%) 10/48 (20.8%) 15/44 (34.1%) 26/34 (76.4%) Rate of Pass in Oral Challenge Test Placebo 1M Active1M Active2M Active3M A Group 22 17 9 B Group 30 26 17 10 Pass1 10 5 11 p=0.019* * χ ２ test

27. Placebo Heated & OM-reduced Whole Egg 1M 2M 3M No 30513820 Side Effect 2(0.2%) 5(0.3%)11 Details (Severity Grade*) Transient abdominal pain1110 Deterioration of AD (2)1100 Oral and pharyngeal Irritation (1)0300 Localized rush (2)0001 Side effects; No treatment was needed. * Grade classification; Sampson H; Pediatrics 2003;111:1601 Side Effect during Oral Immunotherapy

28. Mechanism of Oral Immunotherapy in Egg Allergy Th2 IL-12 IL-10 TGF-  INF-  ↓ IL-4 ↓ Th1 SOCS1 STAT1 Th2 IL-10 SOCS5 STAT6 Dendritic cell IL-10 TGF-  IgG4↑ IgE↓ Th2 Th1 Treg STAT4 SOCS1 Change observed in this study Inhibition Enhance IL-12