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World Allergy Organization (WAO)Diagnosis and Rationale for Action againstCows Milk Allergy (DRACMA) Guidelines

Authorship

Alessandro Fiocchi, MD, Pediatric Division,Department of Child and Maternal Medi-cine, University of Milan Medical School atthe Melloni Hospital, Milan 20129, Italy.

Holger J. Schu nemann, MD,a Department of

Clinical Epidemiology & Biostatistics,McMaster University Health Sciences Cen-tre, 1200 Main Street West, Hamilton, ONL8N 3Z5, Canada.

Sami L. Bahna, MD, Pediatrics & Medicine,Allergy & Immunology, Louisiana StateUniversity Health Sciences Center, Shreve-port, LA 71130.

Andrea Von Berg, MD, Research Institute,Childrens department, Marien-Hospital,Wesel, Germany.

Kirsten Beyer, MD, Charite Klinik fu r Pa diatriem.S. Pneumologie und Immunologie, Augu-stenburger Platz 1, D-13353 Berlin, Germany.

Martin Bozzola, MD, Department of Pediatrics,British Hospital-Perdriel 74-CABA-BuenosAires, Argentina.

Julia Bradsher, PhD, Food Allergy & Anaphy-laxis Network, 11781 Lee Jackson Highway,Suite 160, Fairfax, VA 22033.

Jan Brozek, MD,a Department of ClinicalEpidemiology & Biostatistics, McMasterUniversity Health Sciences Centre, 1200 MainStreetWest,Hamilton,ONL8N3Z5,Canada.

Enrico Compalati, MD,a Allergy & RespiratoryDiseases Clinic, Department of InternalMedicine, University of Genoa, 16132,Genoa, Italy.

Alessandro Fiocchi, (Chair), JanBrozek, Holger Schnemann, (Chair),Sami L. Bahna, Andrea von Berg,Kirsten Beyer, Martin Bozzola, JuliaBradsher, Enrico Compalati, MotohiroEbisawa, Maria Antonieta Guzmn,Haiqi Li, Ralf G. Heine, Paul Keith,Gideon Lack, Massimo Landi, AlbertoMartelli, Fabienne Ranc, HughSampson, Airton Stein, LuigiTerracciano and Stefan Vieths

Keywords: Cow milk allergy; oral food challenge;

epidemiology; DBPCFC; amino acid formula;

hydrolyzed milk formula; hydrolyzed rice formula;

hydrolyzed soy formula; skin prick test; specific IgE;

OIT; SOTI; GRADE

Correspondence to: Alessandro Fiocchi, MD,

Paediatric Division, Department of Child and

Maternal Medicine, University of Milan Medical

School at the Melloni Hospital, Milan 20129, Italy.

E-mail: [email protected].

This supplement is co-published as an article in the

April 2010 issue of the World Allergy OrganizationJournal. Fiocchi A, Brozek J, Schnemann H, Bahna

S, von Berg A et al. World Allergy Organization

(WAO) Diagnosis and Rationale for Action against

Cow's Milk Allergy (DRACMA) Guidelines. World

Allergy Organization Journal2010; 3 (4): 57161.

Pediatr Allergy Immunol 2010: 21 (Suppl. 21): 1125

DOI: 10.1111/j.1399-3038.2010.01068.x

2010 John Wiley & Sons A/S

PEDIATRIC ALLERGY AND

IMMUNOLOGY

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Motohiro Ebisawa, MD, Department of Allergy,Clinical Research Center for Allergy andRheumatology, Sagamihara National Hos-pital, Kanagawa 228-8522, Japan.

Maria Antonieta Guzma n, MD, Immunologyand Allergy Division, Clinical HospitalUniversity of Chile, Santiago, Chile. SantosDumont 999.

Haiqi Li, MD, Professor of Pediatric Division,Department of Primary Child Care, Chil-drens Hospital, Chongqing Medical Uni-versity, China, 400014.

Ralf G. Heine, MD, FRACP, Department ofAllergy & Immunology, Royal ChildrensHospital, University of Melbourne, Mur-doch Childrens Research Institute, Mel-bourne, Australia.

Paul Keith, MD, Allergy and Clinical Immu-nology Division, Department of Medicine,McMaster University, Hamilton, Ontario,

Canada.Gideon Lack, MD, Kings College London,Asthma-UK Centre in Allergic Mechanismsof Asthma, Department of Pediatric Allergy,St Thomas Hospital, London SE1 7EH,United Kingdom.

Massimo Landi, MD, National PediatricHealthcare System, Italian Federation ofPediatric Medicine, Territorial PediatricPrimary Care Group, Turin, Italy.

Alberto Martelli, MD, Pediatric Division,Department of Child and MaternalMedicine, University of Milan Medical

School at the Melloni Hospital, Milan 20129,Italy.

Fabienne Rance , MD, Allergologie, Hopital desEnfants, Pole Me dicochirurgical de Pe diat-rie, 330 av. de Grande Bretagne, TSA 70034,31059 Toulouse CEDEX, France.

Hugh Sampson, MD, Jaffe Food Allergy Insti-tute, Mount Sinai School of Medicine, OneGustave L. Levy Place, NY 10029-6574.

Airton Stein, MD, Conceicao Hospital, PortoAlegre, Brazil.

Luigi Terracciano, MD,a Pediatric Division,Department of Child and Maternal Medi-

cine, University of Milan Medical Schoolat the Melloni Hospital, Milan 20129,Italy.

Stefan Vieths, MD, Division of Allergology,Paul-Ehrlich-Institut, Federal Institute forVaccines 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 Assaad, MD, Division of Allergy andImmunology, Cincinnati Childrens HospitalMedical Center, Cincinnati, Ohio, USA.

Carlos Baena-Cagnani, MD, LIBRA foundationArgentina, Division of Immunology andRespiratory Medicine, Department of Pedi-

atric, Infantile Hospital Cordoba, SantaRosa 381, 5000 Cordoba, Argentina.GR Bouygue, MSc, Pediatric Division, Depart-

ment of Child and Maternal Medicine,University of Milan Medical School at theMelloni Hospital, Milan 20129, Italy.

Walter Canonica, MD, Allergy & RespiratoryDiseases Clinic, Department of InternalMedicine. University of Genoa, 16132,Genoa, Italy.

Christophe Dupont, MD, Service de gastroente -rologie et nutrition, Hopital Saint Vincent dePaul, 82, avenue Denfert-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 andInflammation Branch, National Institute ofAllergy and Infectious Diseases, NIH, 6610Rockledge Dr., Bethesda, MD 20892.

Rosa Elena Huerta Hernandez, MD, PediatricAllergy Clinic, Mexico City, Mexico.

Manuel Martin-Esteban, MD, Allergy Depart-ment, Hospital Universitario La Paz, Ma-drid, Spain.

Anna Nowak-Wegrzyn, MD, Jaffe FoodAllergy Institute, Mount Sinai School ofMedicine, One Gustave L. Levy Place, NY10029-6574.

Ruby Pawankar, MD, Department of Otolar-yngology, Nippon Medical School, 1-1-5Sendagi, Tokyo, 113 Japan.

Susan Prescott, MD, School of Pediatrics andChild Health, University of Western Aus-tralia, Princess Margaret Hospital for Chil-dren, Perth, Australia.

Patrizia Restani, PhD, Department of Pharma-

cological Sciences, Universita` degli Studi diMilano.Teresita Sarratud, MD, Department of Pediat-

rics, University of Carabobo Medical Schoolat the Carabobo Hospital, Valencia, Vene-zuela.

Aline Sprikkelmann, MD, Department of Pedi-atric Respiratory Medicine and Allergy,Emma Childrens Hospital Academic Medi-cal Centre, Amsterdam, The Netherlands.

