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These limitations notwithstanding, this study confirmed that bullying victimization is a common experience in food-allergic patients. Previous studies were conducted exclusively in NorthAmerican population; the fact that similar data emerged from Italy suggests that bullying of food-allergic kids is a universal issue. Any clinician who cares for children or adolescents, in primary care or specialty area, may be in an important position to recognize the effects of bullying. Questions about bullying experiences with peers should be included routinely during visits with patients and parents. Families and schools should be educated about the need to address this risk when it is present. Antonella Muraro, MD, PhDa Laura Polloni, PsyDa Francesca Lazzarotto, MDa Alice Toniolo, BSca Ileana Baldi, PhDb Roberta Bonaguro, MD, PhDa Gianluca Gini, PhDc Matthew Masiello, MD, MPHd From athe Referral Centre for Food Allergy Diagnosis and Treatment, Veneto Region, Department of Women and Child Health, Padua University Hospital, bthe Unit of Biostatistics, Epidemiology and Public Health, Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, and cthe Department of Developmental and Social Psychology, University of Padua, Padua, Italy; and dthe Center for Health Promotion and Disease Prevention, Windber Research Institute, Windber, Pa. E-mail: [email protected]. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

REFERENCES 1. Gini G, Pozzoli T. Bullied children and psychosomatic problems: a meta-analysis. Pediatrics 2013;132:720-9. 2. Ttofi MM, Farrington DP, Losel F, Loeber R. Do the victims of school bullies tend to become depressed later in life? A systematic review and meta-analysis of longitudinal studies. J Aggress Conflict Peace Res 2011;3:63-73. 3. Reijntjes AHA, Kamphuis JH, Prinzie P, Telch MJ. Peer victimization and internalizing problems in children: a meta-analysis of longitudinal studies. Child Abuse Negl 2010;34:244-52. 4. Lieberman JA, Weiss C, Furlong TJ, Sicherer M, Sicherer SH. Bullying among pediatric patients with food allergy. Ann Allergy Asthma Immunol 2010;105:282-6. 5. Shemesh E, Annunziato RA, Ambrose MA, Ravid NL, Mullarkey C, Rubes M, et al. Child and parental reports of bullying in a consecutive sample of children with food allergy. Pediatrics 2013;131:e10-7. 6. Oppenheimer J, Bender B. The impact of food allergy and bullying. Ann Allergy Asthma Immunol 2010;105:410-1. 7. Genta ML, Menesini E, Fonzi A, Costabile A, Smith PK. Bullies and victims in schools in central and southern Italy. Eur J Psychol Educ 1996;11:97-110. 8. Referral Centre for Health Promotion, Veneto Region. Mirandola M, Campostrini S, Furegato M, Baldassari D, editors. Youth in Veneto - Analysis of the results of the Health Behaviour in School-aged Children (HBSC) Study 2010 - World Health Organization. 2011: Cierre Edition. Available at: http://www.crrps.org/download/ HBSC_Giovani_In_Veneto_Indagine2010.pdf. Accessed February 27, 2014. Available online July 18, 2014. http://dx.doi.org/10.1016/j.jaci.2014.05.043

Loss of allergenic proteins during boiling explains tolerance to boiled peanut in peanut allergy To the Editor: Thermal processing has the potential to modify the capacity of a food to elicit an allergic reaction.1-3 Cooking practices may account for the lower reported rates of peanut allergies in countries where boiled rather than roasted peanuts are predominantly consumed.4,5 In vitro data support this premise: roasting either

TABLE I. Serial component resolved diagnostics to peanut in patient 1, pre- and post-boiled peanut desensitization

Specific IgE to peanut (kU/L) Ara h 1 (kU/L) Ara h 2 (kU/L) Ara h 3 (kU/L)

