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World Allergy Organization (WAO) Diagnosis and Rationale for Action against Cow's Milk Allergy (DRACMA) Guidelines

      Keywords

      This article was originally published online on 23 April 2010

      Authorship

      Alessandro Fiocchi, MD, Pediatric Division, Department of Child and Maternal Medicine, University of Milan Medical School at the Melloni Hospital, Milan 20129, Italy.
      Holger Schünemann, MD,a Department of Clinical Epidemiology & Biostatistics, McMaster University Health Sciences Centre, 1200 Main Street West Hamilton, ON L8N 3Z5, Canada.
      Sami L. Bahna, MD, Pediatrics & Medicine, Allergy & Immunology, Louisiana State University Health Sciences Center, Shreveport, LA 71130.
      Andrea Von Berg, MD, Research Institute, Children’s department, Marien-Hospital, Wesel, Germany.
      Kirsten Beyer, MD, Charité Klinik für Pädiatrie m.S. Pneumologie und Immunologie, Augustenburger Platz 1, D-13353 Berlin, Germany.
      Martin Bozzola, MD, Department of Pediatrics, British Hos-pital-Perdriel 74-CABA-Buenos Aires, Argentina.
      Julia Bradsher, PhD, Food Allergy & Anaphylaxis Network, 11781 Lee Jackson Highway, Suite 160, Fairfax, VA 22033.
      Jan Brozek, MD,a Department of Clinical Epidemiology & Biostatistics, McMaster University Health Sciences Centre, 1200 Main Street West Hamilton, ON L8N 3Z5, Canada.
      Enrico Compalati, MD,a Allergy & Respiratory Diseases Clinic, Department of Internal Medicine. University of Genoa, 16132, Genoa, Italy.
      Motohiro Ebisawa, MD, Department of Allergy, Clinical Research Center for Allergy and Rheumatology, Sagami-hara National Hospital, Kanagawa 228-8522, Japan.
      Maria Antonieta Guzman, MD, Immunology and Allergy Division, Clinical Hospital University of Chile, Santiago, Chile. Santos Dumont 999.
      Haiqi Li, MD, Professor of Pediatric Division, Department of Primary Child Care, Children’s Hospital, Chongqing Medical University, China, 400014.
      Ralf G. Heine, MD, FRACP, Department of Allergy & Immunology, Royal Children’s Hospital, University of Melbourne, Murdoch Children’s Research Institute, Melbourne, Australia.
      Paul Keith, MD, Allergy and Clinical Immunology Division, Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
      Gideon Lack, MD, King’s College London, Asthma-UK Centre in Allergic Mechanisms of Asthma, Department of Pediatric Allergy, St Thomas’ Hospital, London SE1 7EH, United Kingdom.
      Massimo Landi, MD, National Pediatric Healthcare System, Italian Federation of Pediatric Medicine, Territorial Pediat-ric Primary Care Group, Turin, Italy.
      Alberto Martelli, MD, Pediatric Division, Department of Child and Maternal Medicine, University of Milan Medical School at the Melloni Hospital, Milan 20129, Italy.
      Fabienne Rancé, MD, Allergologie, Hôpital des Enfants, Pôle Médicochirurgical de Pédiatrie, 330 av. de Grande Bretagne, TSA 70034, 31059 Toulouse CEDEX, France.
      Hugh Sampson, MD, Jaffe Food Allergy Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, NY 10029-6574.
      Airton Stein, MD, Conceicao Hospital, Porto Alegre, Brazil.
      Luigi Terracciano, MD,a Pediatric Division, Department of Child and Maternal Medicine, University of Milan Medical School at the Melloni Hospital, Milan 20129, Italy.
      Stefan Vieths, MD, Division of Allergology, Paul-EhrlichInstitut, Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Str. 51-59, D-63225 Langen, Germany.
      aMember of the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) Working Group

      Revision Panel

      Amal Assa’ad, MD, Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA.
      Carlos Baena-Cagnani, MD, LIBRA foundation Argentina, Division of Immunology and Respiratory Medicine, Department of Pediatric, Infantile Hospital Cordoba, Santa Rosa 381, 5000 Cordoba, Argentina.
      GR Bouygue, MSc, Pediatric Division, Department of Child and Maternal Medicine, University of Milan Medical School at the Melloni Hospital, Milan 20129, Italy.
      Walter Canonica, MD, Allergy & Respiratory Diseases Clinic, Department of Internal Medicine. University of Genoa, 16132, Genoa, Italy.
      Christophe Dupont, MD, Service de gastroentérologie et nutrition, Hôpital Saint Vincent de Paul, 82, avenue Den-fert-Rochereau, 75674, Paris CEDEX 14, France.
      Yehia El-Gamal, MD, Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt.
      Matthew Fenton, MD, Asthma, Allergy and Inflammation Branch, National Institute of Allergy and Infectious Diseases, NIH, 6610 Rockledge Dr., Bethesda, MD 20892.
      Rosa Elena Huerta Hernandez, MD, Pediatric Allergy Clinic, Mexico City, Mexico.
      Manuel Martin-Esteban, MD, Allergy Department, Hospital Universitario La Paz, Madrid, Spain.
      Anna Nowak-Wegrzyn, MD, Jaffe Food Allergy Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, NY 10029-6574.
      Ruby Pawankar, MD, Department of Otolaryngology, Nippon Medical School, 1-1-5 Sendagi, Tokyo, 113 Japan.
      Susan Prescott, MD, School of Pediatrics and Child Health, University of Western Australia, Princess Margaret Hospital for Children, Perth, Australia.
      Patrizia Restani, PhD, Department of Pharmacological Sciences, Università degli Studi di Milano.
      Teresita Sarratud, MD, Department of Pediatrics, University of Carabobo Medical School at the Carabobo Hospital, Valencia, Venezuela.
      Aline Sprikkelmann, MD, Department of Pediatric Respiratory Medicine and Allergy, Emma Children’s Hospital Academic Medical Centre, Amsterdam, The Netherlands.

      Sections

      • 1:
        Introduction, p. 58.
      • 2:
        Methodology, p. 59.
      • 3:
        Epidemiology of CMA, p. 61.
      • 4:
        Allergens of Cow’s Milk, p. 65.
      • 5:
        Immunological Mechanisms of CMA, p. 71.
      • 6:
        Clinical History and Symptoms of CMA, p. 76.
      • 7:
        Diagnosis of CMA According to Preceding Guidelines, p. 85.
      • 8:
        The Elimination Diet in Work-Up of CMA, p. 88.
      • 9:
        Guidelines for Diagnosing CMA, p. 89.
      • 10:
        Oral Food Challenge Procedures in Diagnosis of CMA, p. 100.
      • 11:
        Natural History of CMA, p. 108.
      • 12:
        Treatment of CMA According to Preceding Guidelines, p. 112.
      • 13:
        When Can Milk Proteins Be Eliminated From Diet Without Substituting Cow’s Milk?, p. 117.
      • 14:
        Guidelines for Choosing a Replacement Formula, p. 119.
      • 15:
        Milks From Different Animals for Substituting Cow’s Milk, p. 124.
      • 16:
        Nutritional Considerations in CMA Treatment, p. 128.
      • 17:
        Choosing the Appropriate Substitute Formula in Different Presentations, p. 130.
      • 18:
        Grade Recommendations on Immunotherapy for CMA, p. 131.
      • 19:
        Unmet Needs, Recommendations for Research, Implementation of DRACMA, p. 133.
      Acknowledgements, p. 134
      Appendix 1: Cow’s Milk Allergy Literature Search Algorithms, p. 135
      Appendix 2: Evidence Profiles: Diagnosis of CMA, p. 144
      Appendix 3: Evidence Profiles: Treatment of CMA, p. 154
      Appendix 4: Evidence Profiles: OIT for Treatment of CMA, p. 160

      Section 1: Introduction

      Allergy and clinical immunology societies have issued guidance for the management of food allergy.
      American College of Allergy, Asthma, & Immunology
      Food allergy: a practice parameter.
      ,
      • Mukoyama T.
      • Nishima S.
      • Arita M.
      • Ito S.
      • Urisu A.
      • et al.
      Guidelines for diagnosis and management of pediatric food allergy in Japan.
      Guidelines are now regarded as translational research instruments, designed to provide cutting-edge benchmarks for good practice and bedside evidence for clinicians to use in an interactive learning context with their national or international scientific communities. In the management of cow’s milk allergy (CMA), both diagnosis and treatment would benefit from a reappraisal of the more recent literature, for “current” guidelines summarize the achievements of the preceding decade, deal mainly with prevention,
      • Prescott S.L.
      The Australasian Society of Clinical Immunology and Allergy position statement: Summary of allergy prevention in children.
      ,
      • Muraro A.
      • Dreborg S.
      • Halken S.
      • Høst A.
      • Niggemann B.
      • et al.
      Dietary prevention of allergic diseases in infants and small children. Part III: Critical review of published peer-reviewed observational and interven-tional studies and final recommendations.
      ,
      • Muraro A.
      • Dreborg S.
      • Halken S.
      • Høst A.
      • Niggemann B.
      • et al.
      Dietary prevention of allergic diseases in infants and small children. Part I: immunologic background and criteria for hypoallergenicity.
      • Muraro A.
      • Dreborg S.
      • Halken S.
      • Høst A.
      • Niggemann B.
      • Aalberse R.
      • et al.
      Dietary prevention of allergic diseases in infants and small children. Part II. Evaluation of methods in allergy prevention studies and sensi-tization markers. Definitions and diagnostic criteria of allergic diseases.
      do not always agree on recommendations and date back to the turn of the centu-ry.
      • Høst A.
      • Koletzko B.
      • Dreborg S.
      • Muraro A.
      • Wahn U.
      Dietary products used in infants for treatment and prevention of food allergy. Joint Statement of the European Society for Paediatric Allergology and Clinical Immunology (ESPACI) Committee on Hypoallergenic Formulas and the European Society for Paediatric Gastroenterology, Hepatol-ogy and Nutrition (ESPGHAN) Committee on Nutrition.
      ,
      American Academy of Pediatrics Committee on Nutrition
      Hypoaller-genic infant formulae.
      In 2008, the World Allergy Organization (WAO) Special Committee on Food Allergy identified CMA as an area in need of a rationale-based approach, informed by the consensus reached through an expert review of the available clinical evidence, to make inroads against a burdensome, world-wide public health problem. It is in this context that the WAO Diagnosis and Rationale for Action against Cow’s Milk Allergy (DRACMA) Guidelines was planned to provide physicians everywhere with a management tool to deal with CMA from suspicion to treatment. Targeted (and tapped for their expertise), both on the DRACMA panel or as nonsitting reviewers, were allergists, pediatricians (allergists and generalists), gastroenterologists, dermatologists, epidemiologists, methodologists, dieticians, food chemists, and representatives of allergic patient organizations. Ultimately, DRACMA is dedicated to our patients, especially the younger ones, whose burden of issues we hope to relieve through an ongoing and collective effort of more interactive debate and integrated learning.

      Definitions

      Adverse reactions after the ingestion of cow’s milk can occur at any age from birth and even among infants fed exclusively at the breast, but not all such reactions are of an allergic nature. A revision of the allergy nomenclature was issued in Europe in 2001
      • Johansson S.G.
      • Hourihane J.O.
      • Bousquet J.
      A revised nomenclature for allergy. An EAACI position statement from the EAACI nomenclature task force.
      and was later endorsed by the WAO
      • Johansson S.G.
      • Bieber T.
      • Dahl R.
      Revised nomenclature for allergy for global use: report of the Nomenclature Review Committee of the World Allergy Organization, 2003.
      under the overarching definition of “milk hypersensitivity,” to cover nonallergic hypersensitivity (traditionally termed “cow’s milk intolerance”) and allergic milk hypersen-sitivity (or “cow’s milk allergy”). The latter definition requires the activation of an underlying immune mechanism to fit. In DRACMA, the term “allergy” will abide by the WAO definition (“allergy is a hypersensitivity reaction initiated by specific immunologic mechanisms”). In most children with CMA, the condition can be immunoglobulin E (IgE)-medi-ated and is thought to manifest as a phenotypical expression of atopy, together with (or in the absence of) atopic eczema, allergic rhinitis and/or asthma. A subset of patients, however, have non-IgE mediated (probably cell-mediated) allergy and present mainly with gastro-intestinal symptoms in reaction to the ingestion of cow’s milk.

      Section 2: Methodology

      The outline of the consensus guideline was the result of the considered opinion of the whole panel. Narrative parts, that is, sections 1–8, 9–13, 15–17, and 19 included the relevant CMA literature as searched using the algorithms reported in Appendix 1. For these sections, the relative weight of the suggestions retained for the purpose of DRACMA reflects the expert opinion of the panel. They may contain general indications, but no evidence-based recommendations. The consensus on these indications was expressed by the panelists using a checklist itemizing the clinical questions considered relevant after analysis of the literature. The collective judgment of the panel is expressed as a percentage of agreement among panelists. The panel decided to use a GRADE methodology for defining some treatments and diagnostic questions.
      The DRACMA worked with the GRADE members on this panel the clinical questions and their scope after various fine-tuning stages. The GRADE panelists independently searched the relevant literature for sections 9, 14, 18. Their analysis was independent of the other panel lists. For question formulation, guideline panel members explicitly rated the importance of all outcomes on a scale from 1–9, where the upper end of the scale (7–9) identifies outcomes of critical importance for decision making, ratings of 4–6 represent outcomes that are important but not critical and ratings of 1–3 are items of limited importance. Evidence summaries were prepared following the GRADE Working Group’s ap-proach
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Falck-Ytter Y.
      • Vist G.E.
      • Liberati A.
      • Schunemann H.J.
      Going from evidence to recommendations.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Vist G.E.
      • Falck-Ytter Y.
      • Schunemann H.J.
      What is “quality of evidence” and why is it important to clinicians.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Vist G.E.
      • Kunz R.
      • Falck-Ytter Y.
      • Alonso-Coello P.
      • Schunemann H.J.
      GRADE: an emerging consensus on rating quality of evidence and strength of recommendations.
      ,
      • Schünemann H.J.
      • Fretheim A.
      • Oxman A.D.
      Improving the use of research evidence in guideline development: 9. Grading evidence and recommendations.
      ,
      • Schünemann H.J.
      • Oxman A.D.
      • Fretheim A.
      Improving the use of research evidence in guideline development: 6. Determining which outcomes are important.

