Les laits

% identité par rapport au lait de vache
	alpha lactalbumine	béta lactoglobuline	alpha caséines	bêta caséines	kappa caséines
femme	75	(absence)
chèvre	95	94	89	91	84
brebis	94	93	87	91	84
bufflone	75	98
jument	75	45-60

[1] - Eigenmann PA. The spectrum of cow’s milk allergy. Pediatr Allergy Immunol 2007;18:265-271

Childhood cow's milk allergy is a diagnosis encompassing various syndromes. Antigen-immunoglobulin E (IgE) antibody interaction is classically involved in mast cell degranulation in IgE-mediated food allergy, while non-IgE mediated cow's milk allergy is mostly mediated by cellular mechanisms. The diagnosis of cow's milk allergy largely relies on a good knowledge of the clinical expression of the disease. In this educational review series, we describe three cases of cow's milk allergy, first a 7-yr-old girl with persisting IgE-mediated cow's milk allergy, second a 8-month-old boy with cow's milk induced flares of atopic dermatitis, and third a 6-yr-old boy with sheep and goat milk allergy, in the absence of cow's milk allergy. The cases are discussed and summarized with more general recommendations for the clinical management of cow's milk allergy.

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[2] - Skripak JM, Matsui EC, Mudd K, Wood RA. The natural history of IgE-mediated cow's milk allergy. J Allergy Clin Immunol 2007;120:1172-1177

BACKGROUND: Cow's milk allergy (CMA) is the most common food allergy in infants and young children, affecting 2% to 3% of the general population. Most studies have shown the prognosis of developing tolerance to cow's milk to be good, with most outgrowing their allergy by age 3 years . OBJECTIVE: To define the natural course of CMA and identify the factors that best predict outcome in a large referral population of children with CMA . METHODS: Clinical history, test results, and final outcome were collected on 807 patients with IgE-mediated CMA. Patients were considered tolerant after they passed a challenge or experienced no reactions in the past 12 months and had a cow's milk IgE (cm-IgE) level <3 kU/L . RESULTS: Rates of resolution were 19% by age 4 years, 42% by age 8 years, 64% by age 12 years, and 79% by 16 years. Patients with persistent allergy had higher cm-IgE levels at all ages to age 16 years. The highest cm-IgE for each patient, defined as peak cm-IgE, was found to be highly predictive of outcome (P < .001). Coexisting asthma (P < .001) and allergic rhinitis (P < .001) were also significant predictors of outcome . CONCLUSION: The prognosis for CMA in this population is worse than previously reported. However, some patients developed tolerance during adolescence, indicating that follow-up and re-evaluation of CMA patients is important in their care. cm-IgE level is highly predictive of outcome. CLINICAL IMPLICATIONS: The increasing potential for persistence of CMA, along with cm-IgE level's effect on prognosis, should be considered when counseling families regarding expected clinical course.

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[3] - Järvinen KM, Chatchatee P. Mammalian milk allergy: clinical suspicion, cross-reactivities and diagnosis. Curr Opin Allergy Clin Immunol 2009;9:251-258

PURPOSE OF REVIEW: Cow's milk allergy affects 2-3% of young children, the economic impact of which necessitates search for simple diagnostic tools and affordable milk substitutes. This review examines recent studies on the diagnosis of cow's milk allergy as well as on the allergenicity of milk from other mammalian species. RECENT FINDINGS: Resolution of symptoms during strict milk avoidance and their re-appearance during the double-blind, placebo-controlled milk challenge remains the gold standard for the diagnosis of cow's milk allergy. Allergic eosinophilic esophagitis/gastroenteritis requires confirmatory endoscopic biopsy. There are increasing data in various populations on cut-off points based on positive predictive values for skin prick test and milk-specific IgE measurements to aid in the diagnosis of cow's milk allergy and to decrease the number of unnecessary food challenges. For non-IgE-mediated manifestations, noninvasive diagnostic tests are still largely lacking. The significant homology between milk from cow, sheep and goat results in clinical cross-reactivity. However, mare's or donkey's milk may be tolerated by some individuals. SUMMARY: Data have been accumulating on the utility of diagnostic tools for mostly IgE-mediated milk allergy and allergenicity of milk from other mammalian species, although further studies are sought.

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[4] - Sergeant P, Kanny G, Moneret-Vautrin DA. Régime d'éviction des protéines de lait de vache. Alim'Inter 2006;11:153

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[5] - Moneret-Vautrin DA, Hatahet R, Kanny G. Hydrolysats de protéines: laits hypoallergéniques et formules extensivement hydrolysées. Bases immuno-allergologiques de leur utilisation dans la prévention et le traitement de l'allergie au lait. Arch Pediatr 2001;8:1348-1357

Allergy to milk has an estimated incidence of 2.5%. Hypoallergenic milks are partial hydrolysates of proteins with lactose. Extensive hydrolysates of casein are mainly small peptides and do not include lactose in their formula. A primary allergenicity as well as a cross-sensitivity are shown for all partial hydrolysates. Even extensive hydrolysates have a weak potential of cross-allergy. The prevention of cow's milk allergy in infants at peculiar risk, born from atopic parents, is based on breast-feeding and/or partial or extensive hydrolysates. There is no conclusive evidence for the usefulness of eviction of dairy products during pregnancy or during the period of breast-feeding. The treatment needs exclusively extensive casein hydrolysates ensuring a 90% protection with 95% confidence intervals. Predictive prick tests in certain cases may help the choice of another hydrolysate. Severe cases with failure to thrive or multiple food intolerance require an amino-acid based formula (Neocate). A rational diversification of the diet is advised in order to prevent the onset of a multiple food intolerance syndrome which could avert from the recovery of cow milk allergy.

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[6] - Dupont C, de Boissieu D. Allergie aux hydrolysats poussés de protéines chez le nourrisson: efficacité et durée d'utilisation d'une formule à base d'acides aminés. Alim'Inter 2003;8:72-78

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[7] - Wal JM. Bovine milk allergenicity. Ann Allergy Asthma Immunol 2004;93(5 suppl.3):S2-S11

OBJECTIVE: To provide updated data on the characteristics (eg, structure, function, stability) of the main milk proteins identified as allergens and on the characterization of their epitopes. DATA SOURCES: Basic literature and the most relevant original recent publications on clinical and epidemiologic aspects of milk allergy and the biochemistry and immunochemistry of milk proteins. STUDY SELECTION: The expert opinion of the author was used to select the relevant data for the review . RESULTS: Most milk proteins, even proteins present at low concentrations, are potential allergens. Epitopes on milk proteins are both conformational and linear epitopes, widely spread throughout the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule, that comprise highly conserved sequences responsible for IgE cross-reactivity with corresponding milk proteins of other mammals, including humans. Those sequential epitopes have also been proposed as good markers of persistent allergy to milk proteins and may be of particular clinical significance . CONCLUSIONS: No specific structure or function is associated with allergenicity of milk proteins. Due to the great variability and heterogeneity of the human IgE response, no single allergen or particular structure can account for a major part of milk allergenicity. Furthermore, the available evidence is not sufficient to establish an intake threshold below which allergic reactions are not triggered or to predict reliably the effect of food processing on allergenic potential of milk proteins.

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[8] - Besler M, Eigenmann P, Schwartz RH. Cow's milk (Bos domesticus). Internet Symp Food Allergens 2002;4(1):19-106

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[9] - Malandain H. Transglutaminases : a meeting point for wheat allergy, celiac disease, and food safety. Eur Ann Allergy Clin Immunol 2005;37:397-403

Wheat is the staple cereal in many countries and its uses in manufactured foods are ever growing due to the technological qualities of gluten proteins. Transglutaminases (TG) are ubiquitous enzymes with many functions. They are able to transform proteins by deamidation and/or transamidation. This last reaction can cross-link proteins together. Intestinal tissue TG has been shown to play an important role in two kinds of immune reactions to wheat: celiac disease and wheat-dependent exercise-induced anaphylaxis. In addition, new epitopes have been suspected in cases of anaphylaxis to wheat isolates, a food ingredient consisting mainly of deamidated gluten proteins. As a microbial TG is included in many food technological processes, its safe use should be checked. This assessment must cover not only the safety of the TG itself but also that of the deamidated/cross-linked proteins generated by this enzyme. This article aims at discussing the possible consequences of using TG in food industry in the light of today knowledge about immune reactions to wheat.