Pre-desensitization

Tolerant to boiled but not raw peanut

Tolerant to raw peanut

38.4

7.89

11.4

1.65 17.2 0.2

0.71 8.6 0.2

1.01 12.1 0.21

had no effect6 or increased the IgE reactivity of peanuts,5 while boiling appears to reduce it, possibly through leaching of allergen into the cooking water5 or inducing a change in structure.5-7 However, the reduced potency of boiled peanuts to elicit a reaction in subjects with peanut allergy has yet to be demonstrated. We report 4 children with a diagnosis of peanut allergy who have varying degrees of tolerance to boiled peanut, with evidence that the boiling process significantly reduced in vitro IgE reactivity to the boiled peanut seeds, as determined by immunoblotting. Patient 1 (14 years, female) had multiple food allergies (egg, cow’s milk, cashew, pistachio) and was avoiding peanut on the basis of sensitization with a persistent large skin prick test (9-26 mm), serum-specific IgE (ssIgE) to peanut, and Ara h 2 (Table I), indicative of a >95% likelihood of clinical reactivity. She successfully tolerated phased introduction of increasing doses of daily boiled peanut (initially boiled for 16 hours, reduced to 2 hours and then 30 minutes) into her diet. Over the course of 2 years, she was transitioned to daily raw peanut, and experienced minor allergic symptoms (transient, self-limiting, abdominal pain) twice during this period. A significant decrease was noted in ssIgE to Ara h 2 (Table I) during this time, and she therefore underwent formal oral food challenge (OFC) to a cumulative dose of 4 g of raw peanut protein, which she tolerated (serum was collected at this point for IgE-immunoblot). OFC was repeated after the patient had ceased peanut ingestion for 1 month; she developed urticaria, cough, and wheeze after 1.7 g raw peanut protein, indicating a lack of sustained tolerance induction. Patients 2 and 3 are siblings (12 and 15 years of age, male). Patient 2 reacted to roasted peanut at formal OFC in 2010 (ssIgE to peanut 9.6 kU/L), with persisting sensitization suggestive of ongoing allergy. Patient 3 had a history of acute-onset, generalized urticaria following exposure to a small amount of peanut as an infant, with persisting sensitization thereafter (ssIgE to peanut 7.5 KU/L; Ara h 2 12.3 kU/L in April 2013). Both boys commenced daily ingestion of peanuts (initially boiled for 4 hours, decreasing to 1 hour over 6 months). After 8 months (at the time of serum collection), patient 2 was tolerating 2 g of boiled peanut daily; patient 3 reacted to 2 g of boiled peanut at up-dosing but continues to tolerate 0.5 g boiled peanut daily. Patient 4 (10 years of age, female) had multiple food allergies (egg, milk, and tree nuts) and was avoiding peanut based on persistent large skin prick test. She tolerated 8 g peanut (boiled for 2 hours) at OFC, at which time serum was obtained; ssIgE to peanut (19.5 kU/L) and Ara h 2 (13.2 kU/L) were indicative of a >95% likelihood of clinical reactivity. The serum IgE reactivity patterns of these patients were then profiled using total extracts of raw or boiled (6 hours in a closed vessel) peanuts prepared from defatted flours8 by sonicating for

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FIG 1. Serum IgE reactivity of patients to raw and boiled peanut proteins, together with clinical data at the time of blood sampling. Sodium dodecyl sulfate PAGE analysis (A) of extracts of raw (lane 1) and boiled (lane 2) defatted peanut flours, and boiled peanut cooking water (lane 3) with 5 mg protein loaded per lane.8 Replica immunoblots developed using serum from patients with peanut allergy (B-F). SPT, Skin prick test.

15 minutes at 608C in 2-dimensional polyacrylamide gel electrophoresis (PAGE) extraction buffer (7 M urea, 2 M thiourea, 2% [w/v] CHAPS, 1% (w/v) Dithiothreitol, pH 8.8). Extracts and the cooking water were then analyzed by sodium dodecyl sulfate PAGE using the NuPAGE gel (Life Technologies, Grand Island, NY) system (5 mg protein loaded per lane) and visualized using a Coomassie-based stain. The key peanut allergens were identified in raw peanut using published Mr values (Fig 1).8 This showed a loss of Ara h 1 in the peanut seeds after boiling, which was accompanied by an apparent reduction in polypeptides corresponding to Ara h 2, 6, and 7. Polypeptides of Mr ;3.5-40 kDa could be identified in the cooking water, several of which appeared to correspond to the acidic subunit of Ara h 2, 3, 6, and 7, although many appeared to be novel. IgE immunoblot was undertaken using sera from the above children, together with control sera (positive control, allergic to both raw and boiled [4 hours] peanut at formal OFC, and nonfood-allergic control). Significant IgE binding to raw and boiled peanut proteins was seen in the positive control, including reactivity to higher molecular weight aggregates in the boiled peanut seeds (Fig 1, B, lane 2) as well as proteins in the cooking water (Fig 1, B, lane 3). In contrast, IgE reactivity of the patients tolerant to boiled peanut was much lower and generally restricted to the Ara h 2, 6, and 7 polypeptides in raw peanut (Fig 1, C-F, lane 1), with evidence of binding to the cognate proteins in the cooking water (Fig 1, C-F, lane 3). No IgE binding to proteins in the boiled peanut extract corresponding to the Ara h 2, 6, and