WAO DRACMA Guidelines

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Sections

1: Introduction, p. 32: Methodology, p. 43: Epidemiology of CMA, p. 64: Allergens of Cows Milk, p. 125: Immunological Mechanisms of CMA, p. 186: ClinicalHistory andSymptomsof CMA, p. 25

7: Diagnosis of CMA According to PrecedingGuidelines, p. 378: TheEliminationDietinWork-Upof CMA,p.419: Guidelines for Diagnosing CMA, p. 42

10: Oral Food Challenge Procedures in Diagno-sis of CMA, p. 56

11: Natural History of CMA, p. 6612: Treatment of CMA According to Preceding

Guidelines, p. 7113: When Can Milk Proteins Be Eliminated From

Diet Without Substituting Cows Milk?, p. 7514: Guidelines for Choosing a Replacement

Formula, p. 79

15: Milks From Different Animals for Substi-tuting Cows Milk, p. 84

16: Nutritional Considerations in CMA Treat-ment, p. 89

17: Choosing the Appropriate Substitute For-mula in Different Presentations, p. 92

18: Grade Recommendations on Immuno-therapy for CMA, p. 93

19: Unmet Needs, Recommendations for Re-search, Implementation of DRACMA, p. 95

Acknowledgements, p. 97Appendix 1: Cows Milk Allergy Literature

Search Algorithms, p. 98Appendix 2: Evidence Profiles: Diagnosis of

CMA, p. 104Appendix 3: Evidence Profiles: Treatment of

CMA, p. 114Appendix4: EvidenceProfiles: OITfor Treatment

of CMA, p. 123

Section 1: Introduction

Allergy and clinical immunology societieshave issued guidance for the managementof food allergy.1,2 Guidelines are now regarded

as translational research instruments, designed toprovide cutting-edge benchmarks for good prac-tice and bedside evidence for clinicians to use in aninteractive learning context with their national orinternational scientific communities. In the man-agement of cows milk allergy (CMA), bothdiagnosis and treatment would benefit from areappraisal of the more recent literature, forcurrent guidelines summarize the achievementsof the preceding decade, deal mainly with preven-tion (36), do not always agree on recommenda-

tions and date back to the turn of the century (7,8). In 2008, the World Allergy Organization(WAO) Special Committee on Food Allergyidentified CMA as an area in need of a rationale-based approach, informed by the consensusreached through an expert review of the availableclinical evidence, to make inroads against aburdensome, world-wide public health problem.

It is in this context that the WAO Diagnosis andRationale for Action against Cows Milk Allergy(DRACMA) Guidelines was planned to providephysicians everywhere with a management tool todeal with CMA from suspicion to treatment.Targeted (and tapped for their expertise), both onthe DRACMA panel or as nonsitting reviewers,were allergists, pediatricians (allergists and gener-alists), gastroenterologists, dermatologists, epi-demiologists, methodologists, dieticians, foodchemists, and representatives of allergic patientorganizations. Ultimately, DRACMA is dedi-

cated to our patients, especially the younger ones,whose burden of issues we hope to relieve throughan ongoing and collective effort of more interac-tive debate and integrated learning.

Definitions

Adverse reactions after the ingestion of cows milkcan occur at any age from birth and even amonginfants fed exclusively at the breast, but not allsuch reactions are of an allergic nature. A revisionof the allergy nomenclature was issued in Europein 2001 (9) and was later endorsed by the WAO

(10) under the overarching definition of milkhypersensitivity, to cover nonallergic hypersen-sitivity (traditionally termed cows milk intoler-ance) and allergic milk hypersensitivity (orcows milk allergy). The latter definition re-quires the activation of an underlying immunemechanism to fit. In DRACMA, the termallergy will abide by the WAO definition(allergy is a hypersensitivity reaction initiated byspecific immunologic mechanisms). In most chil-dren with CMA, the condition can be immuno-globulin E (IgE)-mediated and is thought tomanifest as a phenotypical expression of atopy,

together with (or in the absence of) atopic eczema,allergic rhinitis and/or asthma. A subset ofpatients, however, have non-IgE mediated (prob-ably cell-mediated) allergy and present mainlywith gastro-intestinal symptoms in reaction to theingestion of cows milk.

References, Section 11. American College of Allergy, Asthma, &

Immunology. Food allergy: a practice parameter. AnnAllergy Asthma Immunol. 2006;96(Suppl 2):S1S68.


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and made recommendations. We reached con-sensus on all recommendations. Formulatingthe recommendations included explicit consid-eration of the quality of evidence, benefits,harms, burden, cost, and values and prefer-ences described as the Underlying values andpreferences or in the Remarks sections ofeach recommendation as outlined earlier (9).

Statements about the underlying values andpreferences and the remarks are integral partsof the recommendations and serve to facilitateaccurate interpretation of the recommenda-tions. They cannot be omitted when citing ortranslating DRACMA guidelines. In this doc-ument, the expression values and preferencesrefers to the relative weight one attributes toparticular benefits, harms, burdens, and coststo determine their balance. We used thedecision framework described previously todetermine the strength of recommendations

(1, 10).Little information about costs of diagnosis andtreatment of IgE-mediated cows milk allergy wasavailable to the panel and it is very likely that itvaries considerably across geographical areasand jurisdictions. Cost, therefore, plays a limitedrole in these recommendations. However, when-ever we considered cost and resource expendi-ture, we used health system perspective (11). Forindividual patients, cost may not be an issue ifthe service or treatment strategy is provided atreduced price or free of charge. Clinicians andpatients should consider their local resource

implications when interpreting these recommen-dations.

After the GRADE approach we classifiedrecommendations in these guidelines as eitherstrong or conditional (also known asweak)/weak. The strength of recommendationsdepends on a balance between all desirable andall undesirable effects of an intervention (ie, netclinical benefit), quality of available evidence,values and preferences, and cost (resourceutilization) (1). In general, the higher thequality of the supporting evidence, the morelikely it is for the recommendation to be

strong. Strong recommendations based on lowor very low quality evidence are rare, butpossible (12).

For strong recommendations we used wordswe recommend and for conditional recom-mendations, we suggest. We offer the sug-gested interpretation of strong and weakrecommendations in Table 2-1. Understandingthe interpretation of these 2 grades (strong orconditional) of the strength of recommendationsis essential for clinical decision making.

Table 2-1. Interpretation of ''Strong'' and ''Weak'' Recommendations

Implications Strong Recommendation Weak Recommendation

For patients Most individuals in this sit-uation would want the rec-

ommended course of action

and only a small proportionwould not. Formal decision

aids are not likely to beneeded to help individuals

make decisions consistent

with their values and pref-erences.

The majority of individuals inthis situation would want the

suggested course of action,

but many would not.

For clinicians Most individuals should re-

ceive the intervention.Adherence to this recom-

mendation according to the

guideline could be used as a

quality criterion or perfor-mance indicator.

Recognize that different choi-

ces will be appropriate forindividual patients, and thatyou must help each patient

arrive at a management deci-

sion consistent with his or hervalues and preferences. Deci-

sion aids may be useful help-

ing individuals makingdecisions consistent with their

values and preferences.

For policymakers

The recommendation can beadapted as policy in most

situations.

Policy making will requiresubstantial debates and

involvement of various stake-holders.

How to Use These Recommendations

The DRACMA guidelines are not intended toimpose a standard of care for individual coun-tries and jurisdictions. They should, as anyguideline, provide a basis for rational decisionsfor clinicians and their patients about the man-agement of cows milk allergy. Clinicians,

patients, third-party payers, institutional reviewcommittees, other stakeholders, or the courtsshould never view these recommendations asdictates. Strong recommendations based on highquality evidence will apply to most patients forwhom these recommendations are made, butthey may not apply to all patients in allcircumstances. No recommendation can takeinto account all of the often-compelling uniquefeatures of individual clinical circumstances.Therefore, nobody charged with evaluating cli-nicians actions should attempt to apply the

recommendations from the DRACMA guide-lines as rote or in a blanket fashion.

References, Section 21. Guyatt GH, Oxman AD, Kunz R, Falck-YtterY,

Vist GE, Liberati A, Schunemann HJ. Going fromevidence to recommendations. BMJ. 2008: 336: 10491051.

2. Guyatt GH, Oxman AD, Kunz R, Vist GE, Falck-Ytter Y, Schunemann HJ. What is quality of evi-dence and why is it important to clinicians? BMJ.2008: 336: 995998.


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3. Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Alonso-Coello P, Schunemann HJ.GRADE: an emerging consensus on rating quality ofevidence and strength of recommendations. BMJ.2008: 336: 924926.

4. Schunemann HJ, Fretheim A, Oxman AD. Improv-ing the use of research evidence in guideline develop-ment: 9. Grading evidence and recommendations.Health Res Policy Syst. 2006: 4: 21.

5. Schunemann HJ, Oxman AD, Fretheim A. Improv-ing the use of research evidence in guideline develop-ment: 6. Determining which outcomes are important.Health Res Policy Syst. 2006: 4: 18.

6. World Health Organization. Global Programmeon Evidence for Health Policy. Guidelines for WHOGuidelines. EIP/GPE/EQC/2003.1.Geneva, 2003.

7. Schunemann HJ, Hill SR, Kakad M, Vist GE,Bellamy R, et al. Transparent development of theWHO rapid advice guidelines. PLoS Med. 2007: 4:e119.

8. SchunemannHJ,OxmanAD,BrozekJ, GlasziouP,Jaeschke R, et al. Grading quality of evidence andstrength of recommendations for diagnostic tests andstrategies. BMJ. 2008: 336: 11061110.