      World Health Organization. Global Programme on Evidence for Health Policy. Guidelines for WHO Guidelines. EIP/GPE/EQC/2003.1. Geneva, 2003.

      based on systematic reviews done by an independent team of the GRADE Working Group members (JLB and HJS supported by 5 research associates).
      The GRADE approach suggests that before grading the quality of evidence and strength of each recommendation, guideline developers should first identify a recent well-done systematic review of the appropriate evidence answering the relevant clinical question, or conduct one when none is available. This should be followed by preparing a transparent evidence summary, such as creation of GRADE evidence profiles, on which the guideline panel will base their judg-ments.
      • Schünemann H.J.
      • Hill S.R.
      • Kakad M.
      • Vist G.E.
      • Bellamy R.
      • et al.
      Transparent development of the WHO rapid advice guidelines.
      We prepared 3 systematic reviews addressing the clinical questions covered by the guideline (about the diagnosis, use of formula and immunotherapy of the CMA). We searched MEDLINE, EMBASE, and the Cochrane Library (including Cochrane Central Register of Controlled Trials, DARE, NHS EED) for relevant studies. We included studies published up to September 2009. We developed GRADE evidence profiles (summary of findings tables) for the clinical questions based on the systematic reviews. The summaries of evidence were reviewed by the panel members and corrections and comments were incorporated.
      We assessed the quality of the evidence according to the methodology described by the GRADE system.
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Falck-Ytter Y.
      • Vist G.E.
      • Liberati A.
      • Schunemann H.J.
      Going from evidence to recommendations.
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Vist G.E.
      • Falck-Ytter Y.
      • Schunemann H.J.
      What is “quality of evidence” and why is it important to clinicians.
      • Guyatt G.H.
      • Oxman A.D.
      • Vist G.E.
      • Kunz R.
      • Falck-Ytter Y.
      • Alonso-Coello P.
      • Schunemann H.J.
      GRADE: an emerging consensus on rating quality of evidence and strength of recommendations.
      ,
      • Schünemann H.J.
      • Oxman A.D.
      • Brozek J.
      • Glasziou P.
      • Jaeschke R.
      • et al.
      Grading quality of evidence and strength of recommendations for diagnostic tests and strategies.
      In this system quality of supporting evidence is assessed based on explicit methodological criteria and classified as either “high,” “moderate,” “low,” or “very low.”
      The DRACMA guideline panel reviewed the evidence summaries and the draft guidelines, and made recommendations. We reached consensus on all recommendations. Formulating the recommendations included explicit consideration of the quality of evidence, benefits, harms, burden, cost, and values and preferences described as the “Underlying values and preferences” or in the “Remarks” sections of each recommendation as outlined earlier.
      • Schünemann H.J.
      • Munger H.
      • Brower S.
      • O’Donnell M.
      • Crowther M.
      • Cook D.
      • Guyatt G.
      Methodology for guideline development for the Seventh American College of Chest Physicians Conference on Anti-thrombotic and Thrombolytic Therapy: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy.
      Statements about the underlying values and preferences and the remarks are integral parts of the recommendations and serve to facilitate accurate interpretation of the recommendations. They cannot be omitted when citing or translating DRACMA guidelines. In this document, the expression “values and preferences” refers to the relative weight one attributes to particular benefits, harms, burdens, and costs to determine their balance. We used the decision framework described previously to determine the strength of recommendations.
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Falck-Ytter Y.
      • Vist G.E.
      • Liberati A.
      • Schunemann H.J.
      Going from evidence to recommendations.
      ,
      • Schünemann H.J.
      • Jaeschke R.
      • Cook D.J.
      • Bria W.F.
      • El-Solh A.A.
      • et al.
      An official ATS statement: grading the quality of evidence and strength of recommendations in ATS guidelines and recommendations.
      Little information about costs of diagnosis and treatment of IgE-mediated cow’s milk allergy was available to the panel and it is very likely that it varies considerably across geographical areas and jurisdictions. Cost, therefore, plays a limited role in these recommendations. However, whenever we considered cost and resource expenditure, we used health system perspective.
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Jaeschke R.
      • Helfand M.
      • Liberati A.
      • Vist G.E.
      • Schunemann H.J.
      Incorporating considerations ofresources use into grading recommendations.
      For individual patients, cost may not be an issue if the service or treatment strategy is provided at reduced price or free of charge. Clinicians and patients should consider their local resource implications when interpreting these recommendations.
      After the GRADE approach we classified recommendations in these guidelines as either “strong” or “conditional” (also known as weak)/weak. The strength of recommendations depends on a balance between all desirable and all undesirable effects of an intervention (ie, net clinical benefit), quality of available evidence, values and preferences, and cost (resource utilization).
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Falck-Ytter Y.
      • Vist G.E.
      • Liberati A.
      • Schunemann H.J.
      Going from evidence to recommendations.
      In general, the higher the quality of the supporting evidence, the more likely it is for the recommendation to be strong. Strong recommendations based on low or very low quality evidence are rare, but possible.
      • Brozek J.L.
      • Baena-Cagnani C.E.
      • Bonini S.
      • Canonica G.W.
      • Rasi G.
      • et al.
      Methodology for development of the Allergic Rhinitis and its Impact on Asthma guideline 2008 update.
      For strong recommendations we used words “we recommend” and for conditional recommendations, “we suggest.” We offer the suggested interpretation of “strong” and “weak” recommendations in Table 2-1. Understanding the interpretation of these 2 grades (strong or conditional) of the strength of recommendations is essential for clinical decision making.
      Table 2-1Interpretation of “Strong” and “Weak” Recommendations
      ImplicationsStrong RecommendationWeak Recommendation
      For patientsMost individuals in this situation would want the recommended course of action and only a small proportion would not. Formal decision aids are not likely to be needed to help individuals make decisions consistent with their values and preferences.The majority of individuals in this situation would want the suggested course of action, but many would not.
      For cliniciansMost individuals should receive the intervention. Adherence to this recommendation according to the guideline could be used as a quality criterion or performance indicator.Recognize that different choices will be appropriate for individual patients, and that you must help each patient arrive at a management decision consistent with his or her values and preferences. Decision aids may be useful helping individuals making decisions consistent with their values and preferences.
      For policy makersThe recommendation can be adapted as policy in most situations.Policy making will require substantial debates and involvement of various stakeholders.

      How to Use These Recommendations

      The DRACMA guidelines are not intended to impose a standard of care for individual countries and jurisdictions. They should, as any guideline, provide a basis for rational decisions for clinicians and their patients about the management of cow’s milk allergy. Clinicians, patients, third-party payers, institutional review committees, other stakeholders, or the courts should never view these recommendations as dictates. Strong recommendations based on high quality evidence will apply to most patients for whom these recommendations are made, but they may not apply to all patients in all circumstances. No recommendation can take into account all of the often-compelling unique features of individual clinical circumstances. Therefore, nobody charged with evaluating clinicians’ actions should attempt to apply the recommendations from the DRACMA guidelines as rote or in a blanket fashion.

      Section 3: Epidemiology of CMA

      Overview

      There are no surveys of population and geographical trends in food allergy in adults or children (though the situation is different in pediatric asthma and rhinitis) and this unmet need is particularly felt for CMA. The perception of milk allergy is far more frequent than confirmed CMA. Patient reports of CMA range between 1 and 17.5%, 1 and 13.5%, and 1 to 4% in preschoolers, at children 5 to 16 years of age and adults respectively. Cow’s milk-specific IgE sensitization point prevalence progressively decreased from about 4% at 2 years to less than 1% at 10 years of age in the German Multi-Centre Allergy Study. The most reliable data in epidemiology are those from birth cohorts that are free from selection bias. There are 5 such challenge-confirmed studies. The CMA prevalence during infancy ranged from 1.9% in a Finnish study, 2.16% in the Isle of Wight, 2.22% in a study from Denmark, 2.24% in the Netherlands, and up to 4.9% in Norway.
      Patients with CMA develop gastrointestinal symptoms in 32 to 60% of cases, skin symptoms in 5 to 90%, and anaphylaxis in 0.8 to 9% of cases. This frequency of anaphylaxis is the main concern pointed out in many CMA studies. In a review, nearly one third of children with atopic dermatitis (AD) received a diagnosis of CMA after an elimination diet and an oral food challenge, and about 40 to 50% of children less than a year of age with CMA also had AD. Finally, with actual population and geographical trends remaining unknown, allergists are primarily in need of more detailed epidemiological surveys on a global scale. One large such epidemiological study supported by the European Commission is ongoing and aims to furnish the first prevalence data regarding the suspicion of CMA, sensitization to cow’s milk, and oral food challenge-confirmed diagnosis in 10 European birth cohorts.

      Introduction

      Around 11–26 million of the European population are estimated to suffer from food allergy.
      • Eigenmann P.A.
      Future therapeutic options in food allergy.
      If this prevalence was consistent around the world and projected to the 6,659,040,000 people of the world’s population,

      WHO, World Health Statistics 2009. Available at http://www.who.int/whosis/whostat/2009/en/print.html, accessed June 30, 2009.

      it translates into 220–520 million people and represents a major global health burden. Although there are surveys on the natural history and prevalence trends for symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood,
      ISAAC Phase Three Study Group
      Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys.
      we do not have a study assessing the prevalence of food allergy and its time-trends. The problem is complicated by the fact that perceived food allergy (ie, the self-reported feeling that a particular food negatively influences health status) is not actual food allergy. Allergy prevalence is much greater in the public’s belief than it has ever been reported by double-blind studies. Back in the 1980s, the perceived incidence of allergy to food or food additives in mothers with young children was reported between 17

      Good Housekeeping Institute, Consumer Research Department. Child-care findings V, Children and Food. New York: A Good Housekeeping Report. 1989.

      and 27.5%.

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      Thirty percent of women reported that they or some member of their family were allergic to some food product.
      • Sloan A.E.
      • Powers M.E.
      • Sloan A.E.
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      In the after decade, a British study using a food allergy questionnaire reported a 19.9% incidence of food allergy.
      • Young E.
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      From the mid-1990s onwards, self reports began to be compared with challenge-confirmed diagnoses; reported incidence data of between 12.4 and 25% could be confirmed by oral food challenge in only 1.5 to 3.5% of cases, illustrating how reports of adverse reactions overestimate true food allergy.
      • Jansen J.J.
      • Kardinnal A.F.
      • Huijbers G.
      • Vlieg-Boerstra B.J.
      • Martens B.P.
      • Ockhuizen T.
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      ,
      • Bjornsson E.
      • Janson C.
      • Plaschke P.
      • Norrman E.
      • Sjöberg O.
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      This was further confirmed when prevalence figures of 2.3 to 3.6% were confirmed by challenge procedures in unselected patient populations.
      • Osterballe M.
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      ,
      • Zuberbier T.
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      In the 1990s, it was also confirmed that only a minority of subjects who report food-related illness also test positive by skin prick test using the same food.
      • Woods R.K.
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      • Raven J.
      • Walters E.H.
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      Reported adverse reactions overestimate true food allergy in the community.
      Thus, 2 separate “food allergy epidemiologies” can be distinguished:
      • a.
        Self-reported food allergy; although this does not represent actual food allergy epidemiology, it is useful as a proxy measure of the potential demand for allergy medical services, and may guide public health allergy service users between general and specialist medicine,
        • Fiocchi A.
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        and more generally for public health planning.
      • b.
        Actual food allergy (ie, confirmed by a positive oral food challenge) represents the real extent of this clinical problem.
      In general, food allergy is more frequent in the pediat-ric, rather than the adult, population. According to a recent Japanese multicenter trial, the prevalence of CMA is 0.21% in newborns and 0.35% amid extremely premature babies (< 1000 g).
      • Miyazawa T.
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      Food allergies are a cause of particular concern for children. Incidence is estimated to be greater in toddlers (5–8%) than it is in adults (1–2%).
      • Brugman E.
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      Earlier prospective challenge-based studies have shown that in a population of 480 newborns followed up in the setting of a U.S. general pediatric practice through their third birthday, a parental report of 28% food allergy translates into a challenge-confirmed CMA rate of 8%,
      • Bock S.A.
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      ,
      • Bock S.A.
      Prospective appraisal of complaints of adverse reactions to foods in children during the first three years of life.
      with 2.27 to 2.5% occurring in the first 2 years of life.