7 region was observed, with some weak binding to higher Mr aggregates in patients 3 and 4 (Fig 1, E and F, lane 2). Patient 3 had more extensive binding, which may explain his development of allergic symptoms with exposure to 2 g boiled peanut while his brother (patient 2) did not. No IgE binding to the extracts was observed with the non-food-allergic control serum (data not shown). Our data is consistent with previous reports that boiling results in loss of proteins—particularly Ara h 2, 6, and 7—from peanut seeds, although we cannot exclude the possibility that changes in protein structure6,7,9 might have also contributed to the clinical tolerance observed. Furthermore, we have demonstrated that the in vitro reactivity correlates with in vivo clinical reactivity, something that has not been previously reported. While we cannot exclude the possibility that some persons allergic to peanut may be tolerant to boiled peanut de novo, on the basis of this data, we believe that at least for patients 1 and 3, tolerance to boiled peanut was induced through oral desensitization, because these individuals had reactions during dose escalation. Current attempts in specific oral tolerance induction using peanut involve exposure to peanut protein via the oral or sublingual route, which is associated with a significant rate of allergic symptoms, particularly during the up-dosing phase. On the basis of our data, we believe that exposure to boiled peanut may permit the ingestion of low amounts of proteins (including 2S albumins), which may be an alternative means by which desensitization can be induced in patients allergic to peanut, with potential for increased safety and

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decreased breakthrough allergic symptoms. Based on our findings, this option may be particularly appropriate for individuals with allergy who predominantly produce IgE to 2S albumins. A controlled trial of tolerance induction using boiled peanut in patients allergic to peanut is required to investigate this further, before this can be recommended as a potential treatment modality. Paul J. Turner, FRACP, PhDa,b Sam Mehr, FRACPc Rebekah Sayers, MResd Melanie Wong, FRACP, PhDc Mohamed H. Shamji, PhDe Dianne E. Campbell, FRACP, PhDb,c E. N. Clare Mills, PhDd From athe Section of Paediatrics (Allergy and Infectious Diseases) and MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Imperial College London, London, United Kingdom; bthe Division of Paediatrics and Child Health, University of Sydney, Sydney, New South Wales, Australia; cthe Department of Allergy and Immunology, Children’s Hospital at Westmead, Sydney, Australia; dthe Institute of Inflammation and Repair, Manchester Institute of Biotechnology, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom; and ethe Section of Allergy and Clinical Immunology, National Heart and Lung Institute and MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, Imperial College London, London, United Kingdom. E-mail: paulyt@ doctors.org.uk. Disclosure of potential conflict of interest: P. J. Turner has received research support from the UK Medical Research Council and the NHS National Institute for Health Research. R. Sayers has received research support from the Biotechnology and Biological Sciences Research Council (BBSRC) and Campden BRI and has received travel support from the BBSRC. D. E. Campbell has received research support from Nestle. E. N. C. Mills has received research support from the European Union, the Biotechnology and Biological Sciences Research Council, the UK Food Standards Agency, the European Food Safety Authority, the UK Technology Strategy Board, DBV Technology, and Novartis; has board memberships with Novartis, UK Food Standards Agency, PepsiCo International, BBSRC, and Reacta Biotech Ltd; is employed by the University of Manchester and the Institute of Food Research; has stock/stock options in Standard Life and Reacta Biotech Ltd; has received travel support from the International Life Sciences Institute, the European Academy of Allergy and Clinical Immunology, the University of Bologna, Europa Bio, British High Commission, Iceland Allergy Society, Fresenius, EuroFood Tox 2013, and the International Union of Nutritional Science Annual Meeting; has been a lecturer and supervisor of masters students for Imperial College; and has been an external examiner for the University of Birmingham. The rest of the authors declare that they have no relevant conflicts of interest.