9. Schunemann HJ, MungerH, BrowerS, ODonnell

M, Crowther M, Cook D, Guyatt G. Methodologyfor guideline development for the Seventh AmericanCollege of Chest Physicians Conference on Anti-thrombotic and Thrombolytic Therapy: the SeventhACCP Conference on Antithrombotic and Thrombo-lytic Therapy. Chest. 2004: 126: 174S178S.

10. Schunemann HJ, Jaeschke R, Cook DJ, Bria WF,El-SolhAA, et al. An official ATS statement: gradingthe quality of evidence and strength of recommenda-tions in ATS guidelines and recommendations. Am JRespir Crit Care Med. 2006: 174: 605614.

11. Guyatt GH, Oxman AD, Kunz R, Jaeschke R,Helfand M, Liberati A, Vist GE, Schunemann HJ.Incorporating considerations of resources use into

grading recommendations. BMJ. 2008: 336: 11701173.

12. BrozekJL, Baena-CagnaniCE, BoniniS, CanonicaGW,Rasi G, et al. Methodology for development ofthe Allergic Rhinitis and its Impact on Asthmaguideline 2008 update. Allergy. 2008: 63: 3846.

Section 3: Epidemiology of CMA

Overview

There are no surveys of population andgeographical trends in food allergy in adultsor children (though the situation is different inpediatric asthma and rhinitis) and this unmetneed is particularly felt for CMA. The per-ception of milk allergy is far more frequentthanconfirmedCMA. Patient reports of CMArange between 1 and 17.5%, 1 and 13.5%, and1 to 4% in preschoolers, at children 5 to16 years of age and adults respectively. Cow smilk-specific IgE sensitization point preva-lence progressively decreased from about 4%at 2 years to less than 1% at 10 years of age in

the German Multi-Centre Allergy Study. Themost reliable data in epidemiology are thosefrom birth cohorts that are free from selectionbias. There are 5 such challenge-confirmedstudies. The CMA prevalence during infancyranged 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 upto 4.9% in Norway.

Patients with CMA develop gastrointestinalsymptoms in 32 to 60% of cases, skin symp-toms in 5 to 90%, and anaphylaxis in 0.8 to9% of cases. This frequency of anaphylaxis isthe main concern pointed out in many CMAstudies. In a review, nearly one third ofchildren with atopic dermatitis (AD) receiveda diagnosis of CMA after an elimination dietand an oral food challenge, and about 40 to50% of children less than a year of age withCMA also had AD. Finally, with actual

population and geographical trends remainingunknown, allergists are primarily in need ofmore detailed epidemiological surveys on aglobal scale. One large such epidemiologicalstudy supported by the European Commissionis ongoing and aims to furnish the firstprevalence data regarding the suspicion ofCMA, sensitization to cows milk, and oralfood challenge-confirmed diagnosis in 10European birth cohorts.

Introduction

Around 1126 million of the European popula-tion are estimated to suffer from food allergy (1).If this prevalence was consistent around theworld and projected to the 6,659,040,000 peopleof the worlds population (2), it translates into220520 million people and represents a majorglobal health burden. Although there are surveyson the natural history and prevalence trends forsymptoms of asthma, allergic rhinoconjunctivitis,and eczema in childhood (3), we do not have astudy assessing the prevalence of food allergy

and its time-trends. The problem is complicatedby the fact that perceived food allergy (ie, theself-reported feeling that a particular food neg-atively influences health status) is not actual foodallergy. Allergy prevalence is much greater in thepublics belief than it has ever been reported bydouble-blind studies. Back in the 1980s, theperceived incidence of allergy to food or foodadditives in mothers with young children wasreported between 17 (4) and 27.5% (5). Thirtypercent of women reported that they or some


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member of their family were allergic to somefood product (6). In the after decade, a Britishstudy using a food allergy questionnaire reporteda 19.9% incidence of food allergy (7). From themid-1990s onwards, self reports began to becompared 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 ofadverse reactions overestimate true food allergy(8, 9). This was further confirmed when preva-lence figures of 2.3 to 3.6% were confirmed bychallenge procedures in unselected patient pop-ulations (10, 11). In the 1990s, it was alsoconfirmed that only a minority of subjects whoreport food-related illness also test positive byskin prick test using the same food (12).

Thus, 2 separate food allergy epidemiologiescan be distinguished:

a. Self-reported food allergy; although this doesnot represent actual food allergy epidemiology,it is useful as a proxy measure of the potentialdemand for allergy medical services, and mayguide public health allergy service users be-tween general and specialist medicine (13), andmore generally for public health planning.

b. Actual food allergy (ie, confirmed by a posi-tive oral food challenge) represents the realextent of this clinical problem.

In general, food allergy is more frequent in thepediatric, rather than the adult, population.

According to a recent Japanese multicenter trial,the prevalence of CMA is 0.21% in newbornsand 0.35% amid extremely premature babies(


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(0.55%) (49). In the German Multicenter AllergyStudy, 1314 children initially recruited were fol-lowed from birth for 13 years. The longitudinaldata were analyzed for 273 children testingpositive for serum cows milk specific IgE anti-body and were obtained at age 2, 5, 7, and 10. Thepoint prevalence of sensitization to cows milkprogressively decreased from about 4% at 2 years

to less than 1% at 10 years (50).

Epidemiology of Challenge-Confirmed CMA

The epidemiology of oral food challenge-con-firmed CMA of the last 10 years consists of thefollowing 5 studies:

a. In a Danish study of 1,749 newborns followedfor 12 months, 39 (or 2.22%) were confirmedallergic (51)

b. In a study from Finland 6,209 newborns fol-

lowed for 15 months, 118 (1.9%) had positiveDBPCFC (52)c. In a Norwegian study of 193 premature and

416 full-term infants, 27 of 555 (or 4.9%) werediagnosed with an allergic reaction to cowsmilk on the basis of an open challenge but notall children were tested; interestingly, all hadsymptoms before 6 months of age (53)

d. In an Isle of Wight cohort of 969 newbornsfollowed for 12 months, 21 (2.16%) reportedCMA but only 2 (0.21%) were actually withIgE-mediated CMA (54)

e. In a newborn cohort from the Netherlands

1,158 infants prospectively followed through12 months of age reporting cows milk pro-tein intolerance (defined as two positivecows milk elimination/challenge tests) re-ported 26 allergic children (or 2.24%) of 211(or 18.2%) suspected cases (33).

In this series of challenge-based studies, theDanish study further suggested that reproducibleclinical reactions to CMP in human milk werereported in 0.5% of breast-fed infants (55).Data from cross-sectional studies (analyzed byRona and coworkers (2)) demonstrated a rate of

0.6 to 2.5% prevalence in preschoolers, 0.3% at 5to 16 years of age, and of less than 0.5% inadults (23, 5658).

While most of our information on cows milkallergy prevalence comes from northern Europeanand Spanish studies, there are methodological andgeographical differences in clinical evaluation,which must be considered in assessing the epide-miological features we discuss here. Some studiesmay consider only immediate reactions, whileothers include delayed reactions; not all studies

include IgE sensitization assessments; some stud-ies are based on open oral food challenges, someperformed blinded oral food challenge tests.Methods used across studies in this literature oforal food challenges with (59) cows milk are notstandardized (see section on Diagnosis).

Thus, among the unmet needs of epidemiolog-ical research in this field are high-quality com-

munity studies based on patient data objectivelyconfirmed by DBPCFC to close the currentknowledge gap on the prevalence of CMA inthe population. To address this, the EuropeanCommission launched the EuroPrevall Project(http://www.europrevall.org) in 2005 in concertwith more than 60 partners including patientorganizations, the food industry and researchinstitutions from across Europe, Russia, Ghana,India, and China. This translational endeavorinvolves basic and clinical research components,and large epidemiological studies of both chil-

dren and adults (60). The first results, will includedata on suspicion of CMA, on sensitization tocows milk and of oral food challenge-confirmeddiagnosis from 10 birth cohorts (61).

Different Clinical Presentations of CMA

In a Danish birth cohort, 60% of children withCMA presented with gastrointestinal symptoms,50 to 60% with skin issues, and respiratorysymptoms present in 20 to 30% while 9%developed anaphylaxis (62, 63). In the Norwe-gian cohort noted above, young infants experi-

enced pain (48%), gastrointestinal symptoms(32%), respiratory problems (27%), and atopicdermatitis (4.5%) (53). In the Finnish cohort,presentation symptoms included urticaria(45.76%), atopic dermatitis (89.83%), vomitingand/or diarrhea (51.69%), respiratory symptoms(30.50%), and anaphylaxis (2.54%). The samechildren reacted at oral food challenge withsymptoms of urticaria (51.69%), atopic derma-titis (44.06%), vomiting and/or diarrhea(20.33%), respiratory symptoms (15.25%), andanaphylaxis (0.84%) (52). In the British studyquoted above, infants reacted to oral food

challenges with eczema (33%), diarrhea (33%),vomiting (23.8%), and urticaria in 2 childrenwho immediately reacted to the challenge meal(one with wheeze and the other with excessivecrying) (54). Dutch infants with CMA from thestudy noted above developed gastrointestinal(50%), skin (31%), and respiratory (19%)symptoms (33).