      Perceived Cow’s Milk Allergy

      Similar considerations can be applied to cow’s milk allergy perception. Self-report is common. In a large European survey of above 44,000 telephone contacts, 5 million European respondents claimed to be milk-allergic, with adult women as the group making most of these claims. There were also wide national differences ranging from 13.8% of reports from Greece to 52.3% from Finland. In this survey milk was the most often reported offending food in children (38.5% of reports) and the second food most often implicated by adults (26%).
      • Steinke M.
      • Fiocchi A.
      the REDALL group
      Perceived Food allergy in children. A report on a representative telephone survey in 10 European countries.
      In a group of 600 children less than 4 years, CMA was reported by the parents of 18 children (3%).
      • Kilgallen I.
      • Gibney M.J.
      Parental perception of food allergy or intolerance in children under4 years of age.
      Milk reactions were reported by the parents of 2% of children without wheeze and by 16% of wheezers.
      • Sandin A.
      • Annus T.
      • Björkstén B.
      • Nilsson L.
      • Riikjärv M.A.
      • van Hage-Hamsten M.
      • Bråbäck L.
      Prevalence of self-reported food allergy and IgE antibodies to food allergens in Swedish and Estonian schoolchildren.
      In the literature, the bulk of studies based only on self-reports of CMA is staggering, compared with reports that include an objective measure to assess the condition.
      • Rona R.J.
      • Keil T.
      • Summers C.
      • Gislason D.
      • Zuidmeer L.
      • et al.
      The prevalence of food allergy: a meta-analysis.
      Currently, at least a score of studies have evaluated the self-perception of CMA over the last 20 years in preschool-ers,
      • Rancé F.
      • Grandmottet X.
      • Grandjean H.
      Prevalence and main characteristics of schoolchildren diagnosed with food allergies in France.
      ,
      • Dalal I.
      • Binson I.
      Food allergy is a matter of geography after all: sesame as a major cause of severe IgE-mediated food allergic reactions among infants and young children in Israel.
      ,
      • Tariq S.M.
      • Matthews S.M.
      Egg allergy in infancy predicts respiratory allergic disease by 4 years of age.
      ,
      • Garcia Ara M.C.
      • Boyano Martinez M.T.
      Incidence of allergy to cow’s milk protein in the first year of life and its effect on consumption of hydrolyzed formulae.
      ,
      • Kristjansson I.
      • Ardal B.
      • Jonsson J.S.
      • Sigurdsson J.A.
      • Foldevi M.
      • Bjork-sten B.
      Adverse reactions to food and food allergy in young children in Iceland and Sweden.
      ,
      • Eggesbø M.
      • Halvorsen R.
      • Tambs K.
      • Botten G.
      Prevalence of parentally perceived adverse reactions to food in young children.
      ,
      • Høst A.
      • Halken S.A.
      A prospective study of cow milk allergy in Danish infants during the first year of life.
      ,
      • Bival’kevich V.G.
      Allergic diathesis in infants in the first year of life.
      ,
      • Frongia O.
      • Bellomo A.R.
      Food allergies and intolerance in infants and children.
      • Schrander J.J.
      • Van Den Bogart J.P.
      Cow’s milk protein intolerance in infants under 1 year of age: a prospective epidemiological study.
      school-age children (5–16 years),
      • Steinke M.
      • Fiocchi A.
      the REDALL group
      Perceived Food allergy in children. A report on a representative telephone survey in 10 European countries.
      ,
      • Penard-Morand C.
      • Raherison C.
      • Kopferschmitt C.
      • Caillaud D.
      • Lavaud F.
      • et al.
      Prevalence of food allergy and its relationship to asthma and allergic rhinitis in schoolchildren.
      ,
      • Roehr C.C.
      • Edenharter G.
      • Reimann S.
      • Ehlers I.
      • Worm M.
      • et al.
      Food allergy and non-allergic food hypersensitivity in children and adolescents.
      ,
      • Santadusit S.
      • Atthapaisalsarudee S.
      • Vichyanond P.
      Prevalence of adverse food reactions and food allergy among Thai children.
      ,
      • Isolauri E.
      • Huurre A.
      The allergy epidemic extends beyond the past few decades.
      • van Bockel-Geelkerken M.
      • Meulmeester J.F.
      Prevalence of putative hypersensitivity in young children.
      and young adults.
      • Steinke M.
      • Fiocchi A.
      the REDALL group
      Perceived Food allergy in children. A report on a representative telephone survey in 10 European countries.
      ,
      • Pereira B.
      • Venter C.
      • Grundy J.
      • Clayton C.B.
      • Arshad S.H.
      • Dean T.
      Prevalence of sensitization to food allergens, reported adverse reaction to foods, food avoidance, and food hypersensitivity among teenagers.
      ,
      • Gislason D.
      • Bjornsson E.
      • Gislason S.
      Allergy and intolerance to food in an Icelandic urban population 20-44 years of age.
      ,
      • Woods R.K.
      • Abramson M.
      • Bailey M.
      • Walters E.H.
      on behalf of the European Community Respiratory Health Survey (ECRHS)
      International prevalence of reported food allergies and intolerances: comparisons arising from the European Community Respiratory Health Survey (ECRHS) 1991-1994.
      ,
      • Falcao H.
      • Lunet N.
      • Lopes C.
      • Barros H.
      Food hypersensitivity in Portuguese adults.
      ,
      • Altman D.R.
      • Chiaramonte L.T.
      Public perception of food allergy.
      ,
      • Woods R.K.
      • Thien F.
      • Raven J.
      • Walters H.
      • Abramson M.
      Prevalence of food allergies in young adults and their relationship to asthma, nasal allergies, and eczema.
      • Marklund B.
      • Ahlstedt S.
      • Nordstrom G.
      Health-related quality of life among adolescents with allergy-like conditions: with emphasis on food hypersensitivity.
      From these studies, reviewed in the only metaanalysis in the field,
      • Roehr C.C.
      • Edenharter G.
      • Reimann S.
      • Ehlers I.
      • Worm M.
      • et al.
      Food allergy and non-allergic food hypersensitivity in children and adolescents.
      the prevalence of self-reports varies between 1 to 17.5% in preschoolers, 1 and 13.5% in 5 to 16-year-olds, and between 1 and 4% in adults.
      The children from these studies neither underwent sensi-tization testing nor oral food challenge. In a population of 6-year-olds, 1 out of 7 cases was based on self-reports whereas less than one out of 2 children with a positive cow’s milk specific skin prick test was confirmed allergic by DBPCFC, thereby confirming that most parent-reported symptoms of CMA are unreliable.
      • Venter C.
      Prevalence of sensitization reported and objectively assessed food hypersensitivity amongst six-year-old children: a population-based study.
      Not only parents, but also health care professionals, allergists, and nonallergists alike, cite cow’s milk-induced reactions as the most common food allergy affecting children.
      • Wilson B.G.
      • Cruz N.V.
      • Fiocchi A.
      • Bahna S.L.
      American College of Allergy, Asthma & Immunology Adverse Reactions to Food Committee
      Survey of physicians’ approach to food allergy, Part 2: Allergens, diagnosis, treatment, and prevention.
      Thus, the incidence of self-reports of CMA remains of interest for public health authorities, health maintenance organizations and the processed food industry as a metric for policy planning, planning diagnostic services;
      • Hourihane JO’B
      Prevalence and severity of food allergy: need for control.
      tabling labeling legislation and even meeting the demand for milk-free products. However, as such, this proxy cannot represent the full extent of the clinical issues at stake.

      Sensitization to Cow’s Milk Proteins

      The number of studies on CM sensitization in unselected populations is limited. The meta-analysis carried out by Rona and colleagues
      • Rona R.J.
      • Keil T.
      • Summers C.
      • Gislason D.
      • Zuidmeer L.
      • et al.
      The prevalence of food allergy: a meta-analysis.
      identified 7 studies reporting a sensitization rate of 0.5 to 2% of preschoolers, of 0.5% at 5 to 16 years of age, and in less than 0.5% of adults.
      • Rona R.J.
      • Keil T.
      • Summers C.
      • Gislason D.
      • Zuidmeer L.
      • et al.
      The prevalence of food allergy: a meta-analysis.
      ,
      • Dalal I.
      • Binson I.
      Food allergy is a matter of geography after all: sesame as a major cause of severe IgE-mediated food allergic reactions among infants and young children in Israel.
      ,
      • Tariq S.M.
      • Matthews S.M.
      Egg allergy in infancy predicts respiratory allergic disease by 4 years of age.
      ,
      • Garcia Ara M.C.
      • Boyano Martinez M.T.
      Incidence of allergy to cow’s milk protein in the first year of life and its effect on consumption of hydrolyzed formulae.
      ,
      • Kristjansson I.
      • Ardal B.
      • Jonsson J.S.
      • Sigurdsson J.A.
      • Foldevi M.
      • Bjork-sten B.
      Adverse reactions to food and food allergy in young children in Iceland and Sweden.
      ,
      • Eggesbø M.
      • Halvorsen R.
      • Tambs K.
      • Botten G.
      Prevalence of parentally perceived adverse reactions to food in young children.
      ,
      • Høst A.
      • Halken S.A.
      A prospective study of cow milk allergy in Danish infants during the first year of life.
      ,
      • Bival’kevich V.G.
      Allergic diathesis in infants in the first year of life.
      ,
      • Frongia O.
      • Bellomo A.R.
      Food allergies and intolerance in infants and children.
      • Schrander J.J.
      • Van Den Bogart J.P.
      Cow’s milk protein intolerance in infants under 1 year of age: a prospective epidemiological study.
      In a later cohort of 543 children from the Isle of Wight followed-up from birth and tested at 1, 2, and 3 years of age, a positive milk sensitization test was found in 2 infants at 12 months (0.37%), in 5 at 2 years (0.92%), and in 3 at 3 years (0.55%).
      • Dean T.
      • Venter C.
      • Pereira B.
      • Arshad S.H.
      • Grundy J.
      • Clayton C.B.
      • Higgins B.
      Patterns of sensitization to food and aeroallergens in the first 3 years of life.
      In the German Multicenter Allergy Study, 1314 children initially recruited were followed from birth for 13 years. The longitudinal data were analyzed for 273 children testing positive for serum cow’s milk specific IgE antibody and were obtained at age 2, 5, 7, and 10. The point prevalence of sensitization to cow’s milk progressively decreased from about 4% at 2 years to less than 1% at 10 years.
      • Matricardi P.M.
      • Bockelbrink A.
      • Beyer K.
      • Keil T.
      • Niggemann B.
      • Grüber C.
      • Wahn U.
      • Lau S.
      Primary versus secondary immunoglobulin E sensitization to soy and wheat in the Multi-Centre Allergy Study cohort.

      Epidemiology of Challenge-Confirmed CMA

      The epidemiology of oral food challenge-confirmed CMA of the last 10 years consists of the following 5 studies:
      • a.
        In a Danish study of 1,749 newborns followed for 12 months, 39 (or 2.22%) were confirmed allergic
        • Høst A.
        • Halken S.
        • Jacobsen H.P.
        • Christensen A.E.
        • Herskind A.M.
        • Plesner K.
        Clinical course of cow’s milk protein allergy/intolerance and atopic diseases in childhood.
      • b.
        In a study from Finland 6,209 newborns followed for 15 months, 118 (1.9%) had positive DBPCFC
        • Saarinen K.M.
        • Juntunen-Backman K.
        • Järvenpää A.L.
        • Kuitunen P.
        • et al.
        Supplementary feeding in maternity hospitals and the risk of cow’s milk allergy: a prospective study of 6209 infants.
      • c.
        In a Norwegian study of 193 premature and 416 full-term infants, 27 of 555 (or 4.9%) were diagnosed with an allergic reaction to cow’s milk on the basis of an open challenge but not all children were tested; interestingly, all had symptoms before 6 months of age
        • Kvenshagen B.
        • Halvorsen R.
        • Jacobsen M.
        Adverse reactions to milk in infants.
      • d.
        In an Isle of Wight cohort of 969 newborns followed for 12 months, 21 (2.16%) reported CMA but only 2 (0.21%) were actually with IgE-mediated CMA
        • Venter C.
        • Pereira B.
        • Grundy J.
        • Clayton C.B.
        • Roberts G.
        • Higgins B.
        • Dean T.
        Incidence of parentally reported and clinically diagnosed food hyper-sensitivity in the first year of life.
      • e.
        In a newborn cohort from the Netherlands 1,158 infants prospectively followed through 12 months of age reporting “cow’s milk protein intolerance” (defined as two positive cow’s milk elimination/challenge tests) reported 26 allergic children (or 2.24%) of 211 (or 18.2%) suspected cases.
        • Schrander J.J.
        • Van Den Bogart J.P.
        Cow’s milk protein intolerance in infants under 1 year of age: a prospective epidemiological study.
      In this series of challenge-based studies, the Danish study further suggested that reproducible clinical reactions to CMP in human milk were reported in ~0.5% of breast-fed in-fants.
      • Høst A.
      Frequency of cow’s milk allergy in childhood.
      Data from cross-sectional studies (analyzed by Rona and coworkers

      WHO, World Health Statistics 2009. Available at http://www.who.int/whosis/whostat/2009/en/print.html, accessed June 30, 2009.