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[10] - Morgan PE, Treweek TM, Lindner RA, Price WE, Carver JA. Casein Proteins as Molecular Chaperones. J Agric Food Chem 2005;53:2670-2683

Under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. We have investigated the chaperone activity of the major milk proteins alpha(S)-, beta-, and kappa-casein with reduced insulin and the milk whey proteins, alpha-lactalbumin and beta-lactoglobulin, and compared it with that of the mammalian small heat shock protein (sHsp), alpha-crystallin, and clusterin. alpha(S)-Casein exhibited different chaperone behavior under reduction and heat stresses, i.e., chaperone activity increased with increasing temperature (as observed with alpha-crystallin), but under reduction stress, its chaperone activity increased at lower temperatures. beta- and kappa-casein had comparable chaperone ability with each other but were less effective than alpha(S)-casein. Under molecular crowding conditions, precipitation of stressed protein was accelerated, and alpha(S)-casein was a poorer chaperone. Furthermore, at slightly alkaline pH values, alpha(S)-casein was a less effective chaperone than at neutral pH. Detailed fluorescence, size exclusion chromatography, and real-time NMR studies studies indicated that the casein proteins underwent conformational changes and stabilized the partially unfolded whey proteins prior to formation of high molecular weight soluble complexes. These results are consistent with casein proteins acting as molecular chaperones in a manner similar to sHsps and clusterin.

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[11] - Gaudin JC, Rabesona H, Choiset Y, Yeretssian G, Chobert JM, Sakanyan V, et al. Assessment of the immunoglobulin E-mediated immune response to milk-specific proteins in allergic patients using microarrays. Clin Exp Allergy 2008;38:686-693

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available . METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins . RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients . CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.

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[12] - Bernard H, Creminon C, Negroni L, Peltre G, Wal JM. IgE Cross-reactivity with Caseins from Different Species in Humans Allergic to Cow"s Milk. Food Agric Immunol 1999;11:101-111

Fifty-eight sera from humans allergic to cow"s milk proteins were analysed for the specificity of their IgE response to the whole casein fraction of milk from different ruminant and nonruminant species (e.g. cow, sheep, goat, rabbit and rat). IgE-specific responses were determined by an enzyme allergosorbent test using the purified casein fractions as immobilized antigen and an anti-human IgE monoclonal antibody labelled with acetylcholinesterase. Co-and/or cross-sensitizations to caseins of the different ruminant species occurred extensively, though IgE responses to ovine and caprine casein appeared to be lower than that obtained with bovine casein. Cross-reactivity is suggested by the significant reactivity of rat and rabbit casein toward human IgE. In terms of specificity and intensity, the IgE response to caseins demonstrates a great variability. Structural homologies in caseins of such different species, that can share common epitopes for the IgE of some patients, suggest that prevention of cow"s milk allergy cannot be achieved by using milk from other species as substitutes.

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[13] - Bellioni Businco B, Paganelli R, Lucenti P, Giampietro PG, Perborn H, Businco L. Allergenicity of goat's milk in children with cow's milk allergy. J Allergy Clin Immunol 1999;103:1191-1194

Cow's milk allergy (CMA) is a common disease of infancy and childhood. An appropriate cow's milk (CM) substitute is necessary for feeding babies with CMA. CM substitutes are soy formulas and casein- or whey-based extensively hydrolyzed formulas. In several countries, including Italy, goat's milk (GM) formulas are available, and some physicians recommend them for feeding babies with CMA. OBJECTIVE: We sought to investigate, in vitro and in vivo, the allergenicity of GM in 26 children with proven IgE-mediated CMA. METHODS: All the children underwent skin tests with CM and GM; detection of specific serum IgE to CM and GM; and double-blind, placebo-controlled, oral food challenges (DBPCOFCs) with fresh CM, GM, and, as placebo, a soy formula (Isomil, Abbott, Italy). CAP inhibition and immunoblotting inhibition assays were also carried out in 1 of 26 and 4 of 26 children with positive RAST results to both CM and GM, respectively. RESULTS: All the children had positive skin test responses and CAP results to both CM and GM, all had positive DBPCOFC results to CM, and 24 of 26 had positive DBPCOFCs to GM. In CAP inhibition tests, preincubation of serum with CM or GM strongly inhibited IgE either to CM or to GM. In immunoblotting inhibition assays, preincubation with CM completely extinguished reactivity to GM, whereas GM partially inhibited reactivity to CM. CONCLUSIONS: These data strongly indicate that GM is not an appropriate CM substitute for children with IgE-mediated CMA. A warning on the lack of safety of GM for children with CMA should be on the label of GM formulas to prevent severe allergic reactions in babies with CMA.

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[14] - Besler M, Eigenmann P, Schwartz RH. Sheep's Milk (Ovis spp.). Internet Symp Food Allergens 2002;4(2):125-130

The true prevalence of goat's milk and sheep's milk allergy is not known. However, because IgE sensitization to sheep and goat casein has been found to be as high as 93% to 98% in children with IgEmediated cow's milk allergy, it is to be expected that children who are allergic to cow's milk are also allergic to sheep's and goat's milks. Several case reports indicate that IgE-mediated sensitization and allergic reactions to sheep's and goat's milks can occur in children and adults who are tolerant of and not significantly sensitized to cow's milk. Because such sensitization and reactions have occurred with certain types of cheese and there are hundreds of different types of cheese, it is helpful to know the animal from which the cheeses are derived. Feta cheese can be made from cow, ewe, or goat; pecorino and Roquefort from ewe; ricotta from cow or ewe; mozzarella from cow, ewe, or buffalo. Other less common sources of milk used in cheese production include camel, mare, reindeer, and yak. Symptoms of goat's milk and sheep's milk allergy may vary in severity from mild urticaria or localized oral pruritis to severe anaphylactic reactions. The diagnosis of goat's and sheep's milk allergy is based on a thorough history supported by positive skin prick tests and high levels of specific serum IgE to goat's and sheep‚s milk allergens, especially casein, respectively. Because of both the high association with cow's milk allergy and the sometimes isolated occurrence of sheep's and goat's milk allergy, testing with cow's milk can be informative. Oral challenge procedures can be performed when acute anaphylactic reactions are not expected, when the diagnosis is in doubt, or to determine if tolerance is present or has developed. This review presents data on prevalence, symptoms, cross-reacting allergens, and sources in tabular form.

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[15] - Vita D, Passalacqua G, Di Pasquale G, Caminiti L, Crisafulli G, Rulli I, et al. Ass’s milk in children with atopic dermatitis and cow’s milk allergy: Crossover comparison with goat’s milk. Pediatr Allergy Immunol 2007;18:594-598

Cow milk allergy is a common disease of infancy, often associated with atopic dermatitis (AD). Avoidance of cow milk (CM) implies the use of alternative dietary supports such as mammalian milks. In this study, we assessed the tolerability and clinical effect of ass's milk (AM), when compared with the largely used goat's milk (GM) in a single-blind, controlled, randomized crossover. Twenty-eight children with AD and ascertained allergy to CM were enrolled. The children were randomized to AM or GM for 6 months, then switched to the other milk for further 3 months. The SCORAD index (SI) and a visual analog scale (VAS) were evaluated blindly. After termination of the study, food challenges with GM and AM were performed. An SDS-PAGE analysis of different milks was performed. Two children from the GM group dropped out after randomization and 26 completed the study. Ass milk invariantly led to a significant improvement of SI and VAS of symptoms (p < 0.03 vs. baseline and inter-group), whereas GM had no measurable clinical effect. At the end of the study 23 of 26 children had a positive food challenge with GM and one of 26 with AM. Ass's milk had a protein profile closer to human milk than GM. Ass milk is better tolerated and more effective than GM in reducing symptoms of AD. It may represent a better substitute of CM than the currently used GM.

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[16] - Bellioni Businco B, Paganelli R, Lucenti P, Giampietro PG, Perborn H, Businco L. Allergenicity of goat's milk in children with cow's milk allergy. J Allergy Clin Immunol 1999;103:1191-1194

Cow's milk allergy (CMA) is a common disease of infancy and childhood. An appropriate cow's milk (CM) substitute is necessary for feeding babies with CMA. CM substitutes are soy formulas and casein- or whey-based extensively hydrolyzed formulas. In several countries, including Italy, goat's milk (GM) formulas are available, and some physicians recommend them for feeding babies with CMA. OBJECTIVE: We sought to investigate, in vitro and in vivo, the allergenicity of GM in 26 children with proven IgE-mediated CMA. METHODS: All the children underwent skin tests with CM and GM; detection of specific serum IgE to CM and GM; and double-blind, placebo-controlled, oral food challenges (DBPCOFCs) with fresh CM, GM, and, as placebo, a soy formula (Isomil, Abbott, Italy). CAP inhibition and immunoblotting inhibition assays were also carried out in 1 of 26 and 4 of 26 children with positive RAST results to both CM and GM, respectively. RESULTS: All the children had positive skin test responses and CAP results to both CM and GM, all had positive DBPCOFC results to CM, and 24 of 26 had positive DBPCOFCs to GM. In CAP inhibition tests, preincubation of serum with CM or GM strongly inhibited IgE either to CM or to GM. In immunoblotting inhibition assays, preincubation with CM completely extinguished reactivity to GM, whereas GM partially inhibited reactivity to CM. CONCLUSIONS: These data strongly indicate that GM is not an appropriate CM substitute for children with IgE-mediated CMA. A warning on the lack of safety of GM for children with CMA should be on the label of GM formulas to prevent severe allergic reactions in babies with CMA.