Several other studies have assessed the inci-dence of CMA in populations selected forreferral by other care givers to a tertiary


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institution for specialist assessment of theirsymptoms and therefore requires caution ingeneralizing the results of such studies. As acase in point, in a long-term study of 97children with challenge-confirmed CMA, 21%had atopic dermatitis at the final follow-upevaluation (at 8 years) (62). In another follow-up study of 42 infants with IgE-mediated CMA,

57% of children had developed atopic dermati-tis at the median age of 3.7 years (63).

Thus, CMA appears with GI symptoms in 32to 60% of cases, cutaneous symptoms in 5 to90%, anaphylaxis in 0.8 to 9% of cases. Respi-ratory complaints, including asthma, are notrare. Clearly, in most of the populations studied,there are overlapping presenting symptoms andmultiple symptoms are often confirmed duringchallenge.

CMA in Different Clinical Conditions

Reversing the point of view, milk sensitizationand CMA are reported with different frequenciesin different clinical presentations. In 2184 youngchildren aged 13-24 months with atopic dermati-tis, the frequency of positive serum IgE responsesagainst cows milk protein was 3% (64). Among59 breast-fed children with moderate-severe AD,5 (8,5%) were SPT-positive with milk extracts(65). In a consecutive series with moderate atopiceczema referred to a University-affiliated derma-tology department, SPT showed 16% of infantswith IgE against CMP (66). In a group of infants

and children (mean age 17.6 months) with ADand no other allergic manifestations, 20/54 chil-dren (37%) had a diagnosis of CMA (67). Among90 children with IgE-mediated food allergy, 17%were allergic to cows milk (68). Thus, as reviewedsome years ago, nearly one third of AD childrenhave a diagnosis of CMA according to elimina-tion diet and challenge tests, and about 40-50% ofchildren


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prevalence will largely reflect local factors such asexposure to foods, mode of preparation, andcultural attitudes. As an example, in Israelsesame is the third most frequently implicatedoffending food, probably because of its wide-spread consumption. Among young Australianadults, the major offender was peanut, followedby shrimp, wheat, egg, and milk (44). In Iranian

children CM is the most common offenderidentified during diagnostic provocation chal-lenge (77). Thus, it may be said that the mostrepresentative allergen is a hand-maiden to localcustoms.

Table 3-1. Comparison of the Three Main Food Allergens In Children Studies

75

Country 1st 2nd 3rd

USA Egg Cows milk PeanutsGermany Egg Cows milk Wheat

Spain Egg Cows milk FishSwitzerland Egg Cows milk Peanuts

Israel Egg Cows milk SesameJapan Egg Cows milk Wheat

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Section 4: Allergens of Cows Milk

Overview

The main allergens of cows milk are distrib-uted among the whey and casein fractions.

The whey allergens include:a. Alpha-lactalbumin (Bos d 4): its role in

milk allergy is controversial and prevalencedata across studies vary between 0 and 80% ofpatients reacting to this protein.

b. Beta-lactoglobulin (B o s d 5), the mostabundant cows milk whey protein; it occurs inthe milk of many other species but is notpresent in human milk. Thirteen to 76% ofpatients are found to react to this protein.

c. Bovine serum albumin (Bos d 6): involvedin other allergies such as beef; it accounts for

between 0 and 88% of sensitization events,while clinical symptoms occur in up to 20% ofpatients.

d. Bovine immunoglobulins (Bos d 7): areseldom held responsible for clinical symptomsin CMA.

The casein allergens (collectively known asBos d 8) consist of 4 different proteins (alphas1,alphas2, beta, and kappa casein) which sharelittle sequential homology. Despite this, simul-taneous sensitization to these caseins is fre-quently observed. Patients are more oftensensitized to alpha (100%) and kappa caseins

(91.7%).Of clinical relevance, milk allergens of vari-

ous mammalian species cross-react. The great-esthomologyis among cows, sheepsandgoatsmilks protein as Bos(oxen),Ovis (sheep), andCapra (goat) are genera belonging to theBovidae family of ruminants. Proteins in theirmilks have less structural similarity with thosefrom the Suidae (pig), Equidae (horse anddonkey), and Camelidae (camel and drome-dary) families and also from those of humans.Its noteworthy that the milks of camels and

dromedaries (and human milk) do not containBos d 5.All this is relevant for later consider-ations on formula (section 13).

There is no clear relationship between digest-ibility and protein allergenicity. Milk allergensare known to preserve their biologic activityeven after boiling, pasteurization, ultra-high-temperature processing, or evaporation for theproduction of powdered infant formula. Toobtain hypoallergenic formulas, extensivehydrolysis and further processing, such as heattreatment, ultrafiltration, and application ofhigh pressure are necessary. Attempts have

been made to classify formulas into partial andextensively hydrolyzed products according totheir degree of protein fragmentation, but thereis no agreement on the criteria on which to basethis classification. Nevertheless, hydrolyzedformulas have until now proven to be a usefuland widely used protein source for infantssuffering from CMA (section 12).


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Table 4-2. Characteristics of Alpha-Lactalbumin (Bos d 4)

Parameter Description

Allergen nomenclature Bos d 4

Entry name LALBA_BOVINSynonyms Lactose Synthase B protein

Sequence databases Genbank: M18780

PIR: A27360, LABOSwiss-Prot: P00711

Number of aminoacids 123 residues

Molecular weight 14.2 kDaIsoelectric point 4.8

Involvement in allergicsensitization to cows milk

080% CM allergic subjects75% CM allergic children by SPT

Beta-Lactoglobulin (Bos d 5)

Beta-lactoglobulin (BLG) is the most abundantcows milk whey protein; it occurs in the milk ofmany other mammalian species but is not presentin human milk. Bos d5 belongs to the lipocalinallergen family and is synthesized by the mam-malian gland. Its function is unknown, althoughit may be involved in retinol transport, with whichit readily binds (14). Table 4-3 shows its mainphysical and chemical characteristics. It contains2 internal disulphide bonds and one free-SHgroup. Under physiological conditions, BLGexists as an equilibrium mixture of monomerand dimer forms but, at its isoelectric point, thedimers can further associate to octamers. Thereare 2 main isoforms of this protein in cows milk,the genetic variants A and B, which differ only by2 point mutations at amino acids 64 and 118.

Because it is lacking from human milk, BLG haslong been believed to be the most important cowsmilk allergen. The literature indicates that theprevalence of allergic subjects reacting to thisprotein is between 13 and 76% (15).

Table 4-3. Characteristics of Beta-Lactoglobulin (Bos d 5)


Allergen nomenclature Bos d 5Entry name LACB_BOVIN

Synonyms

Sequence databases Genbank: X14712PIR: S10179, LGBO

Swiss-Prot: P02754Number of aminoacids 162 residues

Molecular weight 18.3 kDa

Isoelectric point 5.135.23 (variants)Involvement in allergic

sensitization to cows milk

1376% CM allergic subjects

73.7% CM allergic children by SPT

Bovine Serum Albumin (Bos d 6)

Bovine serum albumin (BSA) is the main proteinof whey. It can bind water, fatty acids, hormones,bilirubin, drugs, and Ca2+, K+, and Na+. Its

main function is the regulation of the colloidalosmotic pressure in blood (15). The tertiarystructure of BSA is stable, and its 3-dimensionalconformation is well documented. The protein isorganized into 3 homologous domains (I to III)and consists of 9 loops connected by 17 covalentdisulphide bridges. Most of the disulphide bondsare well protected in the core of the protein and are

not readily accessible to the solvent. Table 4-4shows some of its characteristics.

Table 4-4. Characteristics of Bovine Serum Albumin (Bos d 6)



Entry name ALBU_BOVINSynonyms BSA

Sequence databases Genbank: M73993

PIR: A38885, ABBOSSwiss-Prot: P02769

Number of aminoacids 583 residues

Molecular weight 67.0 kDa

Isoelectric point 4.95.1Involvement in allergic


088% CM allergic subjects

62.5% CM allergic children

by immunoblotting

Bos d6 is involved not only in milk allergy butalso in allergic reactions to beef (15). It inducedimmediate allergic symptoms (lip edema, urti-caria, cough, and rhinitis) in children allergic tobeef who received the protein in a double-blindplacebo-controlled food challenge (DBPCFC)(16). The prevalence of patients with cows milk

who react to this protein ranges from 0 to 88%,while clinical symptoms may be found in as manyas 20% of patients (17).