      ) demonstrated a rate of 0.6 to 2.5% prevalence in preschoolers, 0.3% at 5 to 16 years of age, and of less than 0.5% in adults.
      • Rona R.J.
      • Keil T.
      • Summers C.
      • Gislason D.
      • Zuidmeer L.
      • et al.
      The prevalence of food allergy: a meta-analysis.
      ,
      • Osterballe M.
      • Hansen T.K.
      • Mortz C.G.
      • Høst A.
      • Bindslev-Jensen C.
      The prevalence of food hypersensitivity in an unselected population of children and adults.
      ,
      • Madrigal B.I.
      • Alfaro A.N.
      Adverse reactions to food in daycare children.
      • Altintas D.
      A prospective study of cow’s milk allergy in Turkish infants.
      While most of our information on cow’s milk allergy prevalence comes from northern European and Spanish studies, there are methodological and geographical differences in clinical evaluation, which must be considered in assessing the epidemiological features we discuss here. Some studies may consider only immediate reactions, while others include delayed reactions; not all studies include IgE sensitization assessments; some studies are based on open oral food challenges, some performed blinded oral food challenge tests. Methods used across studies in this literature of oral food challenges with
      • Martelli A.
      • Bouygue G.R.
      • Fiocchi A.
      • Restani P.
      • Sarratud T.
      • Terracciano L.
      • et al.
      Oral food challenges in children in Italy.
      cow’s milk are not standardized (see section on Diagnosis).
      Thus, among the unmet needs of epidemiological research in this field are high-quality community studies based on patient data objectively confirmed by DBPCFC to close the current knowledge gap on the prevalence of CMA in the population. To address this, the European Commission launched the EuroPrevall Project (www.europrevall.org) in 2005 in concert with more than 60 partners including patient organizations, the food industry and research institutions from across Europe, Russia, Ghana, India, and China. This translational endeavor involves basic and clinical research components, and large epidemiological studies of both children and adults.
      • Clare Mills E.N.
      • Mackie A.R.
      • Burney P.
      • Beyer K.
      • Frewer L.
      • Madsen C.
      • et al.
      The prevalence, cost and basis of food allergy across Europe.
      The first results, will include data on suspicion of CMA, on sensitization to cow’s milk and of oral food challenge-confirmed diagnosis from 10 birth cohorts.
      • Keil T.
      • McBride D.
      • Grimshaw K.
      • Niggemann B.
      • Xepapadaki P.
      • et al.
      The multinational birth cohort of EuroPrevall: background, aims and methods.

      Different Clinical Presentations of CMA

      In a Danish birth cohort, 60% of children with CMA presented with gastrointestinal symptoms, 50 to 60% with skin issues, and respiratory symptoms present in 20 to 30% while 9% developed anaphylaxis.
      • Bishop J.M.
      • Hill D.G.
      • Hosking C.S.
      Natural history of cow milk allergy.
      ,
      • Hill D.J.
      • Bannister D.G.
      • Hosking C.S.
      • Kemp A.S.
      Cow milk allergy within the spectrum of atopic disorders.
      In the Norwegian cohort noted above, young infants experienced pain (48%), gastrointestinal symptoms (32%), respiratory problems (27%), and atopic dermatitis (4.5%).
      • Kvenshagen B.
      • Halvorsen R.
      • Jacobsen M.
      Adverse reactions to milk in infants.
      In the Finnish cohort, presentation symptoms included urticaria (45.76%), atopic dermatitis (89.83%), vomiting and/or diarrhea (51.69%), respiratory symptoms (30.50%), and anaphylaxis (2.54%). The same children reacted at oral food challenge with symptoms of urticaria (51.69%), atopic dermatitis (44.06%), vomiting and/or diarrhea (20.33%), respiratory symptoms (15.25%), and anaphylaxis (0.84%).
      • Saarinen K.M.
      • Juntunen-Backman K.
      • Järvenpää A.L.
      • Kuitunen P.
      • et al.
      Supplementary feeding in maternity hospitals and the risk of cow’s milk allergy: a prospective study of 6209 infants.
      In the British study quoted above, infants reacted to oral food challenges with eczema (33%), diarrhea (33%), vomiting (23.8%), and urticaria in 2 children who immediately reacted to the challenge meal (one with wheeze and the other with excessive crying).
      • Venter C.
      • Pereira B.
      • Grundy J.
      • Clayton C.B.
      • Roberts G.
      • Higgins B.
      • Dean T.
      Incidence of parentally reported and clinically diagnosed food hyper-sensitivity in the first year of life.
      Dutch infants with CMA from the study noted above developed gastrointestinal (50%), skin (31%), and respiratory (19%) symptoms.
      • Schrander J.J.
      • Van Den Bogart J.P.
      Cow’s milk protein intolerance in infants under 1 year of age: a prospective epidemiological study.
      Several other studies have assessed the incidence of CMA in populations selected for referral by other care givers to a tertiary institution for specialist assessment of their symptoms and therefore requires caution in generalizing the results of such studies. As a case in point, in a long-term study of 97 children with challenge-confirmed CMA, 21% had atopic dermatitis at the final follow-up evaluation (at 8 years).
      • Bishop J.M.
      • Hill D.G.
      • Hosking C.S.
      Natural history of cow milk allergy.
      In another follow-up study of 42 infants with IgE-mediated CMA, 57% of children had developed atopic dermatitis at the median age of 3.7 years.
      • Hill D.J.
      • Bannister D.G.
      • Hosking C.S.
      • Kemp A.S.
      Cow milk allergy within the spectrum of atopic disorders.
      Thus, CMA appears with GI symptoms in 32 to 60% of cases, cutaneous symptoms in 5 to 90%, anaphylaxis in 0.8 to 9% of cases. Respiratory complaints, including asthma, are not rare. Clearly, in most of the populations studied, there are overlapping presenting symptoms and multiple symptoms are often confirmed during challenge.

      CMA in Different Clinical Conditions

      Reversing the point of view, milk sensitization and CMA are reported with different frequencies in different clinical presentations. In 2184 young children aged 13–24 months with atopic dermatitis, the frequency of positive serum IgE responses against cow’s milk protein was 3%.
      • Wahn U.
      • Warner J.
      • Simons F.E.
      • de Benedictis F.M.
      • Diepgen T.L.
      • et al.
      IgE antibody responses in young children with atopic dermatitis.
      Among 59 breast-fed children with moderate-severe AD, 5 (8,5%) were SPT-positive with milk extracts.
      • Rennick G.J.
      • Moore E.
      • Orchard D.C.
      Skin prick testing to food allergens in breast-fed young infants with moderate to severe atopic dermatitis.
      In a consecutive series with moderate atopic eczema referred to a University-affiliated dermatology department, SPT showed 16% of infants with IgE against CMP.
      • Hill D.J.
      • Heine R.G.
      • Hosking C.S.
      • Brown J.
      • Thiele L.
      • et al.
      IgE food sensitization in infants with eczema attending a dermatology department.
      In a group of infants and children (mean age 17.6 months) with AD and no other allergic manifestations, 20/54 children (37%) had a diagnosis of CMA.
      • Novembre E.
      • Vierucci A.
      Milk allergy/intolerance and atopic dermatitis in infancy and childhood.
      Among 90 children with IgE-mediated food allergy, 17% were allergic to cow’s milk.
      • Hill D.J.
      • Hosking C.S.
      Food allergy and atopic dermatitis in infancy: an epidemiologic study.
      Thus, as reviewed some years ago, nearly one third of AD children have a diagnosis of CMA according to elimination diet and challenge tests, and about 40–50% of children <1 year of age with CMA have AD.
      • Novembre E.
      • Vierucci A.
      Milk allergy/intolerance and atopic dermatitis in infancy and childhood.
      An exception to the uncertainty of information about epidemiology of CMA is anaphylaxis. In a prospective survey of hospital admissions for food-allergic reactions, conducted through the British Pediatric Surveillance Unit, covering the 13 million children in the United Kingdom and Ireland, 229 cases were reported by 176 physicians in 133 departments, yielding a rate of 0.89 hospital admissions per 100,000 children per year. With a 10% rate, milk was the third most frequent allergenic trigger, after peanut (21%) and tree nuts (16%).
      • Colver A.F.
      • Nevantaus H.
      • Macdougall C.F.
      • Cant A.J.
      Severe food-allergic reactions in children across the UK and Ireland, 1998-2000.
      In the UK, there are 13 million individuals less than 16 years of age, and over the past 10 years 8 children died of anaphylaxis (incidence of 0.006 deaths per 100 000 children 0–15 years per year). Milk caused the greatest number of fatal reactions (four of eight),
      • Macdougall C.F.
      • Cant A.J.
      • Colver A.F.
      How dangerous is food allergy in childhood? The incidence of severe and fatal allergic reactions across the UK and Ireland.
      in line with reports of both the frequency and severity
      • Stewart A.G.
      • Ewan P.W.
      The incidence, aetiology and management of anaphylaxis presenting to an accident and emergency department.
      of reactions to milk.

      Secular Trends of CMA

      In such a leopard-skin epidemiological context, it is hardly surprising that there is no continuum that can be identified across studies regarding time variations in CMA frequency.
      • Madsen C.H.
      Prevalence of food allergy: an overview.
      Is CMA prevalence on the rise? Utilizing surrogate indicators, we can only infer changes in CMA prevalence based on studies of general food allergy. Among those, a British study found that the admission rates per million population between 1990 and 2004 rose form 5 to 26 for anaphylaxis, from 5 to 26 for food allergy, and from 16 to 107 specifically for pediatric food allergy.
      • Gupta R.
      Time trends in allergic disorders in the UK.
      Reinforcing this picture, eczema rose from 13% in 1991 to 16% in 2003.
      ISAAC Phase Three Study Group
      Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys.

      Geographical Trends in CMA

      Is milk the most important offender in food allergy in children? From self-reports, it appears that this may be the case. However, given the paucity of epidemiological studies, we do not have sufficient information to argue the relative importance of CMA in different parts of the world. The maximum information comes from Spain, Scandinavian countries, the UK, and Germany. Inadequate information from different areas in the world are available, including Italy, Australia and North America where many cross-sectional and referral studies come from. Table 3-1 shows the comparison of the 3 main food allergens in the child studies. The pan-European RedAll survey estimated milk as the most frequently reported offender in children (38.5% of reports) and the second in adults (26.2%).
      • Steinke M.
      • Fiocchi A.
      the REDALL group
      Perceived Food allergy in children. A report on a representative telephone survey in 10 European countries.
      In France, 29/182 school-aged children with reported food allergy are milk-allergic in 11.9% of cases.
      • Rancé F.
      • Grandmottet X.
      • Grandjean H.
      Prevalence and main characteristics of schoolchildren diagnosed with food allergies in France.
      Accordingly, the Rona
      • Rona R.J.
      • Keil T.
      • Summers C.
      • Gislason D.
      • Zuidmeer L.
      • et al.
      The prevalence of food allergy: a meta-analysis.
      metanalysis indicates milk as the major food offender in challenge-based studies, followed by egg and fish. However, cow’s milk accounts for less than one third of any food that can be blamed for food allergy among the studies significantly combined (P < 0.001).
      • Thong B.Y.
      • Hourihane J.O.
      Monitoring of IgE-mediated food allergy in childhood.
      Similarly a review of studies of various designs (surveys, reviews, clinico-epidemiological studies) indicated egg as the most frequently found allergen in chil-dren.
      • Ebisawa M.
      • Ikematsu K.
      • Takanori I.
      • Tachimoto H.
      Food allergy in Japan.
      The pattern is repeated in Japan, where CM accounts for 22.6% of children with food allergy.
      • Iikura Y.
      • Imai Y.
      • Imai T.
      • Akasawa A.
      • Fujita K.
      • et al.
      Frequency of immediate-type food allergy in children in Japan.
      The same may not be true in other parts of the world, where the prevalence will largely reflect local factors such as exposure to foods, mode of preparation, and cultural attitudes. As an example, in Israel sesame is the third most frequently implicated offending food, probably because of its widespread consumption. Among young Australian adults, the major offender was peanut, followed by shrimp, wheat, egg, and milk.
      • Woods R.K.
      • Thien F.
      • Raven J.
      • Walters H.
      • Abramson M.
      Prevalence of food allergies in young adults and their relationship to asthma, nasal allergies, and eczema.
      In Iranian children CM is the most common offender identified during diagnostic provocation challenge.
      • Pourpak Z.
      • Farhoudi A.
      • Arshi S.
      • Movahedi M.
      • Gharegozlou M.
      • Yazdani F.
      • Mesdaghi M.
      Common Food Allergens in Iranian Children.
      Thus, it may be said that the most representative allergen is a hand-maiden to local customs.
      Table 3-1Comparison of the Three Main Food Allergens In Children Studies
      • Ebisawa M.
      • Ikematsu K.
      • Takanori I.
      • Tachimoto H.
      Food allergy in Japan.
      Country1st2nd3rd
      USAEggCow’s milkPeanuts
      GermanyEggCow’s milkWheat
      SpainEggCow’s milkFish
      SwitzerlandEggCow’s milkPeanuts
      IsraelEggCow’s milkSesame
      JapanEggCow’s milkWheat

      Section 4: Allergens of Cow’s Milk

      Overview

      The main allergens of cow’s milk are distributed among the whey and casein fractions.
      The whey allergens include:
      • a.
        Alpha-lactalbumin (Bos d4): its role in milk allergy is controversial and prevalence data across studies vary between 0 and 80% of patients reacting to this protein.
      • b.
        Beta-lactoglobulin (Bos d 5), the most abundant cow’s milk whey protein; it occurs in the milk of many other species but is not present in human milk. Thirteen to 76% of patients are found to react to this protein.
      • c.
        Bovine serum albumin (Bos d 6): involved in other allergies such as beef; it accounts for between 0 and 88% of sensitization events, while clinical symptoms occur in up to 20% of patients.
      • d.
        Bovine immunoglobulins (Bos d 7): are seldom held responsible for clinical symptoms in CMA.
      The casein allergens (collectively known as Bos d8) consist of 4 different proteins (alphas1, alphas2, beta, and kappa casein) which share little sequential homology. Despite this, simultaneous sensitization to these caseins is frequently observed. Patients are more often sensitized to alpha (100%) and kappa caseins (91.7%).
      Of clinical relevance, milk allergens of various mammalian species cross-react. The greatest homology is among cow’s, sheep’s and goat’s milks protein as Bos (oxen), Ovis (sheep), and Capra (goat) are genera belonging to the Bovidae family of ruminants. Proteins in their milks have less structural similarity with those from the Suidae (pig), Equidae (horse and donkey), and Camelidae (camel and dromedary) families and also from those of humans. Its noteworthy that the milks of camels and dromedaries (and human milk) do not contain Bos d 5. All this is relevant for later considerations on formula (section 13).
      There is no clear relationship between digestibility and protein allergenicity. Milk allergens are known to preserve their biologic activity even after boiling, pasteurization, ultra-high-temperature processing, or evaporation for the production of powdered infant formula. To obtain hypoallergenic formulas, extensive hydrolysis and further processing, such as heat treatment, ultrafiltration, and application of high pressure are necessary. Attempts have been made to classify formulas into partial and extensively hydro-lyzed products according to their degree of protein fragmentation, but there is no agreement on the criteria on which to base this classification. Nevertheless, hydrolyzed formulas have until now proven to be a useful and widely used protein source for infants suffering from CMA (section 12).