REFERENCES 1. Nowak-Wegrzyn A, Bloom KA, Sicherer SH, Shreffler WG, Noone S, Wanich N, et al. Tolerance to extensively heated milk in children with cow’s milk allergy. J Allergy Clin Immunol 2008;122:342-7. 2. Martos G, Lopez-Exposito I, Bencharitiwong R, Berin MC, Nowak-We˛grzyn A. Mechanisms underlying differential food allergy response to heated egg. J Allergy Clin Immunol 2011;127:990-7. 3. Turner PJ, Wong M, Varese N, Rolland JM, O’Hehir RE, Campbell DE. Tolerance to wheat in whole-grain cereal biscuit in wheat-allergic children. J Allergy Clin Immunol 2013;131:920-3. 4. Shek LP, Cabrera-Morales EA, Soh SE, Gerez I, Ng PZ, Yi FC, et al. A population-based questionnaire survey on the prevalence of peanut, tree nut, and shellfish allergy in 2 Asian populations. J Allergy Clin Immunol 2010; 126:324-31, 331.e1-7. 5. Beyer K, Morrow E, Li XM, Bardina L, Bannon GA, Burks AW, et al. Effects of cooking methods on peanut allergenicity. J Allergy Clin Immunol 2001;107: 1077-81. 6. Blanc F, Vissers YM, Adel-Patient K, Rigby NM, Mackie AR, Gunning AP, et al. Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity. Mol Nutr Food Res 2011;55:1887-94. 7. Vissers YM, Blanc F, Skov PS, Johnson PE, Rigby NM, Przybylski-Nicaise L, et al. Effect of heating and glycation on the allergenicity of 2S albumins (Ara h 2/6) from peanut. PLoS One 2011;6(8):e23998.

8. Marsh J, Rigby N, Wellner K, Reese G, Knulst A, Akkerdaas J, et al. Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis. Mol Nutr Food Res 2008;52(suppl 2):S272-85. 9. Mondoulet L, Paty E, Drumare MF, Ah-Leung S, Scheinmann P, Willemot RM, et al. Influence of thermal processing on the allergenicity of peanut proteins. J Agric Food Chem 2005;53:4547-53. Available online July 25, 2014. http://dx.doi.org/10.1016/j.jaci.2014.06.016

Peanut allergy prevalence among school-age children in a US cohort not selected for any disease To the Editor: What is the prevalence of peanut allergy among US children? Given that 90% of US households consume peanut butter,1 this is an important question. The answer is not straightforward, however, as estimates of peanut allergy prevalence among US children differ by allergy definition, study population, and methodology.2 Previous estimates for US children have been based on self-report3-6 or specific IgE (sIgE) criteria,7 which are thought to be inaccurate.2 Estimates have varied according to whether they were based on telephone surveys,3 electronic surveys,4 or nationally representative surveys such as the National Health and Nutrition Examination Survey (NHANES) (Table I).5-7 One must consider that self-report is hindered by reporting bias, surveys of food allergy are more likely to enlist those with the condition, and nationally representative surveys are limited in the extent of phenotyping possible given their wide scope. It can therefore be difficult to discern how differences in definition, study population, and methodology affect prevalence estimates across studies. Here we report and compare prevalence estimates of childhood peanut allergy according to varying criteria among 7- to 10-year-old children participating in a US birth cohort not selected for any disease. We determined prevalence of childhood peanut allergy based on reported symptoms, sIgE levels, clinical information, and combinations of these variables among participants of Project Viva. Project Viva is a large, observational cohort study based in eastern Massachusetts with enrollment from Harvard Vanguard Medical Associates, a multi-site group medical practice. Participants were not selected for any disease. The study was designed to examine maternal dietary and other factors that could influence child health outcomes, with health broadly defined.8 Enrollment occurred between 1999 and 2002 in early pregnancy and resulted in delivery of 2128 singleton children. Interviews and questionnaires on child health were administered when the children were age 6 months, 1 year, and annually thereafter. We collected outcome data for this study at the mid-childhood in-person visit (mean age 7.9 years). Among the 1277 children who presented for an in-person interview at mid-childhood, 699 (55%) had blood drawn, and 616 (87.7% of those with blood samples) had sIgE measured by ImmunoCAP (Phadia AB, Uppsala, Sweden). Compared with those who did not follow up, participants who did follow up showed higher proportions of maternal white race (69% vs 62%), college or graduate education _ $70,000 (63% vs (69% vs 58%), and annual household income > 58%), but there were no significant differences in parental atopy (P 5 .13). Compared with the general US population, there was a higher proportion of blacks and lower proportion of Hispanics among participants. Further details regarding the comparability