Immunoglobulins (Bos d 7)

Bovine immunoglobulins are present in blood,tissues, fluids, and secretions such as milk. Somecharacteristics of the bovine IgG are shown inTable 4-5. Bovine IgG seldom cause clinicalsymptoms in CMA (18).

Table 4-5. Characteristics of Cow's Milk Immunoglobulin G



Entry name

Synonyms IgGSequence databases

Number of aminoacids Molecular weight 160.0 kDa

Isoelectric point Involvement in allergic


Frequency unknown


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Caseins (Bos d 8)

Most of the casein aggregates as colloidal parti-cles (the casein micelle) and its biologic functionis to transport calcium phosphate to the mam-malian newborn. More than 90% of the calciumcontent of skim milk is attached to or included incasein micelles. Caseins consist of 4 different

proteins (alphas1, alphas2, beta, and kappa case-in) with little sequential homology. Anothergroup, the gamma caseins, are present in verylow quantities in milk and are by-products ofbeta casein proteolysis. A distinguishing featureof all caseins is their low solubility at pH 4.6;another common characteristic is that caseins areconjugated proteins, most with phosphate groupsesterified to the amino acid serine. Caseinscontain no disulphide bonds, while the highnumber of proline residues causes pronouncedbending of the protein chain, which inhibits the

formation of close-packed, ordered secondarystructures. Characteristics ofBos d 8are reportedin Table 4-6.

Table 4-6. Allergenic Characteristics of Caseins

Parameter as1-casein as2-casein b-casein j-casein

Allergen nomenclature Bos d 8 Bos d 8 Bos d 8 Bos d 8

E nt ry n ame CAS1_BOVI N CAS2 _BOVIN CASB_BOVIN CASK_BOVIN

Synonyms None None None None

Sequence databases G X00564/

M33123

G M16644 G M16645/

X06359

G X14908/

M36641

P S22575/

KABOSB

P JQ2008/

KABOS2

P I45873/

KBBOA2

P S02076/

KKBOB

S P02662 S P02663 S P02666 S P02668

No. aminoacids 199 207 209 169

Molecular weight 23.6 kDa 25.2 kDa 24.0 kDa 19.0 kDa

Isoelectric point 4.95.0 5.25.4 5.15.4 5.45.6

Involvement in allergic

sensitization to cows

milk-1. whole casein

65100% 65100% 65100% 65100%

Involvement in allergic

sensitization to cows

milk-2. single casein

54% 54% 39% NT

100% 100% 66.7% 91.7%

Despite the poor sequence homology betweenproteins of the casein fraction, poly-sensitizationto many caseins is frequently observed; this may

be because of cross-sensitization through sharedor closely related epitopes (8). Patients are almostalways sensitized to alpha (100%) and kappacaseins (91.7%) (19).

Cross-Reactivity Between Milk Proteins from Different AnimalSpecies

Cross-reactivity occurs when 2 proteins share partof their amino acid sequence (at least, thesequence containing the epitopic domain) or when

the 3-dimensional conformation makes 2 mole-cules similar in binding capacity to specific anti-bodies. In general, cross-reactivity betweenmammalian proteins reflects the phylogeneticrelationships between animal species and evolu-tionary conserved proteins that are often cross-reactive (20). Table 4-7 shows the sequencesimilarity (expressed in percentages) between milk

proteins from different mammalian species (22).

Table 4-7. Sequence Homology Between Mammalian Milk Proteins (in Per-

centage, Relative To Cows Milk Proteins)

Protein Goat Ewe B uffalo Sow M are Donk ey Drom edary H uman

ALA 95.1 97.2 99.3 74.6 72.4 71.5 69.7 73.9

BLG 94.4 93.9 96.7 63.9 59.4 56.9 Absent Absent

Serum alb. 92.4 79.9 74.5 74.1 76.6

a s1 CAS 87.9 88.3 47.2 42.9 32.4

a s2 CAS 88.3 89.2 62.8 58.3

b CAS 91.1 92.0 97.8 67.0 60.5 69.2 56.5

j CAS 84.9 84.9 92.6 54.3 57.4 58.4 53.2

The greatest homology is between cows,sheeps and goats milk proteins as Bos (oxen),Ovis (sheep), and Capra (goat) that are generabelonging to the Bovidae family of ruminants.The proteins in their milks consequently have lessstructural similarity with those from the Suidae(pig), Equidae (horse and donkey), and Cameli-dae (camel and dromedary) families and alsowith those in human milk. It is noteworthy thatthe milks of camels and dromedaries (as well ashuman milk) do not contain BLG.

However, phylogeny does not explain every-thing. In 1996, a clinical trial in France showed

that 51/55 children with cows milk allergytolerated goats milk for periods ranging from8 days to 1 year (22), but subsequent researchshowed that other subjects allergic to cows milkdid not tolerate goats and sheeps milks (23).This is consistent with the pattern of IgE cross-reactivity shown by several independent studiesin vitro, for instance the cross-reactivity betweenmilk proteins from different mammalian species(including goats milk) (24). Furthermore, selec-tive allergy to goats and sheeps milk but not tocows milk has also been reported in 28 older

children with severe allergic reactions, includinganaphylaxis. In one study, IgE antibodies recog-nized caseins from goats milk but cows milkcaseins were not or scarcely recognized (25). Thisis not an isolated finding (26, 27), however, and acase report of an adult with goats milk allergywithout CMA found specific IgE to caprineALA (28). Finally, allergy to sheeps milk canalso evolve into allergy to cows milk (29).Mares and donkeys milks have proved some-times useful to some patients (3032), but


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uncertainties remain about chemical compositionand hygienic control. The same considerationsapply to Camellidae (camel and dromedaries)milks, which could represent an alternative tocows milk for allergic subjects because of theirlow sequence homology with cows milk and theabsence of BLG, if problems related to avail-ability and technological processing to avoid new

sensitization (33).Figure 4-1 shows the electrophoretic patterns

of milk from several mammalian species. Thepronounced similarity is evident for milk fromcows, goats, and sheep, while the protein profilesof mares, donkeys, and camels milks presentsome specificities. The low cross-immunoreactiv-ity of horse/donkey milk and the absence of BLGin camels and human milk is easily visible inimmunoblots using antibodies against bovineBLG.

Structural Modifications and Cows Milk Protein Allergenicity

The 3-dimensional structure of most antigenicproteins is unknown, even where the amino acidsequence has been precisely identified, becausethe conformation is not immutable but is influ-enced by the surrounding environment. Thisproblem is even more significant for milk proteinssince their organization is complex and thepresence of micelles in caseins makes their inves-tigation difficult. We discuss here the structuralmodifications brought about by gastrointestinaldigestion or technological treatments and their

role in allergenic potential where this is known orcan be inferred.

Digestibility and Cows Milk Protein Allergenicity

Food proteins are digested by gastrointestinalenzymes; it is generally believed that proteinsresistant to proteolysis are the more powerfulallergens. However, it has been shown that thereis no clear relationship between in vitro digest-ibility and protein allergenicity (34). Caseins arethought to be easily digestible, but they coagulatein an acidic medium (at gastric pH). Acidification

increases the solubility of minerals, so that thecalcium and phosphorus contained in the mi-celles gradually become soluble in the aqueousphase. As a result, casein micelles disintegrateand casein precipitates. Whey proteins are moresoluble in saline solution than caseins andtheoretically they should be more easily digestedby proteases that work in aqueous medium.However, the correlation between water solubil-ity and digestibility is not linear. Caseins aredigested faster than whey proteins by the com-

monest food-grade enzymes (eg, pepsin, trypsin,and thermolysin) (35).

Although BSA is very soluble in water and richin amino acids broken-down by gastrointestinalenzymes, it is also relatively resistant to diges-tion. Sequential epitopes were unaffected for atleast 60 minutes when BSA was digested withpepsin (36). Its 9 loops are maintained by

disulphide bonds, which are not easily reducedunder physiological conditions, and slow thefragmentation of BSA into short peptides thathave decreased antigenic activity.