      Introduction

      Milk can give rise to several food hypersensitivities, usually classified as milk allergy or milk intolerance.
      • Bahna S.L.
      Cow’s milk allergy versus cow milk intolerance.
      The mechanism of intolerance to cow’s milk is not IgE antibody-mediated and has been blamed on the functionality of a specific enzyme deficiency, commonly lactose intolerance, attributable to beta-galactosidase (lactase) deficiency. DRACMA will not address lactase deficiency or other cow’s milk-induced hypersensitivity not mediated by immune mechanisms, which have been described in detail else-where.
      • Vesa T.H.
      • Marteau P.
      • Korpela R.
      Lactose intolerance.
      ,
      • Shukla H.
      Lactose Intolerance in health and disease.
      ,

      Swallow DM, Hollox EJ. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill; 2001.

      Cox TM. Food Allergy and Intolerance (chapt 25). London: Saunders; 2002.

      Cow’s milk allergy is an adverse clinical reaction associated with the binding of immunoglobulin (IgE) to antigens capable of eliciting an immune response.
      • Johansson S.G.
      • Bieber T.
      • Dahl R.
      Revised nomenclature for allergy for global use: report of the Nomenclature Review Committee of the World Allergy Organization, 2003.
      Where allergy is not mediated by IgE, other classes of immunoglobulin, immune complexes, or a cell-mediated reaction have been proposed to be involved. In IgE-mediated allergy, circulating antibodies recognize specific molecular regions on the antigen surface (epitopes), which are classified according to their specific amino acid sequence (sequential epitopes)or the folding and configuration of their protein chains (conformational epitopes). In this section, we describe the chemical characteristics of cow’s milk allergens, how they are involved in cross-reactivity among mammalian species, their resistance to digestion and proteolysis and their response to technological processing.

      Chemical Characterization of Cow’s Milk Allergens

      Cow’s milk contains several proteins that could each in principle elicit an allergic reaction in a sensitized individual. Some of these proteins are considered major allergens, some minor ones, while others have rarely or never been associated with reports of clinical reactions. The casein and whey proteins of cow’s milk are listed in Table 4-1. Each of these 2 fractions contains 5 major components.

      International Union of Immunological Societies Allergen Nomenclature Sub-Committee. Allergen Nomenclature. Retrieved from http://www.allergen.org/Allergen.aspx. Accessed 2009.

      ,
      • Wal J-M
      Cow’s milk proteins/allergens.

      Restani P, Ballabio C, Di Lorenzo C, Tripodi S, Fiocchi A. Molecular aspects of milk allergens and their role in clinical events. Anal Bioanal Chem. 2009 Jul 5. [Epub ahead of print]

      The casein fraction contains 80% of the total protein of cow’s milk while alphas1 and beta-casein make up for 70% of this fraction. Whey proteins are less abundant, and beta-lactoglobulin (BLG) accounts for 50% of this fraction. Because BLG is not present in human milk, this protein was previously considered the most important cow’s milk allergen, but it has since been shown that other proteins, such as the caseins, are also critically involved in the etiology of the disease.
      Table 4-1The Proteins of Cow’s Milk
      FractionProteinAllergen10g/L% Total ProteinMW (kDa)# AApI
      CaseinsBos d8~3080
      αs1-casein12–152923.61994.9–5.0
      αs2-casein3–4825.22075.2–5.4
      ß-casein9–112724.02095.1–5.4
      γ1-casein20.61805.5
      γ2-casein1–2611.81046.4
      γ3-casein11.61025.8
      κ-casein3–41019.01695.4–5.6
      Whey proteins~5.020
      Alpha-lactalbuminBos d41–1.5514.21234.8
      Beta-lactoglobulinBos d 53–41018.31625.3
      ImmunoglobulinBos d 70.6–1.03160.0
      BSA
      Bovine serum albumin.
      Bos d 60.1–0.4167.05834.9–5.1
      Lactoferrin0.09Traces800.07038.7
      * Bovine serum albumin.
      By convention, allergens in the international nomenclature are designated by an abbreviation formed by the genus (capitalized; abbreviated to the first 3 letters) and species (reduced to one letter) names of the Linnaean taxonomical system in italics, followed by an Arabic numeral reflecting the chronological order in which the allergen was identified and characterized (eg, Bos d[omesticus] 4).
      • Chapman M.D.
      • Pomés A.
      • Breiteneder H.
      • Ferreira F.
      Nomenclature and structural biology ofallergens.

      Alpha-Lactalbumin (Bosd4)

      Alpha-lactalbumin (A-LA) is a whey protein belonging to the lysozyme superfamily. It is a regulatory subunit of lactose synthase and is, able to modify the substrate specificity of galactosyl-transferase in the mammary gland, making glucose a good acceptor substrate for this enzyme and allowing lactose synthase to synthesize lactose.
      • McKenzie H.A.
      Alpha-lactalbumins and lysozymes.
      ,

      UniProt Knowledgebase, Available online from http://www.uniprot.org/ uniprot/P00711&format=html.

      A-LA is produced by the mammary gland and has been found in all milks analyzed so far. Table 4-2 shows its main chemical characteristics.
      Table 4-2Characteristics of Alpha-Lactalbumin (Bos d 4)
      ParameterDescription
      Allergen nomenclatureBos d4
      Entry nameLALBA_BOVIN
      SynonymsLactose Synthase B protein
      Sequence databasesGenbank: M18780
      PIR: A27360, LABO
      Swiss-Prot: P00711
      Number of aminoacids123 residues
      Molecular weight14.2 kDa
      Isoelectric point4.8
      Involvement in allergic sensitization to cow’s milk0–80% CM allergic subjects 75% CM allergic children by SPT
      A-LA contains 8 cysteine groups, all forming internal disulphide bonds, and 4 tryptophan residues. It contains high-affinity calcium binding sites stabilizing its highly ordered secondary structure. The role of A-LA in milk allergy is controversial and prevalence data across studies vary between 0 and 80% of patients reacting to this protein (reviewed in

      Besler M, Eigenmann P, Schwartz RH. Internet Symposium on Food Allergens. 2002;4:19.

      ). This heterogeneity is probably linked to whether skin prick test, specific IgE determinations, immunoblotting, or other method of sensitization assessment was used.

      Beta-Lactoglobulin (Bos d 5)

      Beta-lactoglobulin (BLG) is the most abundant cow’s milk whey protein; it occurs in the milk of many other mammalian species but is not present in human milk. Bos d 5 belongs to the lipocalin allergen family and is synthesized by the mammalian gland. Its function is unknown, although it may be involved in retinol transport, with which it readily binds.

      UniProt Knowledgebase, Available online from http://www.uniprot.org/uniprot/P02754&format=html

      Table 4-3 shows its main physical and chemical characteristics. It contains 2 internal disulphide bonds and one free–SH group. Under physiological conditions, BLG exists as an equilibrium mixture of monomer and dimer forms but, at its isoelectric point, the dimers can further associate to octamers. There are 2 main isoforms of this protein in cow’s milk, the genetic variants A and B, which differ only by 2 point mutations at amino acids 64 and 118. Because it is lacking from human milk, BLG has long been believed to be the most important cow’s milk allergen. The literature indicates that the prevalence of allergic subjects reacting to this protein is between 13 and 76%.
      • Restani P.
      • Ballabio C.
      • Tripodi S.
      • Fiocchi A.
      Meat allergy.
      Table 4-3Characteristics of Beta-Lactoglobulin (Bos d 5)
      ParameterDescription
      Allergen nomenclatureBos d 5
      Entry nameLACB_BOVIN
      Synonyms
      Sequence databasesGenbank: X14712
      PIR: S10179, LGBO
      Swiss-Prot: P02754
      Number of aminoacids162 residues
      Molecular weight18.3 kDa
      Isoelectric point5.13–5.23 (variants)
      Involvement in allergic sensitization to cow’s milk13–76% CM allergic subjects 73.7% CM allergic children by SPT

      Bovine Serum Albumin (Bos d 6)

      Bovine serum albumin (BSA) is the main protein of whey. It can bind water, fatty acids, hormones, bilirubin, drugs, and Ca2+,K+, and Na+. Its main function is the regulation of the colloidal osmotic pressure in blood.
      • Restani P.
      • Ballabio C.
      • Tripodi S.
      • Fiocchi A.
      Meat allergy.
      The tertiary structure of BSA is stable, and its 3-dimensional conformation is well documented. The protein is organized into 3 homologous domains (I to III) and consists of 9 loops connected by 17 covalent disulphide bridges. Most of the disulphide bonds are well protected in the core of the protein and are not readily accessible to the solvent. Table 4-4 shows some of its characteristics.
      Table 4-4Characteristics of Bovine Serum Albumin (Bos d 6)
      ParameterDescription
      Allergen nomenclatureBos d 6
      Entry nameALBU_BOVIN
      SynonymsBSA
      Sequence databasesGenbank: M73993
      PIR: A38885, ABBOS
      Swiss-Prot: P02769
      Number of aminoacids583 residues
      Molecular weight67.0 kDa
      Isoelectric point4.9–5.1
      Involvement in allergic sensitization to cow’s milk0–88% CM allergic subjects
      62.5% CM allergic children by immunoblotting
      Bos d 6 is involved not only in milk allergy but also in allergic reactions to beef.
      • Restani P.
      • Ballabio C.
      • Tripodi S.
      • Fiocchi A.
      Meat allergy.
      It induced immediate allergic symptoms (lip edema, urticaria, cough, and rhinitis) in children allergic to beef who received the protein in a doubleblind placebo-controlled food challenge (DBPCFC).
      • Fiocchi A.
      • Restani P.
      • Riva E.
      • Qualizza R.
      • Bruni P.
      • Restelli A.R.
      • Galli C.L.
      Meat allergy: I - Specific IgE to BSA and OSA in atopic, beef-sensitive children.
      The prevalence of patients with cow’s milk who react to this protein ranges from 0 to 88%, while clinical symptoms may be found in as many as 20% of patients.
      • Martelli A.
      • De Chiara A.
      • Corvo M.
      • Restani P.
      • Fiocchi A.
      Beef allergy in children with cow’s milk allergy. Cow’s milk allergy in children with beef allergy.