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[17] - Spuergin P, Walter M, Schiltz E, Deichmann K, Forster J, Mueller H. Allergenicity of alpha-caseins from cow, sheep, and goat. Allergy 1997;52:293-298

The allergic potential of alpha-caseins from bovine, ovine, and goat's milk sharing more than 85% identical amino acids was compared. Caseins were purified by anion-exchange chromatography and used for a specific IgE and IgG ELISA with diluted human sera. Sera were from 17 children with immediate-type allergy to cow's milk, from 59 children with atopy but without food allergy, and from 27 healthy children without atopy disease. The sera of cow's milk-allergic children showed a significantly higher IgE and IgG binding to alpha-caseins from all three species than the sera of the other groups. All groups showed an increased antibody binding to bovine alpha-casein compared to the sheep and goat proteins, but the differences were significant only in the groups of atopic children and of healthy controls. Furthermore, inhibition of the IgE binding to bovine alpha-casein with alpha-casein from cow, goat, and sheep revealed that the alpha-casein from these species are highly cross-reactive; on the basis of the small differences in their primary structure. In conclusion, the milk of goat and sheep harbor an allergic potential and is not suitable for the nutrition of milk-allergic patients.

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[18] - Dean TP, Adler BR, Ruge F, Warner JO. In vitro allergenicity of cows'milk substitutes. Clin Exp Allergy 1993;23:205-210

There are numerous alternatives to cows' milk formula for allergic children. We have investigated the allergenicity of several of these using RAST and RAST inhibition on serum from 16 patients with a known history of cows' milk protein intolerance (CMPI) and 16 atopic controls. A RAST grade of > or = 3 for cows' milk was present in all those with CMPI, whilst all the controls gave RAST of < or = 1. Modified cows' milk formula, goats' infant formula, sheep and goats' milk produced similar results to cows' milk. Only two patients had RAST > or = 3 for soya milk and the soy/beef hydrolysate gave positive results in only three patients. One had positive RAST to Nutramigen and two to Pregestimil. Of the whey hydrolysates investigated, Pepti-junior gave seven positive RASTs whilst we were unable to bind Alfare to the sepharose in sufficient quantities to interpret the results which were negative in all cases. RAST inhibition data on pooled sera from the same patients agreed with the RAST results. The inhibition curves showed high inhibition with goats', sheep, modified cows' milk formula and the casein formula, AL110 (50%). Soy and soy/beef hydrolysate showed a much lower inhibition pattern. Casein hydrolysates showed low inhibition while the whey hydrolysate produced higher inhibition. We have shown that despite claims of low allergenicity, some of these alternative formulae are antigenically recognized in vitro by some cows' milk intolerant patients.

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[19] - Sabbah A, Drouet M, Lauret MG, Giffard G, Minkhar M. Etude de la réactivité croisée entre le lait de vache et le lait de chèvre. Allerg Immunol (Paris) 1997;29:203-214

Tolerance of LC may be explained by a great intra and interspecific genetic diversity that leads to a great variety of proteins. In contrast, industrial treatment (UHT sterilisation) introduces configural modifications that may provoke a very great structural homology between the proteins of the two milks. Casein is in high concentration in both milks and shows some homology of the AA sequences between both and it makes a common allergen for a cross- reaction. It has been observed that a good IgE inhibition in vitro does not automatically form a clinical cross-allergenicity. Therefore there may exist an immunological crossed reaction between LC and LV without production of a real crossed allergenicity.

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[20] - Osorio Galindo AM, Martín Muñoz MF, Jurado Palomo J, Pascual Marcos CY, Sánchez Pastor S, Martín Esteban M. Allergy to goat and sheep´s milk: three cases. Allergy 2007;62(suppl. 83):343-344

Background: Allergy to goat and sheep‚s milk is not frequent in paediatric population and it is commonly associated with allergy to cow‚s milk due to cross reactivity with caseins. We studied three children: 1. Girl, nine years old, with other food allergies, asthma and atopic dermatitis. After taking goat cheese she had immediately eyelids and lips oedema, aphonia, disphagia and vomits. 2. Boy, five years old, with some food allergies and hepatoblastome. He handled some sheep cheese and rubbed his eyes with facial oedema, he also presented allergy oral syndrome with his ingestion. 3. Boy, aged seven, had suffered, during last three years, more than 15 episodes of conjunctivitis, eyelids oedema. The last one with vomits and abdominal pain, related to the ingestion of sheep cheese. All of them are proved to tolerate cow‚s milk. METHODS: Prick-test with cow‚s milk and his proteins, goat and sheep‚s milk and casein, meat and epithelium. Specific IgE to cow, goat and sheep‚s milk assess. In patient 3, simple-blind food challenge with goat and sheep´s cheese. With the serum of patient 1, Transblotting with cow, goat and sheep‚s milk and casein, inhibited with goat and sheep‚s milk. RESULTS: Patient 1 Patient 1 Patient 2 Patient 2 Patient 3 Patient 3 Prick-Test (mm) Specific IgE (kUA/L) Prick-Test (mm) Specific IgE (kUA/L) Prick-Test (mm) Specific IgE (kUA/L) Cow´s milk 4x3 < 0.35 0x0 0.41 2x2 < 0.35 ALA 0x0 0x0 < 0.35 1x1 < 0.35 BLG 0x0 0x0 < 0.35 0x0 < 0.35 BSA 0x0 0x0 < 0.35 0x0 < 0.35 Cow´s casein 4x3 < 0.35 0x0 < 0.35 0x0 < 0.35 Sheep´s milk 407 5x3 2.01 3x2 < 0.35 Sheep´s casein 6x5 6x2 Goat´s milk 10x7 435 3x3 2.34 2x2 < 0.35 Goat´s casein 20x11 11x7 6x4 Lysozime 5x5 < 0.35 8x5 < 0.35 0x0 Positive food challenge in patient 3, with sheep cheese. Transblotting shows complete inhibition of all the proteic bands of every mammalian milk and casein studied, when we incubate the serum with goat and sheep milk‚s. CONCLUSION: Our patients present allergy to some allergens that had kept similar linear structure in cow, goat and sheep milk‚s. These resemblances are only preserved in the three-dimensional structure in goat and sheep milk‚s.

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[21] - Rancé F, Kanny G, Dutau G, Moneret-Vautrin DA. Food hypersensitivity in children: clinical aspects and distribution of allergens. Pediatr Allergy Immunol 1999;10:33-38

The aims of this work were to investigate, in children and adolescents, the clinical aspects of food hypersensitivity and the distribution of allergens, in a prospective and descriptive study. Five hundred and forty-four pediatric cases from a series of 703 patients with food allergies, confirmed by food challenge, were studied. Their clinical characteristics and the distribution according to allergen were investigated. There was a family history of atopic disease in 70.5% of patients. Atopic dermatitis was the main symptom (275/544; 50.5% of patients), followed by urticaria and angio-edema (165/544; 30%). There was asthma in 8.6% of patients (47 children) and anaphylaxis in 4.5% (27 patients). The rarest signs were rhinitis (n=2; 0.3%), oral allergy syndrome (n=8; 1.4%), and gastrointestinal signs (n=11; 2%). Five allergens accounted for 78% of food hypersensitivity. These allergens were: eggs (36%), peanuts (24%), cow's milk (8%), mustard (6%), and cod (4%). Peanut was the most common allergen for children over the age of 3 yr. In this selected population, sensitivity of individuals to more than three foods was unusual (5%). Atopic dermatitis was the main symptom of food allergy in children. The symptoms changed over time, with respiratory disorders, oral allergy syndrome and ocular problems occuring later. Anaphylaxis also occured mostly in older children. Five allergens were responsible for more than three-quarters of food allergies in children. However, the number of allergens implicated was higher for the group of children over the age of 6 yr than for younger children.