Heating and Cows Milk Protein Allergenicity

Cows milk is only marketed after it has beensubjected to technological process, usually pas-teurization, which reduces potential pathogenload (70-80C for 15-20 seconds). Ultra-high-temperature (UHT) processing with flash heating

(above 100

C for a few seconds), evaporation forthe production of powdered infant formula (dryblending or wet mixing-spray drying process)have a minor or no effect on the antigenic/allergenic potential of cows milk proteins. Boil-ing milk for 10 minutes reduces the SPT responsein patients who react to BSA and beta-lactoglob-ulin, whereas wheal diameter remains the same inthose sensitized to caseins (37). Comparativestudies have shown no difference in antigenicitybetween raw and heated milks (38), however, andin some cases the aggregation of new proteinpolymers capable of binding specific IgE have

been demonstrated. After boiling BSA at 100Cfor 10 minutes, dimeric, trimeric, and higherpolymeric forms increased, and all maintainedtheir IgE-binding properties (39).

The persistence of allergenicity in heat-treatedmilk is clinically confirmed by the fact that in

Fig. 4-1 SDS-PAGE of mammalian milk samples. Hcas =human casein; HLA = human lactalbumin; Lfe = humanlactoferrin; a-cas = bovine alpha casein; b-cas = bovinebeta casein; BLG = bovineb-lactoglobulin; ALA = bovinea-lactalbumin.


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some children CMA develops after the ingestionof heat-treated milk. Furthermore, heating pro-cesses can only modify conformational epitopes,which might lose their binding capacity tospecific IgE antibody, while sequential epitopesmaintain their allergenic potential even afterheating (40). Milk proteins contain both types ofepitopes and, even though a slight reduction of

antigenicity can be observed with whey proteins,insignificant alterations in binding properties arereported with caseins. To complicate the picture,vigorous heating (such as that used for certainsterilization processes [121C for 20 minutes])but also the less drastic pasteurization process,have also been shown to enhance some aller-genic characteristics (41). Furthermore, milkproteins can be oxidized during industrial treat-ment, resulting in the formation of modified/oxidized amino acid residues, particularly inBLG, which may be responsible for the devel-

opment of new immunologically reactivestructures (42).

Technological Treatments and Cows Milk Protein Allergenicity

Hypoallergenic formulas can be prepared byhydrolysis and further processing, such as heattreatment, ultrafiltration, and application ofhigh pressure. Attempts have been made toclassify formulas into partial and extensivelyhydrolyzed products according to the degree ofprotein fragmentation, but there is no agreementon the criteria on which to base this classifica-

tion (see section CM hydrolyzed formula).Nevertheless, hydrolyzed formulas have untilnow proved a useful and widely used proteinsource for infants suffering from CMA. Becauseundigested protein can still be present as residueat the end of proteolysis (43), further processingis necessary in combination with e enzymatictreatment. Another attempt to eliminate antige-nicity involves the use of proteolysis combinedwith high pressure. Different authors haveshown increased fragmentation of BLG if pro-teolysis occurs after or during the application ofhigh pressure (44). The partial ineffectiveness of

proteolysis under ordinary atmospheric condi-tions may be because of the inability of enzymesto reach epitopes that are less exposed. Heattreatment is also often combined with proteol-ysis to unfold the protein and modify the 3-dimensional structure of conformational epi-topes. However, thermal denaturation can alsocause the formation of aggregates with greaterresistance to hydrolytic attack, as is the casewith BLG (45).

References, Section 41. Bahna SL. Cows milk allergy versus cow milk intol-

erance. Ann Allergy Asthma Immunol. 2002: 89 (Suppl1): 5660.

2. Vesa TH, Marteau P, Korpela R. Lactose intoler-ance. J Am Coll Nutrit. 2000: 19: 165S175S.

3. Shukla H. Lactose Intolerance in health and disease.Nutr Food Sci. 1997: 2: 6670.

4. Swallow DM, Hollox EJ The Metabolic and

Molecular Bases of Inherited Disease. New York:McGraw-Hill; 2001.

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

6. Johansson SG, Bieber T, Dahl R. Revised nomen-clature for allergy for global use: report of theNomenclature Review Committee of the World AllergyOrganization, 2003. J Allergy Clin Immunol. 2004: 113:832836.

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

8. WalJ-M. Cows milk proteins/allergens. Ann AllergyAsthma Clin Immunol. 2002: 89 (Suppl 9): 310.

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

10. Chapman MD, Pomes A, Breiteneder H, FerreiraF. Nomenclature and structural biology of allergens. JAllergy Clin Immunol. 2007: 119: 414420.

11. McKenzie HA. Alpha-lactalbumins and lysozymes.EXS. 1996: 75: 365409.

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

13. Besler M, Eigenmann P, Schwartz RH. InternetSymposium on Food Allergens. 2002: 4: 19.

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

15. Restani P, Ballabio C, Tripodi S, Fiocchi A. Meatallergy. Curr Opin Allergy Clin Immunol. 2009: 9:265269.

16. FiocchiA, RestaniP, RivaE, QualizzaR, BruniP,RestelliAR, GalliCL. Meat allergy: I - Specific IgEto BSA and OSA in atopic, beef-sensitive children. JAm Coll Nutr. 1995: 14: 239244.

17. Martelli A, De Chiara A, Corvo M, Restani P,Fiocchi A. Beef allergy in children with cows milkallergy. Cows milk allergy in children with beefallergy. Ann Allergy, Asthma & Immunology. 2002:89: S38S43.

18. Bernhisel-Broadbent J, Yolken RH, Sampson HA.Allergenicity of orally administered immunoglobulinpreparations in food-allergic children. Pediatrics. 1991:

87: 208214.19. RestaniP, Velona T,PlebaniA, UgazioAG, Poiesi

C, Muraro A, Galli CL. Evaluation by SDS-PAGEand immunoblotting of residual antigenicity inhydrolysed protein formulas. Clin Exp Allergy. 1995:25: 651.

20. Spitzauer S. Allergy to mammalian proteins: at theborderline between foreign and self? Int Arch AllergyImmunol. 1999: 120: 259269.

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23. Bellioni-Businco B, Paganelli R, Lucenti P,Giampietro PG,PerbornH,BusincoL. Allergenicityof goats milk in children with cows milk allergy.J Allergy Clin Immunol. 1999: 103: 11911194.

24. Restani P, Gaiaschi A, Plebani A, Beretta B,Cavagni G, et al. Cross reactivity between milk pro-teins from different animal species. Clin Exp Allergy.1999: 29: 9971004.

25. Ah-Leung S, Bernard H, Bidat E, Paty E, RanceF, Scheinmann P. Allergy to goat and sheep milkwithout allergy to cows milk. Allergy. 2006: 61:13581365.

26. Bidat E, Rance F, Baranes T, Goulamhoussen S.Goats milk and sheeps milk allergies in children in theabsence of cows milk allergy. Rev Fr Allergol Immu-nol Clin. 2003: 43: 273277.

27. Alvarez MJ, Lombardero M. IgE-mediated ana-phylaxis to sheeps and goats milk. Allergy. 2002: 57:10911092.

28. Tavares B, Pereira C, Rodrigues F, Loureiro G,Chieira C. Goats milk allergy. Allergol Immunopa-thol. (Madr) 2007: 35: 113116.

29. FiocchiA, DecetE, MirriGP,TravainiM, RivaE.Allergy to ewes milk can evolve into allergy to cowsmilk. Allergy. 1999: 54: 401402.

30. VitaD, PassalacquaG,DiPasquale G,CaminitiL,Crisafulli G, Rulli I. Asss milk in children withatopic dermatitis and cows milk allergy: crossovercomparison with goats milk. Pediatr Allergy Immu-nol. 2007: 18: 594598.

31. Monti G, Bertino E, Muratore MC, Coscia A,Cresi F, Silvestro L. Efficacy of donkeys milk intreating highly problematic cows milk allergic chil-dren: an in vivo and in vitro study. Pediatr Allergy

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D,Alabrese L, Iacono G. Intolerance to hydrolysedcows milk proteins in infants: clinical characteristicsand dietary treatment. Clin Exp Allergy. 2000: 30:15971603.

33. RestaniP, BerettaB, FiocchiA, BallabioC, GalliCL. Cross-reactivity between mammalian proteins.Ann Allergy Asthma Immunol. 2002: 89 (6 Suppl 1):1115.

34. Fu TJ, Abbott UR, Hatzos C. Digestibility of foodallergens and nonallergenic proteins in simulated gas-tric fluid and simulated intestinal fluid-a comparativestudy. J Agric Food Chem. 2002: 50: 71547160.

35. Bonomi F, Fiocchi A, Frokiaer H, et al. Reduction

of immunoreactivity of bovine beta-lactoglobulin uponcombined physical and proteolytic treatment. J DairyRes. 2003: 70: 5159.