      Immunoglobulins (Bos d 7)

      Bovine immunoglobulins are present in blood, tissues, fluids, and secretions such as milk. Some characteristics of the bovine IgG are shown in Table 4-5. Bovine IgG seldom cause clinical symptoms in CMA.
      • Bernhisel-Broadbent J.
      • Yolken R.H.
      • Sampson H.A.
      Allergenicity of orally administered immunoglobulin preparations in food-allergic children.
      Table 4-5Characteristics of Cow’s Milk Immunoglobulin G
      ParameterDescription
      Allergen nomenclatureBos d 7
      Entry name
      SynonymsIgG
      Sequence databases
      Number of aminoacids
      Molecular weight160.0 kDa
      Isoelectric point
      Involvement in allergic sensitization to cow’s milkFrequency unknown

      Caseins (Bos d 8)

      Most of the casein aggregates as colloidal particles (the casein micelle) and its biologic function is to transport calcium phosphate to the mammalian newborn. More than 90% of the calcium content of skim milk is attached to or included in casein micelles. Caseins consist of 4 different proteins (alphas1, alphas2, beta, and kappa casein) with little sequential homology. Another group, the gamma caseins, are present in very low quantities in milk and are by-products of beta casein proteolysis. A distinguishing feature of all caseins is their low solubility at pH 4.6; another common characteristic is that caseins are conjugated proteins, most with phosphate groups esterified to the amino acid serine. Caseins contain no disulphide bonds, while the high number of proline residues causes pronounced bending of the protein chain, which inhibits the formation of close-packed, ordered secondary structures. Characteristics of Bos d 8are reported in Table 4-6.
      Table 4-6Allergenic Characteristics of Caseins
      Parameteras1-caseinas2-caseinß-casein?-casein
      Allergen nomenclatureBos d 8Bos d 8Bos d 8Bos d 8
      Entry nameCAS1_BOVINCAS2_BOVINCASB_BOVINCASK_BOVIN
      SynonymsNoneNoneNoneNone
      Sequence databasesG X00564/M33123G M16644G M16645/X06359G X14908/M36641
      P S22575/KABOSBP JQ2008/KABOS2P I45873/KBBOA2P S02076/KKBOB
      S P02662S P02663S P02666S P02668
      No. aminoacids199207209169
      Molecular weight23.6 kDa25.2 kDa24.0 kDa19.0 kDa
      Isoelectric point4.9–5.05.2-5.45.1–5.45.4–5.6
      Involvement in allergic sensitization to cow’s milk–1. whole casein65–100%65-100%65–100%65–100%
      Involvement in allergic sensitization to cow’s milk–2. single casein54%54%39%NT
      100%100%66.7%91.7%
      Despite the poor sequence homology between proteins of the casein fraction, poly-sensitization to many caseins is frequently observed; this may be because of cross-sensitization through shared or closely related epitopes.
      • Wal J-M
      Cow’s milk proteins/allergens.
      Patients are almost always sensitized to alpha (100%) and kappa caseins (91.7%).
      • Restani P.
      • Velonà T.
      • Plebani A.
      • Ugazio A.G.
      • Poiesi C.
      • Muraro A.
      • Galli C.L.
      Evaluation by SDS-PAGE and immunoblotting of residual antigenicity in hydrolysed protein formulas.

      Cross-Reactivity Between Milk Proteins from Different Animal Species

      Cross-reactivity occurs when 2 proteins share part of their amino acid sequence (at least, the sequence containing the epitopic domain) or when the 3-dimensional conformation makes 2 molecules similar in binding capacity to specific antibodies. In general, cross-reactivity between mammalian proteins reflects the phylogenetic relationships between animal species and evolutionary conserved proteins that are often cross-reactive.
      • Spitzauer S.
      Allergy to mammalian proteins: at the borderline between foreign and self.
      Table 4-7 shows the sequence similarity (expressed in percentages) between milk proteins from different mammalian species.

      Freund G. Proceeding of the meeting Interest nutritionnel et dietetique dulait de chevre Niort, France. 7 November 1996, INRA Paris France p. 119.

      Table 4-7Sequence Homology Between Mammalian Milk Proteins (in Percentage, Relative To Cow’s Milk Proteins)
      ProteinGoatEweBuffaloSowMareDonkeyDromedaryHuman
      ALA95.197.299.374.672.471.569.773.9
      BLG94.493.996.763.959.456.9AbsentAbsent
      Serum alb.92.479.974.574.176.6
      a s1 CAS87.988.347.242.932.4
      a s2 CAS88.389.262.858.3
      ß CAS91.192.097.867.060.569.256.5
      ? CAS84.984.992.654.357.458.453.2
      The greatest homology is between cow’s, sheep’s and goat’s milk proteins as Bos (oxen), Ovis (sheep), and Capra (goat) that are genera belonging to the Bovidae family of ruminants. The proteins in their milks consequently have less structural similarity with those from the Suidae (pig), Equidae (horse and donkey), and Camelidae (camel and dromedary) families and also with those in human milk. It is noteworthy that the milks of camels and dromedaries (as well as human milk) do not contain BLG.
      However, phylogeny does not explain everything. In 1996, a clinical trial in France showed that 51/55 children with cow’s milk allergy tolerated goat’s milk for periods ranging from 8 days to 1 year,

      Freund G. Proceeding of the meeting Interest nutritionnel et dietetique dulait de chevre Niort, France. 7 November 1996, INRA Paris France p. 119.

      but subsequent research showed that other subjects allergic to cow’s milk did not tolerate goat’s and sheep’s milks.
      • Bellioni-Businco B.
      • Paganelli R.
      • Lucenti P.
      • Giampietro P.G.
      • Perborn H.
      • Businco L.
      Allergenicity of goat’s milk in children with cow’s milk allergy.
      This is consistent with the pattern of IgE cross-reactivity shown by several independent studies in vitro, for instance the cross-reactivity between milk proteins from different mammalian species (including goat’s milk).
      • Restani P.
      • Gaiaschi A.
      • Plebani A.
      • Beretta B.
      • Cavagni G.
      • et al.
      Cross reactivity between milk proteins from different animal species.
      Furthermore, selective allergy to goat’s and sheep’s milk but not to cow’s milk has also been reported in 28 older children with severe allergic reactions, including anaphylaxis. In one study, IgE antibodies recognized caseins from goat’s milk but cow’s milk caseins were not or scarcely recognized.
      • Ah-Leung S.
      • Bernard H.
      • Bidat E.
      • Paty E.
      • Rance F.
      • Scheinmann P.
      Allergy to goat and sheep milk without allergy to cow’s milk.
      This is not an isolated finding,
      • Bidat E.
      • Rancé F.
      • Baranès T.
      • Goulamhoussen S.
      Goat’s milk and sheep’s milk allergies in children in the absence of cow’s milk allergy.
      ,
      • Alvarez M.J.
      • Lombardero M.
      IgE-mediated anaphylaxis to sheep’s and goat’s milk.
      however, and a case report of an adult with goat’s milk allergy without CMA found specific IgE to caprine ALA.
      • Tavares B.
      • Pereira C.
      • Rodrigues F.
      • Loureiro G.
      • Chieira C.
      Goat’s milk allergy.
      Finally, allergy to sheep’s milk can also evolve into allergy to cow’s milk.
      • Fiocchi A.
      • Decet E.
      • Mirri G.P.
      • Travaini M.
      • Riva E.
      Allergy to ewe’s milk can evolve into allergy to cow’s milk.
      Mare’s and donkey’s milks have proved sometimes useful to some patients,
      • Vita D.
      • Passalacqua G.
      • Di Pasquale G.
      • Caminiti L.
      • Crisafulli G.
      • Rulli I.
      Ass’s milk in children with atopic dermatitis and cow’s milk allergy: crossover comparison with goat’s milk.
      ,
      • Monti G.
      • Bertino E.
      • Muratore M.C.
      • Coscia A.
      • Cresi F.
      • Silvestro L.
      Efficacy of donkey’s milk in treating highly problematic cow’s milk allergic children: an in vivo and in vitro study.
      • Carroccio A.
      • Cavataio F.
      • Montalto G.
      • D’Amico D.
      • Alabrese L.
      • Iacono G.
      Intolerance to hydrolysed cow’s milk proteins in infants: clinical characteristics and dietary treatment.
      but uncertainties remain about chemical composition and hygienic control. The same considerations apply to Camellidae (camel and dromedaries) milks, which could represent an alternative to cow’s milk for allergic subjects because of their low sequence homology with cow’s milk and the absence of BLG, if problems related to availability and technological processing to avoid new sensitization.
      • Restani P.
      • Beretta B.
      • Fiocchi A.
      • Ballabio C.
      • Galli C.L.
      Cross-reactivity between mammalian proteins.
      Figure 4-1 shows the electrophoretic patterns of milk from several mammalian species. The pronounced similarity is evident for milk from cows, goats, and sheep, while the protein profiles of mare’s, donkey’s, and camel’s milks present some specificities. The low cross-immunoreactivity of horse/donkey milk and the absence of BLG in camel’s and human milk is easily visible in immunoblots using antibodies against bovine BLG.
      Figure thumbnail gr1
      Figure 4-1SDS-PAGE of mammalian milk samples. Hcas = human casein;HLA = human lactalbumin;Lfe = human lactoferrin;a-cas = bovine alpha casein;ß-cas = bovine beta casein; BLG = bovine ß-lactoglobulin;ALA = bovine a-lactalbumin.

      Structural Modifications and Cow’s Milk Protein Allergenicity

      The 3-dimensional structure of most antigenic proteins is unknown, even where the amino acid sequence has been precisely identified, because the conformation is not immutable but is influenced by the surrounding environment. This problem is even more significant for milk proteins since their organization is complex and the presence of micelles in caseins makes their investigation difficult. We discuss here the structural modifications brought about by gastrointestinal digestion or technological treatments and their role in allergenic potential where this is known or can be inferred.

      Digestibility and Cow’s Milk Protein Allergenicity

      Food proteins are digested by gastrointestinal enzymes; it is generally believed that proteins resistant to proteolysis are the more powerful allergens. However, it has been shown that there is no clear relationship between in vitro digestibility and protein allergenicity.
      • Fu T.J.
      • Abbott U.R.
      • Hatzos C.
      Digestibility of food allergens and nonallergenic proteins in simulated gastric fluid and simulated intestinal fluid-a comparative study.
      Caseins are thought to be easily digestible, but they coagulate in an acidic medium (at gastric pH). Acidification increases the solubility of minerals, so that the calcium and phosphorus contained in the micelles gradually become soluble in the aqueous phase. As a result, casein micelles disintegrate and casein precipitates. Whey proteins are more soluble in saline solution than caseins and theoretically they should be more easily digested by proteases that work in aqueous medium. However, the correlation between water solubility and digestibility is not linear. Caseins are digested faster than whey proteins by the commonest food-grade enzymes (eg, pepsin, trypsin, and thermolysin).
      • Bonomi F.
      • Fiocchi A.
      • Frokiaer H.
      • et al.
      Reduction of immunoreactivity of bovine beta-lactoglobulin upon combined physical and proteolytic treatment.
      Although BSA is very soluble in water and rich in amino acids broken-down by gastrointestinal enzymes, it is also relatively resistant to digestion. Sequential epitopes were unaffected for at least 60 minutes when BSA was digested with pepsin.
      • Beretta B.
      • Conti A.
      • Fiocchi A.
      • Gaiaschi A.
      • Galli C.L.
      • et al.
      Antigenic determinants of bovine serum albumin.
      Its 9 loops are maintained by disulphide bonds, which are not easily reduced under physiological conditions, and slow the fragmentation of BSA into short peptides that have decreased antigenic activity.

      Heating and Cow’s Milk Protein Allergenicity

      Cow’s milk is only marketed after it has been subjected to technological process, usually pasteurization, which reduces potential pathogen load (70–80°C for 15–20 seconds). Ultra-high-temperature (UHT) processing with flash heating (above 100°C for a few seconds), evaporation for the production of powdered infant formula (dry blending or wet mixing–spray drying process) have a minor or no effect on the antigenic/allergenic potential of cow’s milk proteins. Boiling milk for 10 minutes reduces the SPT response in patients who react to BSA and beta-lactoglobulin, whereas wheal diameter remains the same in those sensitized to caseins.
      • Norgaard A.
      • Bernard H.
      • Wal J.M.
      • Peltre G.
      • Skov P.S.
      • Poulsen L.K.
      • Bindslev-Jensen C.
      Allergenicity of individual cow milk proteins in DBPCFC-positive milk allergic adults.
      Comparative studies have shown no difference in antigenicity between raw and heated milks,
      • Werfel S.J.
      • Cooke S.K.
      • Sampson H.A.
      Clinical reactivity to beef in children allergic to cow’s milk.
      however, and in some cases the aggregation of new protein polymers capable of binding specific IgE have been demonstrated. After boiling BSA at 100°C for 10 minutes, dimeric, trimeric, and higher polymeric forms increased, and all maintained their IgE-binding properties.
      • Restani P.
      • Ballabio C.
      • Cattaneo A.
      • Isoardi P.
      • Terracciano L.
      • Fiocchi A.
      Characterization of bovine serum albumin epitopes and their role in allergic reactions.
      The persistence of allergenicity in heat-treated milk is clinically confirmed by the fact that in some children CMA develops after the ingestion of heat-treated milk. Furthermore, heating processes can only modify conformational epitopes, which might lose their binding capacity to specific IgE antibody, while sequential epitopes maintain their allergenic potential even after heating (Fig. 4-2).
      • Sampson H.A.
      Update on food allergy.
      Milk proteins contain both types of epitopes and, even though a slight reduction of antigenicity can be observed with whey proteins, insignificant alterations in binding properties are reported with caseins. To complicate the picture, vigorous heating (such as that used for certain sterilization processes [121°C for 20 minutes]) but also the less drastic pasteurization process, have also been shown to enhance some allergenic characteristics.

      Roth-Walter F, Berin MC, Arnaboldi P, Escalante CR, Dahan S, Rauch J, Jensen-Jarolim E, Mayer L. Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer’s patches. Allergy. 63:882-890.

      Furthermore, milk proteins can be oxidized during industrial treatment, resulting in the formation of modified/oxidized amino acid residues, particularly in BLG, which may be responsible for the development of new immunologically reactive structures.
      • Fenaille F.
      • Parisod V.
      • Tabet J-C
      • Guy P.A.
      Carbonylation of milk powder proteins as a consequence of processing conditions.