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[22] - Besler M, Eigenmann P, Schwartz RH. Goat's Milk (Capra spp.). Internet Symp Food Allergens 2002;4(2):119-124

The true prevalence of goat's milk and sheep's milk allergy is not known. However, because IgE sensitization to sheep and goat casein has been found to be as high as 93% to 98% in children with IgEmediated cow's milk allergy, it is to be expected that children who are allergic to cow's milk are also allergic to sheep's and goat's milks. Several case reports indicate that IgE-mediated sensitization and allergic reactions to sheep's and goat's milks can occur in children and adults who are tolerant of and not significantly sensitized to cow's milk. Because such sensitization and reactions have occurred with certain types of cheese and there are hundreds of different types of cheese, it is helpful to know the animal from which the cheeses are derived. Feta cheese can be made from cow, ewe, or goat; pecorino and Roquefort from ewe; ricotta from cow or ewe; mozzarella from cow, ewe, or buffalo. Other less common sources of milk used in cheese production include camel, mare, reindeer, and yak. Symptoms of goat's milk and sheep's milk allergy may vary in severity from mild urticaria or localized oral pruritis to severe anaphylactic reactions. The diagnosis of goat's and sheep's milk allergy is based on a thorough history supported by positive skin prick tests and high levels of specific serum IgE to goat's and sheep‚s milk allergens, especially casein, respectively. Because of both the high association with cow's milk allergy and the sometimes isolated occurrence of sheep's and goat's milk allergy, testing with cow's milk can be informative. Oral challenge procedures can be performed when acute anaphylactic reactions are not expected, when the diagnosis is in doubt, or to determine if tolerance is present or has developed. This review presents data on prevalence, symptoms, cross-reacting allergens, and sources in tabular form.

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[23] - Carroccio A, Cavataio F, Iacono G. Cross-reactivity between milk proteins of different animals. Clin Exp Allergy 1999;29:1014-1016

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[24] - Pessler F, Nejat M. Anaphylactic reaction to goat’s milk in a cow’s milk-allergic infant. Pediatr Allergy Immunol 2004;15:183-185

Goat's milk has been used as a substitute for cow's milk in cow's milk-allergic individuals. It is currently advocated for this purpose in writings and internet resources directed at the lay person, and infant formula based on goat's milk is available in many countries. Clinically significant cross-allergenicity between cow's and goat's milk has been noted, but a life-threatening cross-reaction has not been reported. We describe a 4-month-old infant with immunologically documented cow's milk protein allergy but no prior exposure to goat's milk who developed anaphylaxis after the ingestion of commercial goat's milk. Subsequent skin prick testing demonstrated a specific reaction to goat's milk. Thus, the cross-allergenicity between cow's and goat's milk proteins can lead to life-threatening complications. Individuals allergic to cow's milk protein should avoid goat's milk and goat's milk products.

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[25] - Lara-Villoslada F, Olivares M, Jimenez J, Boza J, Xaus J. Goat Milk is Less Immunogenic than Cow Milk in a Murine Model of Atopy. J Pediatr Gastroenterol Nutr 2004;39:354-360

OBJECTIVES:: Cow milk protein allergy occurs in 2% to 6% of infants population. Goat milk has been used as an alternative to cow milk, but there is probably some cross-reactivity between the milks. Little is known about the allergenicity of goat milk per se. The aim of this study is to compare cow and goat milk allergenicity in a mouse atopy model. METHODS:: Balb/C mice were intragastrically sensitized to cow or goat milk by five doses administrated weekly. Six weeks after the first dose mice were killed, sera were collected and spleens removed for analysis. RESULTS:: The number of mice with diarrhea was significantly higher in the cow milk-sensitized group than in the goat milk-sensitized group. Serum cow milk-specific immunoglobulin G1 and histamine levels were also significantly higher in cow milk-sensitized mice. Cytokine production by spleen derived T cells showed a Th2 response with high levels of interleukin-4 production and low levels of interferon-gamma in cow milk-sensitized mice. In addition, goat milk induced a lower lymphocyte sensitization as a result of a significant decrease in the specific proliferation ratio of these cells. CONCLUSION:: Goat milk, when used as the first source of protein after a breast-feeding period, is less allergenic than cow milk in mice. Further studies are needed to clarify if goat milk is suitable as an alternative to cow milk in milk based formulas for infant nutrition.

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[26] - Barnig C, Schulmeister U, Swoboda I, Bessot JC, Spitzauer S, Pauli G. Allergie aux protéines du lait de vache sans allergie associée au lait de brebis chez l'adulte. Rev Fr Allergol Immunol Clin 2005;45:608-611

L'allergie IgE médiée aux protéines de lait de vache est fréquente chez le nourrisson et le jeune enfant, mais peut également apparaître à l'âge adulte. L'allergie aux protéines de lait de vache est souvent associée à une allergie aux laits de chèvre et de brebis. L'allergie isolée aux protéines dérivées de lait de brebis sans allergie associée au lait de vache semble plus exceptionnelle. Nous rapportons l'observation originale d'une patiente présentant une allergie aux protéines du lait de vache apparue à l'âge adulte, sans allergie associée aux protéines dérivées du lait de brebis. Les résultats des tests cutanés, des dosages d'IgE spécifiques (RAST et dot-blot) sont en faveur d'une sensibilisation à différentes protéines présentes dans les laits de vache, de chèvre et de brebis, mais avec une réaction très positive et spécifique vis-à-vis des caséines d'origine bovine (a-caséine et -caséine).

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[27] - Senti G, Leser C, Wal JM, Bernard H, Wuthrich B. Severe asthma and anaphylaxis in a cow's milk-allergic patient. Sheep's, goat's or horse's milk as an alternative for milk-protein-allergic persons. Allergologie 2002;25:333-337

A 24-year-old man was suffering from severe asthma, urticaria and generalized pruritus since the age of 14 after eating all kinds of cow's milk products. Even the ingestion of small amounts of cheese and butter produced the same symptoms. Serologic testing showed a sensitization to casein (CAP class 5, 68.2 kU/L). Prick testing showed a strongly positive reaction to cow's milk and cheese. Prick- to-prick testing with feta cheese was negative so an oral challenge test with feta cheese was done, started with 5 g. 15 minutes after ingestion he presented a conjunctivitis and a running nose, followed by edema and a severe asthma attack. The following examination showed highly elevated specific IgE to casein, especially to the fraction of alpha(1)-casein to cow's, sheep's and goat's milk. The patient was told to follow a milk-free diet. In case of an accidental ingestion of milk the patient was given emergency medicaments (antihistamines, steroids and adrenaline for subcutaneous injection). Cross reactions can explain allergic reactions to other milk species, so goat's, sheep's and horse's milk is definitely no generalized alternative. In any case testing and if uncertainties remain, double blind oral food challenge is necessary.

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[28] - Fiocchi A, Restani P, Decet E, Travaini M, Mirri GP, Bernardo L, et al. Evolution from ewe's milk to cow's milk allergy. Allergy 1999;54:401-402

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[29] - Lamblin C, Bourrier T, Orlando JP, Sauvage C, Wallaert B. Allergie aux laits de chèvre et de brebis sans allergie associée au lait de vache. Rev Fr Allergol Immunol Clin 2001;41:165-168

L'allergie aux laits de chèvre et de brebis sans allergie associée au lait de vache est rare et de description récente. Nous rapportons trois observations de patients qui ont présenté des manifestations allergiques sévères après l'ingestion de fromages de chèvre et/ou de brebis alors que les produits à base de lait de vache sont bien tolérés. L'enquête allergologique cutanée et/ou biologique complétée de tests de provocation labiale est positive pour les laits de chèvre et de brebis, permettant de retenir le diagnostic d'allergie aux laits de chèvre et de brebis. Une étroite homologie structurale au niveau des caséines présentes dans le lait de chèvre et celles du lait de brebis pourrait expliquer l'apparition d'une allergie isolée aux laits de brebis et de chèvre sans allergie associée au lait de vache.

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[30] - Umpierrez A, Quirce S, Marañón F, Cuesta J, Garcia Villamuza Y, Lahoz C, et al. Allergy to goat and sheep cheese with good tolerance to cow cheese. Clin Exp Allergy 1999;29:1064-1068

BACKGROUND: We report on a patient who experienced allergic reactions after eating goat cheese and after touching goat and sheep cheese, but not after consuming cow's milk dairy products . OBJECTIVE: To assess the allergenicity and IgE-binding capacity of the caseins from the three different species . METHODS: Skin prick tests were carried out using whole milk and caseins from three different species (goat, sheep and cow), and whey fractions of cow's milk. Total serum IgE and specific IgE to cow's milk proteins were measured by CAP system and specific IgE against caseins and whole milk were determined by ELISA technique. To evaluate allergenic cross-reactivity, inhibition of the IgE ELISA activity to goat's milk and goat casein was tested for the three caseins. SDS-PAGE and immunoblotting was used to determine IgE binding bands in caseins . RESULTS: Skin tests were positive to sheep and goat's milk, sheep and goat casein, as well as to sheep and goat cheese. Total serum IgE was 66 kU/L and IgE determinations by CAP were negative. IgE ELISA against the caseins from goat and sheep was strongly positive, whereas it was negative to cow casein. ELISA inhibition assays revealed a high degree of cross-reactivity between goat casein and sheep casein. Immunoblotting showed three IgE-binding bands in goat casein at 31, 27 and 22 kDa, which may correspond to alpha-, beta- and gamma-caseins. A band at about 31 kDa was observed in sheep casein and another band at 34 kDa was recognized in cow casein . CONCLUSION: This patient developed allergy to goat and sheep cheese with good tolerance to cow's milk. We identified goat casein as the main allergen causing sensitization in this patient as demonstrated by in vivo and in vitro tests. A high degree of cross-reactivity between goat and sheep casein was observed.