36. Beretta B, Conti A, Fiocchi A, Gaiaschi A, GalliCL, et al. Antigenic determinants of bovine serumalbumin. Intern Arch Allergy Immunol. 2001: 126:188195.

37. NorgaardA, BernardH, Wal JM, PeltreG, SkovPS,PoulsenLK, Bindslev-JensenC. Allergenicity ofindividual cow milk proteins in DBPCFC-positive milkallergic adults. J Allergy Clin Immunol. 1996;97(Pt3):237.

38. Werfel SJ, Cooke SK, Sampson HA. Clinical reac-tivity to beef in children allergic to cows milk. J Al-lergy Clin Immunol. 1997: 99: 293300.

39. Restani P, Ballabio C, Cattaneo A, Isoardi P,Terracciano L, Fiocchi A. Characterization of bo-vine serum albumin epitopes and their role in allergicreactions. Allergy. 2004: 59 (Suppl 78): 2124.

40. Sampson HA. Update on food allergy. J Allergy ClinImmunol. 2004: 113: 805819.

41. Roth-Walter F, Berin MC, Arnaboldi P, Esca-lante CR, Dahan S, Rauch J, Jensen-Jarolim E,Mayer L. Pasteurization of milk proteins promotesallergic sensitization by enhancing uptake throughPeyers patches. Allergy. 63: 882890.

42. Fenaille F, Parisod V, Tabet J-C, Guy PA. Car-bonylation of milk powder proteins as a consequence ofprocessing conditions. Proteomics. 2005: 5: 30973104.

43. RestaniP, Velona T,PlebaniA, UgazioAG, PoiesiC, Muraro A, Galli CL. Evaluation by SDS-PAGEand immunoblotting of residual antigenicity in hy-drolysed protein formulas. Clin Exp Allergy. 1995: 25:651658.

44. Penas E, Restani P, Ballabio C, Prestamo G,Fiocchi A, Gomez R. Evaluation of the residual

antigenicity of dairy whey hydrolysates obtained bycombination of enzymatic hydrolysis and high-pres-sure treatment. J Food Prot. 2006: 69: 17071712.

45. Restani P, Ballabio C, Fiocchi A Milk allergens:chemical characterization, structure modifications andassociated clinical aspects. In: Pizzano R ed. Immu-nochemistry in dairy research. Research Signpost,Kerala. 2006;6176.

Section 5: Immunological Mechanisms of Cows MilkAllergy

Overview

CMA designates objectively reproduciblesymptoms or signs initiated by exposure tocows milk protein at doses tolerated bynormal persons. CMA can be either anti-body-mediated or cell-mediated; occasionallyboth mechanisms may be involved. CMA maybe mediated by any of the 4 basic types ofimmunologic reactions, as outlined by Gelland Coombs: 1) Type I or IgE-mediatedhypersensitivity, 2) Type II (cytotoxic reac-tions), 3) Type III (Arthus-type reactions),and 4) Type IV (delayed T cell reactions).Type I reactions are the best characterized andrepresent the classic immediate allergic reac-tions. The 3 other types, collectively describedas non-IgE-mediated allergy, are less wellunderstood.

The suppression of adverse immune re-sponses to nonharmful ingested food antigensis termed oral tolerance. Ingested milk pro-teins are normally degraded by gastric acid


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foods (5) Dual sugar intestinal permeabilitystudies (lactulose/mannitol) showed that inbreast-fed infants with atopy, gut barrier func-tion improved when breast-feeding was stoppedand hypoallergenic formula started (6).

Oral Tolerance

The mucosa allows nutrients to be transferredfrom the intestinal lumen to the systemic circu-lation, while protecting against pathogens byinducing immune responses. Any down-regula-tion of immune responses to nonharmful in-gested antigens is termed oral tolerance (7).Normally, mature lymph node lymphocytesbecome hyporesponsive after oral administrationof these antigens (8).

Ingested milk proteins are degraded and theirconformational epitopes are destroyed by gastricacid and luminal digestive enzymes, which often

results in the destruction of immunogenic epi-topes. In animal models, disrupting the processof digestion can inhibit milk tolerance and leadto hypersensitivity. Untreated bovine serumalbumin (BSA) is immunogenic when adminis-tered to mice by means of ileal injection, butadministering a peptic digest of the protein in thesame manner results in immune tolerance (9).

Regulatory events after mucosal exposure toantigen have not been well characterized andremain controversial. In general, the acquisitionof tolerance to milk is seen as a TH1-skewedresponse, which on the one hand may prevent

harmful mucosal immune reactions but on theother may contribute to adverse responses in asusceptible individual. The process starts with thecontact of milk allergens with the intestinalmucosa. Here they interact with mucosal T andB cells either directly or through antigen-pre-senting cells (APCs): macrophages, dendriticcells, or microfold cells (M cells). T cell recogni-tion 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 folliclesmigrate first via the lymphatic system and then

via the circulation to any of several target organsincluding the gastrointestinal tract, the respira-tory system, the skin, and the central nervoussystem, a process referred to as homing. Iftolerance is not achieved, T and B cells willactivate at a homing site upon contact with theirspecific food antigen and release their cytokines,vasoactive peptides and antibodies, giving rise toan inflammatory reaction in the affected organand resulting in the clinical manifestations offood hypersensitivity (10).

In this context, dendritic cells play a centralrole in taking up milk proteins and migrating tothe draining mesenteric lymph nodes, where theyinduce regulatory CD4 T-cell differentiation. Theprimary mechanisms by which tolerance may bemediated include deletion, anergy, suppression,ignorance, and apoptosis of T-cells (11).

The balance between tolerance (suppression)

and sensitization (priming) depends on severalfactors, such as: 1) genetic background, 2) natureand dose of antigen, 3) frequency of administra-tion, 4) age at first antigen exposure, 5) immu-nologic status of the host, 6) antigentransmission via breast milk, and others.

Overall, there is evidence in rodents thatmultiple low-dose feeds are likely to induceregulatory cytokines (eg, TGF-b, IL-10, IL-4)in part secreted by CD4+ CD25+ T-regulatorycells. Despite the powerful suppressive effects oforal autoantigen exposure observed in experi-

mental models of autoimmune diseases (includ-ing bystander suppression), their translation intoclinical trials of autoimmune diseases has not yetyielded the expected beneficial results. The samecan be said for CMA (12).

In normal individuals with tolerance, systemicand secretory food-specific IgA antibodies aregenerally absent, indicating that mucosal IgAproduction is regulated similarly to that ofsystemic immunity (13). However, mucosal IgAresponse to foreign antigens remains active (14).In population surveys, more allergic sensitizationwas seen in subjects with an IgA level at the

lower end of the normal range (1517). Thesignificance of IgM, IgG, and IgG subclassantibodies (eg, the role of IgG4) in food allergyis less well understood and remains controversial.It has long been known that milk-specific IgMand IgG antibodies are produced after single orrepeated feedings of relatively large doses of milkproteins in both healthy and allergic persons (18).

Thus, unresponsiveness of the immune systemto milk antigens (oral tolerance) is believed toinvolve the deletion or switching off (anergy) ofreactive antigen-specific T cells and the produc-tion of regulatory T cells (Treg) that suppress

inflammatory responses to benign antigens (19,20).

Innate Immunity and Tolerance Development

The innate immune system has the ability tomodulate adaptive immune responses to foodproteins. In this process, dendritic cells (DC) playa central role (21). In addition, TLR directlyinteract with innate immune cells. TLR recognizefood antigens, and specific bacterial surface


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markers, so-called PAMP (21). However, theexact mechanisms by which TLR influence Tregresponses are incompletely understood. Regula-tory T-cells are involved in the control ofimmune responses to food antigens via theproduction of tolerogenic cytokines, includingIL-10 and TGF-b (22, 23). Intestinal microbiotamay have a diverse effect on TLR and immune

responses. Several types of intestinal Bifidobac-teria have been shown to promote tolerogenicimmune responses. The type of gastrointestinalmicrobiota of the newborn infant is crucial in thiscontext. The probiotic effects of complex oligo-saccharides in human milk promote the estab-lishment of a bididogenic microbiota which, inturn, induces a milieu of tolerogenic immuneresponses to foods. Several probiotic bacterialstrains have been shown to have similar proper-ties. For example, Lactobacillus paracaseiinhib-its TH1 and TH2 cytokine production, and

induces CD4

(+)

T cells to produce TGF- andIL-10, that is, induces a tolerogenic response(24). It appears possible that the recent decreasein exposure to early childhood infections andharmless environmental microorganisms in thewesternized environment has contributed to anincrease in T-cell dysregulatory disorders andautoimmunity (25, 26).