      Technological Treatments and Cow’s Milk Protein Allergenicity

      Hypoallergenic formulas can be prepared by hydrolysis and further processing, such as heat treatment, ultrafiltration, and application of high pressure. Attempts have been made to classify formulas into partial and extensively hydrolyzed products according to the degree of protein fragmentation, but there is no agreement on the criteria on which to base this classification (see section “CM hydrolyzed formula”). Nevertheless, hydrolyzed formulas have until now proved a useful and widely used protein source for infants suffering from CMA. Because undigested protein can still be present as residue at the end of proteolysis,
      • Restani P.
      • Velonà T.
      • Plebani A.
      • Ugazio A.G.
      • Poiesi C.
      • Muraro A.
      • Galli C.L.
      Evaluation by SDS-PAGE and immunoblotting of residual antigenicity in hydrolysed protein formulas.
      further processing is necessary in combination with e enzymatic treatment. Another attempt to eliminate antigenicity involves the use of proteolysis combined with high pressure. Different authors have shown increased fragmentation of BLG if proteolysis occurs after or during the application of high pressure.
      • Penas E.
      • Restani P.
      • Ballabio C.
      • Prestamo G.
      • Fiocchi A.
      • Gomez R.
      Evaluation of the residual antigenicity of dairy whey hydrolysates obtained by combination of enzymatic hydrolysis and high-pressure treatment.
      The partial ineffectiveness of proteolysis under ordinary atmospheric conditions may be because of the inability of enzymes to reach epitopes that are less exposed. Heat treatment is also often combined with proteolysis to unfold the protein and modify the 3-dimensional structure of conformational epitopes. However, thermal denaturation can also cause the formation of aggregates with greater resistance to hydrolytic attack, as is the case with BLG.

      Restani P, Ballabio C, Fiocchi A. Milk allergens: chemical characterization, structure modifications and associated clinical aspects. In: Pizzano R, ed. Immunochemistry in dairy research. Research Signpost, Kerala. 2006;61-76.

      Section 5: Immunological Mechanisms of Cow’s Milk Allergy

      Overview

      CMA designates objectively reproducible symptoms or signs initiated by exposure to cow’s milk protein at doses tolerated by normal persons. CMA can be either antibody-mediated or cell-mediated; occasionally both mechanisms may be involved. CMA may be mediated by any of the 4 basic types of immunologic reactions, as outlined by Gell and Coombs: 1) Type I or IgE-mediated hypersensitivity, 2) Type II (cytotoxic reactions), 3) Type III (Arthus-type reactions), and 4) Type IV (delayed T cell reactions). Type I reactions are the best characterized and represent the classic immediate allergic reactions. The 3 other types, collectively described as non-IgE-mediated allergy, are less well understood.
      The suppression of adverse immune responses to nonharmful ingested food antigens is termed oral tolerance. Ingested milk proteins are normally degraded by gastric acid and luminal digestive enzymes. The exact mechanisms involved in tolerance development remain unclear. The primary immunologic mechanisms include deletion, anergy, suppression, “ignorance,” and apoptosis of T-cells. The balance between tolerance (suppression) and sensitization (priming) depends on several factors, including: 1) genetic background, 2) nature and dose of the antigen, 3) frequency of administration, 4) age at first antigen exposure, 5) immunologic status of the host, and 6) antigen transmission via breast milk.
      The acquisition of tolerance to milk is seen as a TH1 (T helper cells type 1)-skewed immune response. After intestinal mucosal exposure to cow’s milk antigens, antigen-presenting cells (APCs) interact with subepithelial T and B lymphocytes. Recognition of antigens by the T cell receptors (TCR) involves major histocompatibility complex (MHC) molecules. Activated T and B cells of lymphoid follicles migrate via the lymphatic system, and then via the circulation to several target organs, including the gastrointestinal tract, respiratory system or skin. If tolerance is not achieved, T and B cells will be activated and give rise to an inflammatory reaction in the target organ, resulting in the clinical manifestations of CMA.
      The innate immune system has the ability to modulate adaptive immune responses to food proteins. In this process, dendritic cells (DC) and Toll-like receptors (TLR) play a central role. Intestinal microbiota have been shown to exert diverse effects on TLRs and regulatory T cell responses. TLR can recognize specific pathogen-associated molecular patterns (PAMP). The mechanisms by which TLRs influence Treg responses are incompletely understood. Treg promote tolerance to milk antigens via the production of tolerogenic cytokines, including interleukin (IL)-10 and transforming growth factor beta (TGF-ß).
      CMA is believed to result from either the failure to develop normal tolerogenic processes, or their later breakdown. In the case of IgE-mediated CMA, activation of milk-specific T helper cells type-2 (TH2) leads to the production of milk-specific IgE. Non-IgE-mediated reactions may be because of TH1-mediated inflammation. Decreased Treg activity has been identified as a factor in both allergy mechanisms. The development of tolerance in children with a history of CMA was associated with the up-regulation of Treg responses.
      The events after intestinal allergen exposure are complex as digestion and cooking may modify the aller-genicity of bovine proteins. Intact allergenic epitopes on food proteins will interact with the mucosal immune system. Dietary proteins that escape proteolysis can be taken up by intestinal epithelial cells. Early exposure to relatively large doses of soluble protein is thought to promote tolerance. Factors that modulate the risk of sensitization include: 1) nature and dose of the antigen, 2) efficiency of protein digestion, 3) immaturity of the host, 4) rate of absorption of milk proteins, 5) antigen processing in the gut, and 6) the immunosuppressive milieu of Peyer’s patches. The type of gut microbiota may also modulate the risk of sensitization in young infants.

      Introduction

      Acquired immunologic tolerance of environmental agents is an active mechanism of adaptive immunity that is mediated by polarized cells of the T helper type I lymphocyte subset but when, in an atopic individual, the predisposition to secrete IgE antibody to cow’s milk antigen goes into overdrive, homeostasis breaks down and mast cells can become sensitized anywhere in the body, thereby expressing an often baffling array of symptoms in one or more organs which the clinician identifies as CMA.
      • Crittenden R.G.
      • Bennett L.E.
      Cow’s milk allergy: a complex disorder.
      A basic understanding the underlying cellular and mediator mechanisms of CMA is therefore necessary to be proactive about diagnostic and treatment options.

      Gut Barrier

      The mucosal immune system must adapt and be able to discriminate between pathogens and harmless antigens and respond accordingly, that is, to protect the neonate from enteric pathogens while establishing a state of tolerance to dietary proteins and commensal bacteria. This important task is undertaken by cells of the gut-associated lymphoid tissue, the largest immunologic organ in the body.
      • van Wijk F.
      • Knippels L.
      Initiating mechanisms of food allergy: Oral tolerance versus allergic sensitization.
      Many studies have reported increased macromolecular transport across the gut barrier in children with atopy
      • Majamaa H.
      • Isolauri E.
      Evaluation of gut mucosal barrier: evidence for increased antigen transfer in children with atopic eczema.
      ,
      • Pike M.G.
      • Heddle R.J.
      • Boulton P.
      Increased intestinal permeability in atopic eczema.
      which is thought to be because of mucosal damage induced by local hypersensitivity reaction to foods.
      • Strobel S.
      • Brydon W.G.
      • Ferguson A.
      Cellobiose/mannitol sugar permeability test complements biopsy histopathology in clinical investigation of the jejunum.
      Dual sugar intestinal permeability studies (lactulose/mannitol) showed that in breast-fed infants with atopy, gut barrier function improved when breast-feeding was stopped and hypoallergenic formula started.
      • Arvola T.
      • Moilanen E.
      • Vuento R.
      • Isolauri E.
      Weaning to hypoallergenic formula improves gut barrier function in breast-fed infants with atopic eczema.

      Oral Tolerance

      The mucosa allows nutrients to be transferred from the intestinal lumen to the systemic circulation, while protecting against pathogens by inducing immune responses. Any down-regulation of immune responses to nonharmful ingested antigens is termed oral tolerance.
      • Shah U.
      • Walker W.A.
      Pathophysiology of intestinal food allergy.
      Normally, mature lymph node lymphocytes become hyporesponsive after oral administration of these antigens.
      • Burks A.W.
      • Laubach S.
      • Jones S.M.
      Oral tolerance, food allergy, and immunotherapy: implications for future treatment.
      Ingested milk proteins are degraded and their conformational epitopes are destroyed by gastric acid and luminal digestive enzymes, which often results in the destruction of immunogenic epitopes. In animal models, disrupting the process of digestion can inhibit milk tolerance and lead to hypersensitivity. Untreated bovine serum albumin (BSA) is immunogenic when administered to mice by means of ileal injection, but administering a peptic digest of the protein in the same manner results in immune tolerance.
      • Michael J.G.
      The role of digestive enzymes in orally induced immune tolerance.
      Regulatory events after mucosal exposure to antigen have not been well characterized and remain controversial. In general, the acquisition of tolerance to milk is seen as a TH1-skewed response, which on the one hand may prevent harmful mucosal immune reactions but on the other may contribute to adverse responses in a susceptible individual. The process starts with the contact of milk allergens with the intestinal mucosa. Here they interact with mucosal T and B cells either directly or through antigen-presenting cells (APCs): macrophages, dendritic cells, or microfold cells (M cells). T cell recognition of antigen by T cell receptors (TCR) involves the major histocompatibility complex (MHC) molecules (class I and II) of APCs. Activated T and B cells of lymphoid follicles migrate first via the lymphatic system and then via the circulation to any of several target organs including the gastrointestinal tract, the respiratory system, the skin, and the central nervous system, a process referred to as “homing.” If tolerance is not achieved, T and B cells will activate at a homing site upon contact with their specific food antigen and release their cytokines, vasoactive peptides and antibodies, giving rise to an inflammatory reaction in the affected organ and resulting in the clinical manifestations of food hypersensitivity.
      • Sabra A.
      • Bellanti J.A.
      • Rais J.M.
      • Castro H.J.
      • de Inocencio J.M.
      • Sabra S.
      IgE and non-IgE food allergy.
      In this context, dendritic cells play a central role in taking up milk proteins and migrating to the draining mesenteric lymph nodes, where they induce regulatory CD4 T-cell differentiation. The primary mechanisms by which tolerance may be mediated include deletion, anergy, suppression, “ignorance,” and apoptosis of T-cells.
      • Chehade M.
      • Mayer L.
      Oral tolerance and its relation to food hypersensitivities.
      The balance between tolerance (suppression) and sensitization (priming) depends on several factors, such as: 1) genetic background, 2) nature and dose of antigen, 3) frequency of administration, 4) age at first antigen exposure, 5) immunologic status of the host, 6) antigen transmission via breast milk, and others.
      Overall, there is evidence in rodents that multiple low-dose feeds are likely to induce regulatory cytokines (eg, TGF-ß, IL-10, IL-4) in part secreted by CD4+CD25+ T-regulatory cells. Despite the powerful suppressive effects of oral autoantigen exposure observed in experimental models of autoimmune diseases (including bystander suppression), their translation into clinical trials of autoimmune diseases has not yet yielded the expected beneficial results. The same can be said for CMA.
      • Strobel S.
      Oral tolerance, systemic immunoregulation, and autoimmu-nity.
      In normal individuals with tolerance, systemic and secretory food-specific IgA antibodies are generally absent, indicating that mucosal IgA production is regulated similarly to that of systemic immunity.
      • Strobel S.
      • Mowat A.M.
      Oral tolerance and allergic responses to food proteins.
      However, mucosal IgA response to foreign antigens remains active.
      • Mestecky J.
      • McGhee J.R.
      Immunoglobulin A (IgA): molecular and cellular interactions involved in IgA biosynthesis and immune response.
      In population surveys, more allergic sensitization was seen in subjects with an IgA level at the lower end of the normal range.
      • Ludviksson B.R.
      • Eiriksson T.H.
      • Ardal B.
      • Sigfusson A.
      • Valdimarsson H.
      Correlation between serum immunoglobulin A concentrations and allergic manifestations in infants.
      ,
      • Walker-Smith J.
      Cow’s milk allergy: a new understanding from immunology.
      • Kokkonen J.
      • Haapalahti M.
      • Laurila K.
      • Karttunen T.J.
      • Maki M.
      Cow’s milk protein sensitive enteropathy at school age.
      The significance of IgM, IgG, and IgG subclass antibodies (eg, the role of IgG4) in food allergy is less well understood and remains controversial. It has long been known that milk-specific IgM and IgG antibodies are produced after single or repeated feedings of relatively large doses of milk proteins in both healthy and allergic persons.
      • Rothberg R.M.
      • Farr R.S.
      Anti-bovine serum albumin and antialpha lactalbumin in the serum of children and adults.
      Thus, unresponsiveness of the immune system to milk antigens (“oral tolerance”) is believed to involve the deletion or switching off (anergy) of reactive antigen-specific T cells and the production of regulatory T cells (Treg) that suppress inflammatory responses to benign antigens.
      • Schmidt-Weber C.B.
      • Blaser K.
      T-cell tolerance in allergic response.
      ,
      • Curotto de Lafaille M.A.
      • Lafaille J.J.
      CD4 regulatory T cells in autoimmunity and allergy.