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[31] - Wüthrich B, Johansson SGO. Allergy to cheese produced from sheep's and goat's milk but not to cheese produced from cow's milk. J Allergy Clin Immunol 1995;96:270-273

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[32] - Pierart F, Moneret-Vautrin DA. Anaphylaxie pré-léthale aux protéines de brebis. Alim'Inter 2001;6:187-189

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[33] - Moneret-Vautrin DA, Frérot I, Leduc V, Guérin L. Allergie au lait de chèvre sans allergie au lait de vache. Communication: 10èmes Journées Parisiennes d'Allergie - Paris 9-11 janvier 2002

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[34] - Alvarez MJ, Lombardero M. IgE-mediated anaphylaxis to sheep's and goat's milk. Allergy 2002;57:1091-1092

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[35] - Tauler E, Pineda F. Allergy to goat and sheep cheese with tolerance to cow milk and its derivatives. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°612

Background: Allergy to cow milk proteins is common during early life. Although cross reactivity between cow, goat and sheep milk proteins have already been shown, there are some particularities that need to be studied. Case report: We describe the case of a 13 months old boy that presented an anaphylactic reaction (hives, angioedema in face, hands and genitals, and dyspneas with audible sibilances) immediately after a first time sheep cheese ingestion. The intensity of the medical signs required hospital assistance. He reiterated a similar episode when the parents wanted to verify the causality of the cheese in these symptoms. The patient had received breastfeeding for the first three months followed by artificial lactation with cow milk, yoghurt and cheese without problems. Methods: The patient was tested with cow, sheep milk and two diferents cheeses of cow and goat by skin prick-testing.Total and specific IgE was measured by CAP FEIA (Pharmacia, Uppsala, Sweden).Proteins in extracts of cow, sheep and goat cheeses were separated by SDS PAGE. IgE antibody-binding to allergens was analised by inmunoblotting using serum from the allergic patient. Results: Skin prick tests to goat milk and cheese and to sheep cheese were positive, while they were negative to cow milk, a-lactoalbumin, b-lactoglobulin, casein and cow cheese. Specific IgE was more than five times higher for sheep milk (7.78 kU/l) than cow milk (1.4 kU/l). SDS-PAGE and Inmunoblot experiments show that IgEs from the patient recognized a band of approximately 19 kDa, that could be related to a protein of a molecular weight similar to that of a-lactoalbumin, not detected in cow milk derivatives. Conclusions: Although the cross-reactivity between milks and derivatives from cow, goat and sheep has previously been documented, there are differences which can cause the sensitisation to goat or sheep cheese, while preserving tolerance to cow milk and its by-products. On the other hand, we have not come across any case of allergy to sheep and / or goat cheese in a toddler of comparable age in the scientific literature. Due to the fact that the reaction took place with the first intake of sheep cheese, we believe that it was the result of a prenatal or breastfeeding sensitisation phenomenon.

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[36] - Munoz Martin T, de La Hoz Caballer B, Maranon Lizana F, Gonzalez Mendiola R, Prieto Montano P, Sanchez Cano M. Selective allergy to sheep's and goat's milk proteins. Allergol Immunopathol (Madr) 2004;32:39-42

Background: Cow's milk proteins are amongst the most common causes of food allergy in infants, and caseins are probably the main allergens. The existence of a high degree of cross-reactivity between milk caseins from different animals has been reported. We describe a 2-year-old boy who experienced allergic reactions after eating and touching sheep's cheese, but who tolerated cow's milk and cow's milk dairy products. He had never ingested milk or milk derivatives from sheep or goat. Methods: Skin prick tests were carried out using whey fractions of cow's milk, whole milk and casein from goat, sheep and cow. We also performed skin prick tests with enzymes used in cheese production. Prick-by-prick tests with cheese made from cow, sheep and goat and their corresponding whole milk were also performed. Total serum IgE and specific IgE to cow's milk proteins, whole cow's milk and sheep's milk were determined. Specific IgE against casein and whole milk from the three different species were determined by ELISA. Inhibition of IgE binding to bovine casein was tested for casein and whole milk from all three species. The proteins of three types of casein and whole milk from cow, sheep and goat were separated by SDS-PAGE and were incubated with the patient's serum. Results: Skin tests were positive to sheep's milk and goat and sheep casein and were negative to all cow's milk proteins and whole cow's and goat's milk. Prick-by-prick tests were positive to goat's and sheep's cheese and were negative to cow's cheese. In ELISA-inhibition, sheep's milk and goat and sheep casein were able to inhibit > 50 % of specific IgE binding to sheep casein. The results of immunoblotting showed that the patient's circulating IgEs recognized only one band in the lanes corresponding to sheep and goat casein. Conclusions: We report a patient with allergy to sheep's and goat's milk proteins but not to cow's milk proteins. Sheep casein was probably the main allergen causing sensitization in this patient. The results suggest that sheep casein shows a high degree of cross-reactivity with goat casein but not with cow casein. Our patient presented allergic symptoms caused by sheep and goat milk and cheese proteins. However, he was able to tolerate cow's milk and cow's milk dairy products without any ill effects.

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[37] - Martins P, Borrego LM, Pires G, Pinto PL, Afonso AR, Rosado-Pinto J. Sheep and goat's milk allergy – a case study. Allergy 2005;60:129-130

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[38] - Attou D, Caherec A, Bensakhria S, Dookna P, Faverge B. Allergie aux laits de chèvre et de brebis sans allergie associée au lait de vache : revue générale à propos d'une observation à rebondissements. Rev Fr Allergol Immunol Clin 2005;45:601-607

Nous rapportons une observation d'allergie sévère après ingestion de fromages de chèvre et de brebis mais sans allergie associée au lait de vache chez une jeune adolescente de 11 ans et demi. Le diagnostic a été établi sur une histoire clinique convaincante et des tests cutanés et biologiques concordants. Cette allergie de plus en plus préoccupante a toutes les caractéristiques d'une allergie sévère. L'homologie extensive entre la structure des caséines des laits de chèvre et de brebis expliquerait la coexistence de ces deux allergies. Durant l'évolution, la patiente a présenté un épisode d'anaphylaxie induit par l'effort et après l'ingestion d'un produit laitier à base de lait de vache. Le test de provocation orale au lait de vache après un effort physique a permis de reproduire les symptômes. Si l'éviction reste le meilleur moyen de prévention, la prescription d'une trousse d'urgence est dans tous les cas une nécessité.

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[39] - Barnig C, Schulmeister U, Swoboda I, Bessot JC, Spitzauer S, Pauli G. Allergie aux protéines du lait de vache sans allergie associée au lait de brebis chez l'adulte. Rev Fr Allergol Immunol Clin 2005;45:608-611

L'allergie IgE médiée aux protéines de lait de vache est fréquente chez le nourrisson et le jeune enfant, mais peut également apparaître à l'âge adulte. L'allergie aux protéines de lait de vache est souvent associée à une allergie aux laits de chèvre et de brebis. L'allergie isolée aux protéines dérivées de lait de brebis sans allergie associée au lait de vache semble plus exceptionnelle. Nous rapportons l'observation originale d'une patiente présentant une allergie aux protéines du lait de vache apparue à l'âge adulte, sans allergie associée aux protéines dérivées du lait de brebis. Les résultats des tests cutanés, des dosages d'IgE spécifiques (RAST et dot-blot) sont en faveur d'une sensibilisation à différentes protéines présentes dans les laits de vache, de chèvre et de brebis, mais avec une réaction très positive et spécifique vis-à-vis des caséines d'origine bovine (a-caséine et -caséine).