Dysfunctional Tolerance

CMA is believed to result from the failure todevelop normal tolerogenic processes or their

later breakdown. In the case of IgE-mediatedCMA, a deficiency in regulation and a polariza-tion of milk-specific effector T cells toward type-2T helper cells (TH2) both lead to B-cell signalingto produce milk protein-specific IgE (27, 28).Non-IgE-mediated reactions may be because ofTH1-mediated inflammation (29). DysfunctionalTregcell activity has been identified as a factor inboth allergy mechanisms (30). Additionally, theinduction of tolerance in children who haveoutgrown their CMA has been shown to beassociated with the development of Tregcells (31,

32). Much research is currently focused onmanipulating the activity of dendritic cells (spe-cialized antigen-presenting cells important inprogramming immune responses) to induce Tregcells and/or to redress TH1/TH2 imbalances topromote tolerance to allergenic foods.

Allergen Exposure and Sensitization

The events after allergen exposure in the gut arecomplex. Digestion (33) and cooking preparation

(34, 35) slightly modifies the allergenicity ofbovine proteins. Proteins that are not digestedand processed in the lumen of the gut will comein contact with the epithelium and mucosalimmune system in various ways. In the gut,dendritic cells can sample antigens by extendingprocesses through the epithelium and into thelumen. M cells that overlie Peyers patches can

take up particulate antigens and deliver them tosubepithelial dendritic cells. Soluble antigenspossibly cross the epithelium through transcellu-lar or paracellular routes to encounter T cells ormacrophages in the lamina propria. Dietaryproteins that escape proteolysis in the gut canbe taken up by intestinal epithelial cells. Theepithelial cells can act as nonprofessional APCsand can present antigen to primed T cells. Thus,food allergens (and microorganisms and nonvi-able particulate antigens) reach CD4+ andCD8+ T cells in the Peyers patch, resulting in

active immune responses (36). Early gastrointes-tinal encounters with relatively large doses ofsoluble protein almost always induce tolerance(37). Data from rodent models suggest that theeffect of milk allergen exposure on the hostdepends on many factors, including:

a. Nature and dose of the antigenb. Efficiency of digestionc. Immaturity of the hostd. Rate of absorption of milk proteinse. Antigen processing in the gutf. The immunosuppressive milieu of the Peyer

patch (38).

All of these factors can favor the induction ofperipheral tolerance to dietary proteins ratherthan systemic hypersensitivity. In this context,the presence of commensal flora in the gut canlower the production of serum milk-specific IgEduring the primary immune response; also, IgEproduction persists longer in germ-free mice.Conversely, the absence of gut microbiota sig-nificantly increases the milk-specific immuneresponse in mice (39). This raises the possibilityof prevention and treatment of milk allergy

through the manipulation of the gastrointestinalflora.

Milk Allergy

An effect of dysfunctional tolerance, milkallergy designates objectively reproduciblesymptoms or signs initiated by exposure tocows milk at a dose tolerated by normalpersons (40). The term CMA is appropriate


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when specific immunologic mechanisms havebeen demonstrated (see definitions in intro-ductory section). Milk allergy can be eitherantibody-mediated or cell-mediated, or occa-sionally both may be involved. If IgE isinvolved in the reaction, the term atopic foodallergy is appropriate. If immunologic mecha-nisms other than IgE are predominantly in-

volved, the term non-IgE-mediated foodallergy should be used. All other reactionsshould be regarded to as nonallergic foodhypersensitivity (41).

Enhanced immune-mediated reactivity maycome about though any, or a combination of,the 4 basic types of immunologic reactionsoutlined by Gell and Coombs:

a. Type I or IgE-mediated hypersensitivity leadsto immediate symptoms, such as urticaria,angioedema and/or other anaphylactic reac-

tion.b. In type II (cytotoxic) reactions, the antigenbinds to the cell surface and the presence ofantibodies (IgG, IgM, or IgA) disrupts themembrane, leading to cell death.

c. In type III (Arthus-type) reactions, antigen-antibody-complement immune complexes(IgG, IgM, IgA, and IgE antibodies) gettrapped in small blood vessels or renalglomeruli.

d. Type IV (delayed) reactions are mediated bysensitized T lymphocytes.

Type I reactions are the best understood, andthey are often referred to as the most commonand classic allergic reactions. The 3 other types,collectively described as non-IgE-mediated al-lergy, are more difficult to investigate and henceless well understood. In an individual, severaltypes of immune responses may be activated,although IgE-mediated reactions are more usu-ally 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 diag-nosed. Since the onset of symptoms is rapid,occurring within minutes to an hour after aller-gen exposure, IgE-mediated allergy is oftenreferred to as immediate hypersensitivity. (42)It occurs in 2 stages. The first, sensitization,occurs when the immune system is aberrantlyprogrammed to produce IgE antibodies to milkproteins. These antibodies attach themselves tothe surface of mast cells and basophils, arming

them with an allergen-specific trigger. Subse-quent exposure to milk proteins leads to acti-vation when the cell-associated IgE binds theallergenic epitopes on the milk proteins andtriggers the rapid release of powerful inflamma-tory mediators.

IgE-mediated, acute onset CM allergies canaffect several target organs: the skin (urticaria,

angioedema), respiratory tract (rhinitis/rhinor-rhea, asthma/wheeze, laryngoedema/stridor),gastrointestinal tract (oral allergy syndrome,nausea, vomiting, pain, flatulence, and diarrhea),and/or the cardiovascular system (anaphylacticshock) (43, 44). Life-threatening anaphylacticreactions to cows milk may occur, but arefortunately rare (45). Since reactions to cowsmilk proteins can occur on contact with the lipsor mouth, strategies to reduce allergenicity byimproving protein digestibility in the gut areunlikely to be effective for all allergic individuals.

Simple diagnostic procedures, such as skin-pricktests (SPT) and specific serum IgE determina-tions (immuno-CAP), can be used to identifyindividuals with IgE-mediated CMA, althougheither of these tests can produce false-positiveresults (46). Food elimination and challengetesting are sometimes required to confirm milkallergy, and double-blind, placebo-controlled,food challenge (DBPCFC) testing remains thegold standard for diagnosis. IgE-mediated CMAmay occur in neonates on first postnatal expo-sure to the food (47). IgE-mediated reactionsaccount for about half of the CMA cases in

young children (48), but are rare in adults (49,50). In contrast to adults, atopic CMA inchildhood (often a part of the allergic march)resolves in more than 85% of cases (51, 52).

Non-Ige-Mediated CMA (DELAYED HYPERSENSITIVITY)

A significant proportion of infants and themajority of adults with CMA do not havecirculating milk protein-specific IgE and shownegative results in skin prick tests and serum IgEdeterminations (immune-CAP) (53, 54). Thesenon-IgE-mediated reactions tend to be delayed,

with the onset of symptoms occurring from1 hour to several days after ingestion of milk.Hence, they are often referred to as delayedhypersensitivity. As with IgE-mediated reac-tions, a range of symptoms can occur, but aremost commonly gastrointestinal or cutaneous(55). The gastrointestinal symptoms, such asnausea, bloating, intestinal discomfort, and diar-rhea, mimic many symptoms of lactose intoler-ance and may lead to diagnostic mislabeling.Anaphylaxis is not a feature of non-IgE mediated


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mechanisms. IgE- and non-IgE-mediated reac-tions are not mutually exclusive and reactions tomilk can involve a mixture of immunologicmechanisms.

The precise immunologic mechanisms of non-IgE-mediated CMA remain unclear. A numberof mechanisms have been suggested, includingTH1-mediated reactions (Fig. 5-1) (5663), the

formation of immune complexes leading to theactivation of complement (64, 65), or T-cell/mastcell/neuron interactions inducing functionalchanges in smooth muscle action and intestinalmotility (1, 66, 67). A necessarily incompletepicture of such mechanisms indicates that T cellsact through secretion of cytokines such as IL-3,IL-4, IL-5, IL-13, and GM-CSF, activatingeosinophils, mastocytes, basophils, and macro-phages. Macrophages, activated by CM proteinallergens by cytokines, are able to secrete in turnvasoactive mediators (PAF, leukotriens) and

cytokines (IL-1, IL-6, IL-8, GM-CSF, TNF-a)that are able to increase the cellular phlogosis.This involves epithelial cells, which release cyto-kines (IL-1, IL-6, IL-8, IL-11, GM-CSF), chemo-kines (RANTES, MCP-3, MCP-4, eotaxin) andother mediators (leukotrienes, PGs, 15-HETE,endothelin-1). This mechanism results in chroniccellular inflammation (at GI, cutaneous, andrespiratory levels) and ultimately in CMA symp-toms. When

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