      Innate Immunity and Tolerance Development

      The innate immune system has the ability to modulate adaptive immune responses to food proteins. In this process, dendritic cells (DC) play a central role.
      • Duez C.
      • Gosset P.
      • Tonnel A.B.
      Dendritic cells and toll-like receptors in allergy and asthma.
      In addition, TLR directly interact with innate immune cells. TLR recognize food antigens, and specific bacterial surface markers, so-called PAMP.
      • Duez C.
      • Gosset P.
      • Tonnel A.B.
      Dendritic cells and toll-like receptors in allergy and asthma.
      However, the exact mechanisms by which TLR influence Treg responses are incompletely understood. Regulatory T-cells are involved in the control of immune responses to food antigens via the production of tolerogenic cytokines, including IL-10 and TGF-ß.
      • Rautava S.
      • Kalliomaki M.
      • Isolauri E.
      New therapeutic strategy for combating the increasing burden of allergic disease: probiotics-a Nutrition, Allergy, Mucosal Immunology and Intestinal Microbiota (NAMI) Research Group report.
      ,
      • Akdis M.
      • Verhagen J.
      • Taylor A.
      • Karamloo F.
      • Karagiannidis C.
      • Crameri R.
      Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells.
      Intestinal micro-biota may have a diverse effect on TLR and immune responses. Several types of intestinal Bifidobacteria have been shown to promote tolerogenic immune responses. The type of gastrointestinal microbiota of the newborn infant is crucial in this context. The probiotic effects of complex oligosaccha-rides in human milk promote the establishment of a bidido-genic microbiota which, in turn, induces a milieu of tolerogenic immune responses to foods. Several probiotic bacterial strains have been shown to have similar properties. For example, Lactobacillus paracasei inhibits TH1 and TH2 cytokine production, and induces CD4(+) T cells to produce TGF- and IL-10, that is, induces a tolerogenic response.
      • von der Weid T.
      • Bulliard C.
      • Schiffrin E.J.
      Induction by a lactic acid bacterium of a population of CD4(+) T cells with low proliferative capacity that produce transforming growth factor beta and interleukin-10.
      It appears possible that the recent decrease in exposure to early childhood infections and harmless environmental microorganisms in the westernized environment has contributed to an increase in T-cell dysregulatory disorders and autoimmunity.
      • Romagnani S.
      The increased prevalence of allergy and the hygiene hypothesis: missing immune deviation, reduced immune suppression, or both.
      ,
      • Rook G.A.
      • Brunet L.R.
      Microbes, immunoregulation, and the gut.

      Dysfunctional Tolerance

      CMA is believed to result from the failure to develop normal tolerogenic processes or their later breakdown. In the case of IgE-mediated CMA, a deficiency in regulation and a polarization of milk-specific effector T cells toward type-2 T helper cells (TH2) both lead to B-cell signaling to produce milk protein-specific IgE.
      • Beyer K.
      • Castro R.
      • Birnbaum A.
      • Benkov K.
      • Pittman N.
      • Sampson H.A.
      Human milk-specific mucosal lymphocytes of the gastrointestinal tract display a Th2 cytokine profile.
      ,
      • Schade R.P.
      • Tiemessen M.M.
      • Knol E.F.
      • Bruijnzeel-Koomen C.A.
      • van Hoffen E.
      The cow’s milk protein-specific T cell response in infancy and childhood.
      Non-IgE-mediated reactions may be because of TH1-mediated inflammation.
      • Tiemessen M.M.
      • van Hoffen E.
      • Knulst A.C.
      • van der Zee J.A.
      • Knol E.F.
      • Taams L.S.
      CD4_CD25_ regulatory T cells are not functionally impaired in adult patients with IgE-mediated cows milk allergy.
      Dysfunctional Treg cell activity has been identified as a factor in both allergy mechanisms.
      • Perez-Machado M.A.
      • Ashwood P.
      • Thomson M.A.
      • Latcham F.
      • Sim R.
      • Walker-Smith J.A.
      • Murch S.H.
      Reduced transforming growth factor-beta1-producing T cells in the duodenal mucosa of children with food allergy.
      Additionally, the induction of tolerance in children who have outgrown their CMA has been shown to be associated with the development of Treg cells.
      • Karlsson M.R.
      • Rugtveit J.
      • Brandtzaeg P.
      Allergen-responsive CD4_CD25_ regulatory T cells in children who have outgrown cow’s milk allergy.
      ,
      • Tiemessen M.M.
      • Van Ieperen-Van Dijk A.G.
      • Bruijnzeel-Koomen C.A.
      • Garssen J.
      • Knol E.F.
      • Van Hoffen E.
      Cow’s milk-specific T-cell reactivity of children with and without persistent cow’s milk allergy: key role for IL-10.
      Much research is currently focused on manipulating the activity of dendritic cells (specialized antigen-presenting cells important in programming immune responses) to induce Treg cells and/or to redress TH1/TH2 imbalances to promote tolerance to allergenic foods.

      Allergen Exposure and Sensitization

      The events after allergen exposure in the gut are complex. Digestion
      • Fiocchi A.
      • Restani P.
      • Riva E.
      • Restelli A.R.
      • Biasucci G.
      • Galli C.L.
      • Giovannini M.
      Meat allergy: II: effects of food processing and enzymatic digestion on the allergenicity of bovine and ovine meats.
      and cooking preparation
      • Fiocchi A.
      • Restani P.
      • Riva E.
      • Mirri G.P.
      • Santini I.
      • Bernardo L.
      • Galli C.L.
      Heat treatment modifies the allergenicity of beef and bovine serum albumin.
      ,
      • Fiocchi A.
      • Bouygue G.R.
      • Sarratud T.
      • Terracciano L.
      • Martelli A.
      • Restani P.
      Clinical tolerance of processed foods.
      slightly modifies the allergenicity of bovine proteins. Proteins that are not digested and processed in the lumen of the gut will come in contact with the epithelium and mucosal immune system in various ways. In the gut, dendritic cells can sample antigens by extending processes through the epithelium and into the lumen. M cells that overlie Peyer’s patches can take up particulate antigens and deliver them to subepithelial dendritic cells. Soluble antigens possibly cross the epithelium through transcellular or paracellular routes to encounter T cells or macrophages in the lamina propria. Dietary proteins that escape proteolysis in the gut can be taken up by intestinal epithelial cells. The epithelial cells can act as nonprofessional APCs and can present antigen to primed T cells. Thus, food allergens (and microorganisms and nonviable particulate antigens) reach CD4+ and CD8+ T cells in the Peyer’s patch, resulting in active immune responses.
      • Kelsall B.L.
      • Strober W.
      Host defenses at mucosal surfaces.
      Early gastrointestinal encounters with relatively large doses of soluble protein almost always induce tolerance.
      • Strobel S.
      • Mowat A.M.
      Immune responses to dietary antigens: oral tolerance.
      Data from rodent models suggest that the effect of milk allergen exposure on the host depends on many factors, including:
      • a.
        Nature and dose of the antigen
      • b.
        Efficiency of digestion
      • c.
        Immaturity of the host
      • d.
        Rate of absorption of milk proteins
      • e.
        Antigen processing in the gut
      • f.
        The immunosuppressive milieu of the Peyer patch.
        • Kellermann S-A
        • McEvoy L.M.
        The Peyer’s patch microenvironment suppresses T cell responses to chemokines and other stimuli.
      All of these factors can favor the induction of peripheral tolerance to dietary proteins rather than systemic hypersensitivity. In this context, the presence of commensal flora in the gut can lower the production of serum milk-specific IgE during the primary immune response; also, IgE production persists longer in germ-free mice. Conversely, the absence of gut microbiota significantly increases the milk-specific immune response in mice.
      • Hazebrouck S.
      • Przybylski-Nicaise L.
      • Ah-Leung S.
      • Adel-Patient K.
      • Corthier G.
      • Wal J.M.
      • Rabot S.
      Allergic sensitization to bovine beta-lactoglobulin: comparison between germ-free and conventional BALB/c mice.
      This raises the possibility of prevention and treatment of milk allergy through the manipulation of the gastrointestinal flora.

      Milk Allergy

      An effect of dysfunctional tolerance, “milk allergy” designates objectively reproducible symptoms or signs initiated by exposure to cow’s milk at a dose tolerated by normal persons.
      • Johansson S.G.
      • Bieber T.
      • Dahl R.
      Revised nomenclature for allergy for global use: report of the Nomenclature Review Committee of the World Allergy Organization, 2003.
      The term CMA is appropriate when specific immunologic mechanisms have been demonstrated (see “definitions” in introductory section). Milk allergy can be either antibody-mediated or cell-mediated, or occasionally both may be involved. If IgE is involved in the reaction, the term “atopic food allergy” is appropriate. If immunologic mechanisms other than IgE are predominantly involved, the term “non-IgE-mediated food allergy” should be used. All other reactions should be regarded to as nonallergic food hyper-sensitivity.
      • Ortolani C.
      • Bruijnzeel-Koomen C.
      • Bengtsson U.
      • Bindslev-Jensen C.
      • Bjorksten B.
      • et al.
      Controversial aspects of adverse reactions to food.
      Enhanced immune-mediated reactivity may come about though any, or a combination of, the 4 basic types of immunologic reactions outlined by Gell and Coombs:
      • a.
        Type I or IgE-mediated hypersensitivity leads to immediate symptoms, such as urticaria, angioedema and/or other anaphylactic reaction
      • b.
        In type II (cytotoxic) reactions, the antigen binds to the cell surface and the presence of antibodies (IgG, IgM, or IgA) disrupts the membrane, leading to cell death
      • c.
        In type III (Arthus-type) reactions, antigen-antibodycomplement immune complexes (IgG, IgM, IgA, and IgE antibodies) get trapped in small blood vessels or renal glomeruli.
      • d.
        Type IV (delayed) reactions are mediated by sensitized T lymphocytes.
      Type I reactions are the best understood, and they are often referred to as the most common and classic allergic reactions. The 3 other types, collectively described as non-IgE-medi-ated allergy, are more difficult to investigate and hence less well understood. In an individual, several types of immune responses may be activated, although IgE-mediated reactions are more usually measured.

      Ige-Mediated CMA (Immediate Hypersensitivity)

      IgE-mediated allergy is the best understood allergy mechanism and, in comparison to non-IgE-mediated reactions, is relatively easily diagnosed. Since the onset of symptoms is rapid, occurring within minutes to an hour after allergen exposure, IgE-mediated allergy is often referred to as “immediate hypersensitivity.”

      Roitt I, Brostoff J, Male D. Immunology. 6th ed., New York: Mosby; 2001.

      It occurs in 2 stages. The first, “sensitization,” occurs when the immune system is aberrantly programmed to produce IgE antibodies to milk proteins. These antibodies attach themselves to the surface of mast cells and basophils, arming them with an allergen-specific trigger. Subsequent exposure to milk proteins leads to “activation” when the cell-associated IgE binds the allergenic epitopes on the milk proteins and triggers the rapid release of powerful inflammatory mediators.
      IgE-mediated, acute onset CM allergies can affect several target organs: the skin (urticaria, angioedema), respiratory tract (rhinitis/rhinorrhea, asthma/wheeze, laryngoedema/ stridor), gastrointestinal tract (oral allergy syndrome, nausea, vomiting, pain, flatulence, and diarrhea), and/or the cardiovascular system (anaphylactic shock).
      • Sicherer S.H.
      Food allergy.
      ,
      • Hill D.J.
      • Hosking C.S.
      • Zhie C.Y.
      • Leung R.
      • Baratwidjaja K.
      • et al.
      The frequency of food allergy in Australia and Asia.
      Life-threatening anaphylactic reactions to cow’s milk may occur, but are fortunately rare.
      • Eigenmann P.A.
      Anaphylaxis to cow’s milk and beef meat proteins.
      Since reactions to cow’s milk proteins can occur on contact with the lips or mouth, strategies to reduce allergenicity by improving protein digestibility in the gut are unlikely to be effective for all allergic individuals. Simple diagnostic procedures, such as skin-prick tests (SPT) and specific serum IgE determinations (immuno-CAP), can be used to identify individuals with IgE-mediated CMA, although either of these tests can produce false-positive re-sults.
      • Fiocchi A.
      • Bouygue G.R.
      • Restani P.
      • Bonvini G.
      • Startari R.
      • Terracciano L.
      Accuracy of skin prick tests in IgE-mediated adverse reactions to bovine proteins.
      Food elimination and challenge testing are sometimes required to confirm milk allergy, and double-blind, placebo-controlled, food challenge (DBPCFC) testing remains the gold standard for diagnosis. IgE-mediated CMA may occur in neonates on first postnatal exposure to the food.
      • van Asperen P.P.
      • Kemp A.S.
      • Mellis C.M.
      Immediate food hypersensitivity reactions on the first known exposure to the food.
      IgE-mediated reactions account for about half of the CMA cases in young children,
      • Heine R.G.
      • Elsayed S.
      • Hosking C.S.
      • Hill D.J.
      Cow’s milk allergy in infancy.
      but are rare in adults.
      • Woods R.K.
      • Thien F.
      • Raven J.
      • Walters E.H.
      • Abramson M.A.
      Prevalence of food allergies in young adults and their relationship to asthma, nasal allergies, and eczema.
      ,
      • Zuberbier T.
      • Edenharter G.
      • Worm M.
      • Ehlers I.
      • Reimann S.
      • et al.
      Prevalence of adverse reactions to food in Germany: a population study.
      In contrast to adults, atopic CMA in childhood (often a part of the “allergic march”) resolves in more than 85% of cases.
      • Thong B.Y.
      • Hourihane J.O.
      Monitoring of IgE-mediated food allergy in childhood.
      ,
      • Fiocchi A.
      • Terracciano L.
      • Bouygue G.R.
      • Veglia F.
      • Sarratud T.
      • Martelli A.
      • Restani P.
      Incremental prognostic factors associated with cow’s milk allergy outcomes in infant and child referrals: the Milan Cow’s Milk Allergy Cohort study.