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[40] - Vereda A, Sampson MA, Ibanez M, Bardina L, Thanik E, Sampson HA. Selective Allergy To Sheep's And Goat's Milk Proteins: 4 Cases. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°181

RATIONALE: Cow milk [CM] allergic patients usually react to goat and/or sheep milk [GSM] because of the high degree of sequence homology among ruminant milk proteins. We investigated IgE and IgG4 milk protein-specific binding in four children who reacted to GSM products, but did not react to CM METHODS: Specific IgE antibody concentrations to cow, goat and sheep milks were measured [UniCAP; Pharmacia Diagnostics]. IgE antibody binding to proteins of cow, sheep and goat milks were analyzed by immunoblot analyses and protein bands bound only by IgE antibodies of GSM-allergic patients were analyzed by MS/MS. In addition, epitopespecific IgE and IgG4 binding to cow alpha s1, alpha s2, beta and kappacaseins and beta-lactoglobulin was performed by peptide microarray immunoassay RESULTS: The mean milk-specific IgE levels from the 4 patients was 1.27 kU/L for cow (2/4 patients had CM-specific IgE), 47.99 kU/L for sheep and 47.71 kU/L for goat. IgE antibodies from patients allergic to CM or GSM similarly bound proteins in a range of 20-30 kDa of the sheep and goat extracts. However, the GSM allergic patients recognized a 55 kDa band in the sheep milk extract, which based on MS/MS analysis, is a sheep casein, beta-lactoglobulin or immunoglobulin. IgE antibodies from both GSM and CM allergic patients bound similar peptides on the cow milk protein peptide microarrays CONCLUSIONS: The lack of clinical reactivity of GSM-allergic patients to CM cannot be explained by an absence of binding to CM allergenic epitopes, but may be due to recognition of unique GSM proteins Funding: Mount Sinai

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[41] - Ibañez D, Gonzalez G, Jimenez G, Escudero C, Laso T, Moneo I. Allergy To Sheep's And Goat's Milk Cheese With Good Tolerance to Cow's Milk. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°182

RATIONALE: Cow's milk (CM) allergy is common in children, although there are few reports of allergy to sheep´s (SM) and goat´s milk (GM) and with good tolerance to CM METHODS: Patients: 2 y.o. female and a 10 y.o. male who presented inmediate systemic allergic reactions after ingestion of sheep´s cheese and another cheese of unknown origin, respectively. Both patients have always tolerated cow´s milk.Skin prick test, CAP-FEIA and immunoblotting were performed RESULTS: Patient 1: Prick test (PT):CM, BSA, ALA and BLG: negative Prick-prick (PPmm): SM (15), GM (13), cow´s casein (CC) (3), sheep´s casein (SC) (8), goat´s casein (GC) (5). Specific IgE (CAP kU/L): CM:0.53, CC:0.79, SM:81.90; GM >100; BSA, ALA and BLG:<0,35. Patient 2: PT: CM(3), BSA, BLG and ALA: negative. PP: SM(18), GM(12), CC(3), SC(8), GC(8). CAP: CM:2,75, CC:2.94, SM >100, GM>100; ALA, and BLG< 0.35 Both patients had strong IgE-binding to a protein with a MW about 31 kDa in SM, GM, SC and GC extracts. Both patients had weak IgE-binding to other proteins in a range of 19-60 kDa in SM and GM. Patient 2 show weak IgE-binding to other proteins in a range of 19- 60 kDa in CM, SC and GC. Serum from patient 1 did not recognize any band in CM or CC CONCLUSIONS: An IgE-mediated allergy to sheep´s and goat´s milk was demonstrated in two patients without allergy to cow´s milk. Sheep´s and goat´s casein seem to be the main allergen in these cases.

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[42] - Rodriguez Plata E, Garcia Robaina J, Martin Conde L, Sanchez Machin I, Carres J, Lombardero M, et al. Sensitisation to goat and sheep milk with good tolerance to cow milk. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°582

Introduction: Allergy to milk is a well-studied form of food allergy. Milk contains several allergens that may cause allergic reactions. The two main components of milk are whey and casein. The whey fraction (20%) contains mainly alpha-lactalbumin and beta-lactaglobulin and is most likely to induce sensitisation. Whey proteins are altered by high heat. Casein (80%) is heat stable and is the most important allergen in cheese. Several degrees of cross-reactivity have been suggested among cow, goat and sheep milk. It has been suggested that goat's milk is tolerated by only 40% of children who are allergic to cow's milk. Herein we report of a 13 year old patient who had previously suffered multiple anaphylactic reactions and was attended in our Allergy Department. He could not identify the cause of the attacks, apart from a cured cheese sandwich which he had eaten when travelling on an airplane. He has tolerated and continues tolerating cow milk and other lactate products. Material and Methods: Skin test were performed with a panel of common aero-allergens, vegetables, fish, shellfish, fruit and nuts, milk and cheese, and latex. Standardized extracts were used when available. Total and specific IgE to milk and cheese and Immunoblots to cow, goat and sheep milk were also performed. RESULTS: Skin test were positive for goat and sheep milk and cheese and negative for cow milk and alfa-lactoglobulin, beta-lactalbumin and casein. Skin test were also negative for other foods and aero-allergens. The total IgE was 121 kU/L. Specific IgE for goat milk: 55.8; sheep milk: 57.6. Cow milk, alfalactoglobulin, beta-lactalbumin and casein: < 0,35 kU/l. The Inmmunoblots showed multiple bands in goat and sheep milk. No specific recognition to cow milk was seen and only a band of about 30 kDa was observed, which could correspond to casein. CONCLUSION: We describe a case of specific sensitisation to goat and sheep milk with good tolerance to cow milk. In the absence of a specific immunotherapy treatment, severe avoidance of goat and sheep milks was recommended.

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[43] - Cabral Duarte M, Costa A, Santos AS, Santos MP, Barbosa MP. Allergy to Goat's and Sheep's Milk without Allergy to Cow's Milk. J Allergy Clin Immunol 2008;121:S187

RATIONALE: Cow‚s milk (CM) allergy is the main cause of food allergy in childhood. Allergy to goat‚s and sheep‚s milk (GSM) without allergy to CM is rare. METHODS: We describe a case-report of a patient (male-17 years old), who has had 5 episodes since he was 6-7 years old, characterized by malaise, loss of consciousness, itching and pharyngeal pressure, vomiting, diffuse itching erythema, lip and tongue oedema, after eating GSM or their products. The last three episodes occurred with masked allergens. The first four episodes had spontaneous resolution without any treatment. In the last episode, at 17 years old, he was admitted to the hospital and had a clinical resolution after 2-3 hours of treatment. RESULTS: Skin prick tests (SPT) were positive to sheep‚s milk (SM) and goat‚s milk (GM) and negative to CM. Skin prick-to-prick tests were positive to GSM and cheese and negative to CM, cow beef, cow‚s and buffalo‚s cheese. Serum total IgE -169 KU/L and specific IgE (KU/L, class)- GM 0.42(1), SM 1.3(2) and negative for CM proteins. Oral challenge tests were performed in hospital setting: CM-negative and GM/SM-positive. Goat‚s and sheep‚s immunoblotting showed IgE-binding bands at 28 kDa, 33-38 kDa and 74-77 kDa. Immunoblotting inhibition showed a total inhibition of SM/GM and partial inhibition of GM/SM. CONCLUSIONS: Allergy to GSM is different from CM and egg allergy. Periodic allergological evaluation is recommended to detect new sensitivities, because of the high risk of cross-reactivity between milk proteins of different mammals.

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[44] - Garcia-Ara C, Cases B, Pedrosa M, Boyano T, Pastor C, Pérez M et al. Allergy to Goat's and Sheep's Milk. Identification of Allergenic Proteins. J Allergy Clin Immunol 2009;123:S27

RATIONALE: Cow‚s milk (CM) allergy is frequent in children and some of them react also to goat‚s and sheep‚s milk (GSM). There are reports that show IgE mediated allergy to GSM but not to CM. We report 3 children who presented GSM allergy but tolerated CM and its dairy products. Studies were performed in order to characterise the proteins that were involved in the GSM allergy. METHODS: Three children (2males,1 female), aged 4-5 years, who presented immediate allergic manifestations (urticaria/angioedema, cough, stridor and dysnea) upon contact or after ingestion of minimal doses of GSM products but tolerated CM are studied. Skin prick test (whole milk and casein of CM, SM and GM) and specific IgE (CAP Pharmacia) to whole CM and its proteins, and whole SM and GM were carried out. SDS- PAGE of caseins and milk and inmunoblot assays were performed. Identification of protein bands by MALDI-TOFMS or MS-MS was carried out. RESULTS: SPT and IgE determination confirmed that GSM proteins but not CM proteins were involved in allergic symptoms of these three patients. In the inmunoblotting assays, patients‚ IgE reacted with several proteins bands (ranging from 15 to 60Kda)from the caseins and milk of both sheep and goat. The analysis of these proteins turned out to be caseins, corresponding to a-S1 casein, a-S2 casein and k-casein. CONCLUSIONS: We report the identification of a-S1 casein, a-S2 casein and k-casein as allergens involved inGSMallergy with good tolerance to CM.

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[45] - Vitte J, Bongrand P. Anaphylaxie fatale au lait de brebis : place de la biologie. Arch Pediatr 2008;15:1300-1303

L‚allergie isolée au lait de brebis ou de chèvre sans allergie au lait de vache peut induire des tableaux systémiques préoccupants. Le diagnostic et le suivi sont facilités par les marqueurs biologiques. En particulier, le dosage de la tryptase sérique, reflet d‚une dégranulation mastocytaire, doit être associé à la recherche des immunoglobulines E dans les suspicions de choc anaphylactique. Les dosages biologiques sont réalisables sur des prélèvements post-mortem dans le cadre d‚une enquête étiologique. Un cas d‚anaphylaxie mortelle au lait de brebis sans allergie au lait de vache est rapporté ici et la conduite à tenir est rappelée.

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[46] - Lamblin C, Bourrier T, Orlando JP, Sauvage C, Wallaert B. Allergie aux laits de chèvre et de brebis sans allergie associée au lait de vache. Rev Fr Allergol Immunol Clin 2001;41:165-168

L'allergie aux laits de chèvre et de brebis sans allergie associée au lait de vache est rare et de description récente. Nous rapportons trois observations de patients qui ont présenté des manifestations allergiques sévères après l'ingestion de fromages de chèvre et/ou de brebis alors que les produits à base de lait de vache sont bien tolérés. L'enquête allergologique cutanée et/ou biologique complétée de tests de provocation labiale est positive pour les laits de chèvre et de brebis, permettant de retenir le diagnostic d'allergie aux laits de chèvre et de brebis. Une étroite homologie structurale au niveau des caséines présentes dans le lait de chèvre et celles du lait de brebis pourrait expliquer l'apparition d'une allergie isolée aux laits de brebis et de chèvre sans allergie associée au lait de vache.

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[47] - Tavares B, Pereira C, Rodrigues F, Loureiro G, Chieira C. Goat’s milk allergy. Allergol Immunopathol (Madr) 2007;35:113-116

BACKGROUND: Goat's milk (GM) allergy not associated with allergy to cow's milk (CM) is a rare disorder. Caseins have been implicated has the major allergens eliciting symptoms . METHODS: We report the case of a 27 years-old female patient that experienced two episodes of urticaria related to ingestion of goat's cheese (GC). She tolerated CM, dairy products and sheep cheese. Skin prick tests were performed with GM, CM, bovine casein and alpha -lactalbumin and fresh milk and GC. Serum specific IgE to GM, CM and its fractions, and GM and CM immunobloting assays with inhibition were also evaluated . RESULTS: Skin tests were positive to GM and GC and negative to CM. GM immunoblot showed an IgE-binding 14 kDa band that was totally inhibited after serum pre-incubation with GM . CONCLUSIONS: Allergens other than casein can be involved in allergy to GM. Even small quantities of protein can elicit symptoms.

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[48] - Tavares B, Pereira C, Rodrigues F, Loureiro G, Chieira C. Goat’s milk allergy. Allergol Immunopathol (Madr) 2007;35:113-116

BACKGROUND: Goat's milk (GM) allergy not associated with allergy to cow's milk (CM) is a rare disorder. Caseins have been implicated has the major allergens eliciting symptoms . METHODS: We report the case of a 27 years-old female patient that experienced two episodes of urticaria related to ingestion of goat's cheese (GC). She tolerated CM, dairy products and sheep cheese. Skin prick tests were performed with GM, CM, bovine casein and alpha -lactalbumin and fresh milk and GC. Serum specific IgE to GM, CM and its fractions, and GM and CM immunobloting assays with inhibition were also evaluated . RESULTS: Skin tests were positive to GM and GC and negative to CM. GM immunoblot showed an IgE-binding 14 kDa band that was totally inhibited after serum pre-incubation with GM . CONCLUSIONS: Allergens other than casein can be involved in allergy to GM. Even small quantities of protein can elicit symptoms.

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[49] - Alonso-Lebrero E, Fuentes V, Zapatero L, Pérez-Bustamante S, Pineda F, Martinez-Molero MI. Goat's milk allergies in children following specific oral tolerance induction to cow's milk. Allergol Immunopathol (Madr) 2008;36:180-181

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[50] - Lain S, Pérez-Bustamante S, Davila G, Fuentes V, Zapatero L, Alonso E, et al. Anaphylaxis due to sheep and goat’s milk cheese. Allergy 2008;63(suppl. 88):123

Background: Cow‚s milk allergy is the most frequent cause of food allergy in children. The existence of a high degree of crossreactivity between caseins from cow‚s, sheep‚s and goat ‚s milk has been reported. However, sheep‚s and goat‚s milk allergy is uncommon and does not usually involve allergic cross-reactivity to cow‚s milk. We describe two different cases of anaphylaxis due to sheep and goat‚s milk cheese. Methods: CASE 1: A 3-year-old non atopic girl experienced an anaphylactic reaction after eating sheep and goat‚s milk cheese. She presented generalised itching, eyelid swelling, dry cough and wheezy dyspnea. After the episode, she had tolerated cow‚s milk and cow‚s milk dairy products. CASE 2: A 6-year-old girl with antecedents of cow‚s milk allergy was treatment successfully with an specific oral tolerance induction treatment. One year later, fifteen minutes after the ingestion of sheep‚s milk cheese she presented rhinitis, dry cough, wheezy dyspnea and generalised itching.- Skin prick tests (SPT) were carried out using cow‚s milk and its proteins. Prick by prick tests with cow‚s milk, cow‚s, sheep‚s and goat‚s milk cheese, specific IgE measurements, SDS-PAGE immunoblotting, and inhibition tests were performed to study the pattern of IgE-binding proteins and the potential cross-reactivity between caseins. Results: 1. Skin prick tests and Specific IgE.(Table). SPT (mm) SPT (mm) Prick by PricK (mm) Prick by Prick (mm) Specific IgE (KU/L) Specific IgE (KU/L) Case 1 Case 2 Case 1 Case 2 Case 1 Case 2 Cow casein 3_2 4_3 3.64 20.5 ALA 4_2 3_2 0.88o0.35 BLG Negative Negative 0.71 0.6 Cow‚s milk Negative 3_1 Negative 7_5 3.02 3.23 Sheep‚s milk ND ND 4100 18.6 Goat‚s milk ND ND 4 100 20.5 Cow cheese Negative Negative Sheep cheese 20_15 10_5 Goat cheese 14_8 11_4 2. Case 1: Immunoblotting showed two Ig E-binding bands in goat casein at 20 and 14 kDa, several bands between 14 and 45 kDa in sheep‚s milk and no bands in cow‚s milk. 3. Case 2: SDS-PAGE Immunoblotting was carried out using cow‚s milk and sheep‚s milk, showing multiple bands for both. In the SDS-PAGE-Immunoblotting inhibition with sheep‚s milk proteins using cow‚s milk, cow‚s casein, and sheep‚s milk as inhibitory phase showed a band of 30 kDa compatible with sheep‚s casein molecular weight to which was only inhibited by the sheep‚s milk. Conclusion: We report two cases of food allergy due to sheep‚s and goat‚s milk with good tolerance to cow‚s milk. Specific caseins were probably the main allergens causing sensitisation in our patients.

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[51] - Fiocchi A, Restani P, Decet E, Travaini M, Mirri GP, Bernardo L, et al. Evolution from ewe's milk to cow's milk allergy. Allergy 1999;54:401-402

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[52] - Attou D, Caherec A, Bensakhria S, Dookna P, Faverge B. Allergie aux laits de chèvre et de brebis sans allergie associée au lait de vache : revue générale à propos d'une observation à rebondissements. Rev Fr Allergol Immunol Clin 2005;45:601-607

Nous rapportons une observation d'allergie sévère après ingestion de fromages de chèvre et de brebis mais sans allergie associée au lait de vache chez une jeune adolescente de 11 ans et demi. Le diagnostic a été établi sur une histoire clinique convaincante et des tests cutanés et biologiques concordants. Cette allergie de plus en plus préoccupante a toutes les caractéristiques d'une allergie sévère. L'homologie extensive entre la structure des caséines des laits de chèvre et de brebis expliquerait la coexistence de ces deux allergies. Durant l'évolution, la patiente a présenté un épisode d'anaphylaxie induit par l'effort et après l'ingestion d'un produit laitier à base de lait de vache. Le test de provocation orale au lait de vache après un effort physique a permis de reproduire les symptômes. Si l'éviction reste le meilleur moyen de prévention, la prescription d'une trousse d'urgence est dans tous les cas une nécessité.

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[53] - Ibañez D, Gonzalez G, Jimenez G, Escudero C, Laso T, Moneo I. Allergy To Sheep's And Goat's Milk Cheese With Good Tolerance to Cow's Milk. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°182

RATIONALE: Cow's milk (CM) allergy is common in children, although there are few reports of allergy to sheep´s (SM) and goat´s milk (GM) and with good tolerance to CM METHODS: Patients: 2 y.o. female and a 10 y.o. male who presented inmediate systemic allergic reactions after ingestion of sheep´s cheese and another cheese of unknown origin, respectively. Both patients have always tolerated cow´s milk.Skin prick test, CAP-FEIA and immunoblotting were performed RESULTS: Patient 1: Prick test (PT):CM, BSA, ALA and BLG: negative Prick-prick (PPmm): SM (15), GM (13), cow´s casein (CC) (3), sheep´s casein (SC) (8), goat´s casein (GC) (5). Specific IgE (CAP kU/L): CM:0.53, CC:0.79, SM:81.90; GM >100; BSA, ALA and BLG:<0,35. Patient 2: PT: CM(3), BSA, BLG and ALA: negative. PP: SM(18), GM(12), CC(3), SC(8), GC(8). CAP: CM:2,75, CC:2.94, SM >100, GM>100; ALA, and BLG< 0.35 Both patients had strong IgE-binding to a protein with a MW about 31 kDa in SM, GM, SC and GC extracts. Both patients had weak IgE-binding to other proteins in a range of 19-60 kDa in SM and GM. Patient 2 show weak IgE-binding to other proteins in a range of 19- 60 kDa in CM, SC and GC. Serum from patient 1 did not recognize any band in CM or CC CONCLUSIONS: An IgE-mediated allergy to sheep´s and goat´s milk was demonstrated in two patients without allergy to cow´s milk. Sheep´s and goat´s casein seem to be the main allergen in these cases.

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[54] - Alvarez MJ, Lombardero M. IgE-mediated anaphylaxis to sheep's and goat's milk. Allergy 2002;57:1091-1092

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[55] - Martinez D, Castillo R. Allergy to cheese produced from sheep's and goat's milk in a teenager unaffected by cow's milk protein allergy. Allergy 2007;62(suppl. 83):354

A 15 year old female affected by atopic dermatitis that treated by intermitent pimecrolimus, no affected by rhinitis or Asthma, developed episodes of generalizated urticaria immediately after ingestion of pizza, lasagna and cheese, without clinical manifestation after drinking milk or other products made from cow's milk, she has never drinking another types of milk (sheep, goat, mare∑etc) Skin test were carried out using a battery of commons inhalants (duste mite, mold, pollens, animals) and foods allergens (milk, egg, fruits, dried fruits, types of flour, shellfish) were only positive to cat (8mm) and were also negative to whole milk, a- lactalbumin, ßlactoglobulin and cow's casein. Skin prick by prick test with types or cheese made of cow's, sheep's and goat's milk were positive to goat and sheep. (table 1). Total Ig E level were normal (91.80UI/ml), blood differential count showed 200 eosinophils/mm3 (normal) and specific Ig E (CAP Pharmacia) to cow's casein 1.26 L/l, cheddar cheese 0.96Ku/l, sheep's milk 7.41 Ku/l, and negative to a-lactalbumin, ß- lactaglobulin, cambrie cheese, cow's serum and mare's milk. (table 2) Oral challenge was not performed for ethical reason, the patient actually have and exclusion diet of goat's and sheep's milk without clinical urticaria episodes. Allergy to sheep's or goat's milk cheese has already been described in children affected by cow's milk allergy whereas allergy to sheep or goat without cow's protein is quite rare; the first case report by Johansson in 1995; the hypothesize that sheep's milk may have some antigenic determinant in common with goat's milk but different from that in cow's milk, moreover goat casein seems to be responsible for most of cross reactivity with sheep casein. Goat's and sheep's milk is preferred in the treatment of children affected by cow's milk allergy, is considered more natural and less allergenic; at that time is neccesary to discard that is not sensitized to that proteins before autorized since there are patients allergic to other mammals without clinical to cow's protein or with asintomatic sensitized to cow's milk that tolerated drinking or eating these dairy products but not goat or sheep.

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[56] - Lain S, Pérez-Bustamante S, Davila G, Fuentes V, Zapatero L, Alonso E, et al. Anaphylaxis due to sheep and goat’s milk cheese. Allergy 2008;63(suppl. 88):123

Background: Cow‚s milk allergy is the most frequent cause of food allergy in children. The existence of a high degree of crossreactivity between caseins from cow‚s, sheep‚s and goat ‚s milk has been reported. However, sheep‚s and goat‚s milk allergy is uncommon and does not usually involve allergic cross-reactivity to cow‚s milk. We describe two different cases of anaphylaxis due to sheep and goat‚s milk cheese. Methods: CASE 1: A 3-year-old non atopic girl experienced an anaphylactic reaction after eating sheep and goat‚s milk cheese. She presented generalised itching, eyelid swelling, dry cough and wheezy dyspnea. After the episode, she had tolerated cow‚s milk and cow‚s milk dairy products. CASE 2: A 6-year-old girl with antecedents of cow‚s milk allergy was treatment successfully with an specific oral tolerance induction treatment. One year later, fifteen minutes after the ingestion of sheep‚s milk cheese she presented rhinitis, dry cough, wheezy dyspnea and generalised itching.- Skin prick tests (SPT) were carried out using cow‚s milk and its proteins. Prick by prick tests with cow‚s milk, cow‚s, sheep‚s and goat‚s milk cheese, specific IgE measurements, SDS-PAGE immunoblotting, and inhibition tests were performed to study the pattern of IgE-binding proteins and the potential cross-reactivity between caseins. Results: 1. Skin prick tests and Specific IgE.(Table). SPT (mm) SPT (mm) Prick by PricK (mm) Prick by Prick (mm) Specific IgE (KU/L) Specific IgE (KU/L) Case 1 Case 2 Case 1 Case 2 Case 1 Case 2 Cow casein 3_2 4_3 3.64 20.5 ALA 4_2 3_2 0.88o0.35 BLG Negative Negative 0.71 0.6 Cow‚s milk Negative 3_1 Negative 7_5 3.02 3.23 Sheep‚s milk ND ND 4100 18.6 Goat‚s milk ND ND 4 100 20.5 Cow cheese Negative Negative Sheep cheese 20_15 10_5 Goat cheese 14_8 11_4 2. Case 1: Immunoblotting showed two Ig E-binding bands in goat casein at 20 and 14 kDa, several bands between 14 and 45 kDa in sheep‚s milk and no bands in cow‚s milk. 3. Case 2: SDS-PAGE Immunoblotting was carried out using cow‚s milk and sheep‚s milk, showing multiple bands for both. In the SDS-PAGE-Immunoblotting inhibition with sheep‚s milk proteins using cow‚s milk, cow‚s casein, and sheep‚s milk as inhibitory phase showed a band of 30 kDa compatible with sheep‚s casein molecular weight to which was only inhibited by the sheep‚s milk. Conclusion: We report two cases of food allergy due to sheep‚s and goat‚s milk with good tolerance to cow‚s milk. Specific caseins were probably the main allergens causing sensitisation in our patients.

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[57] - Vereda A, Sampson MA, Ibanez M, Bardina L, Thanik E, Sampson HA. Selective Allergy To Sheep's And Goat's Milk Proteins: 4 Cases. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°181

RATIONALE: Cow milk [CM] allergic patients usually react to goat and/or sheep milk [GSM] because of the high degree of sequence homology among ruminant milk proteins. We investigated IgE and IgG4 milk protein-specific binding in four children who reacted to GSM products, but did not react to CM METHODS: Specific IgE antibody concentrations to cow, goat and sheep milks were measured [UniCAP; Pharmacia Diagnostics]. IgE antibody binding to proteins of cow, sheep and goat milks were analyzed by immunoblot analyses and protein bands bound only by IgE antibodies of GSM-allergic patients were analyzed by MS/MS. In addition, epitopespecific IgE and IgG4 binding to cow alpha s1, alpha s2, beta and kappacaseins and beta-lactoglobulin was performed by peptide microarray immunoassay RESULTS: The mean milk-specific IgE levels from the 4 patients was 1.27 kU/L for cow (2/4 patients had CM-specific IgE), 47.99 kU/L for sheep and 47.71 kU/L for goat. IgE antibodies from patients allergic to CM or GSM similarly bound proteins in a range of 20-30 kDa of the sheep and goat extracts. However, the GSM allergic patients recognized a 55 kDa band in the sheep milk extract, which based on MS/MS analysis, is a sheep casein, beta-lactoglobulin or immunoglobulin. IgE antibodies from both GSM and CM allergic patients bound similar peptides on the cow milk protein peptide microarrays CONCLUSIONS: The lack of clinical reactivity of GSM-allergic patients to CM cannot be explained by an absence of binding to CM allergenic epitopes, but may be due to recognition of unique GSM proteins Funding: Mount Sinai

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[58] - Lamblin C, Bourrier T, Orlando JP, Sauvage C, Wallaert B. Allergie aux laits de chèvre et de brebis sans allergie associée au lait de vache. Rev Fr Allergol Immunol Clin 2001;41:165-168

L'allergie aux laits de chèvre et de brebis sans allergie associée au lait de vache est rare et de description récente. Nous rapportons trois observations de patients qui ont présenté des manifestations allergiques sévères après l'ingestion de fromages de chèvre et/ou de brebis alors que les produits à base de lait de vache sont bien tolérés. L'enquête allergologique cutanée et/ou biologique complétée de tests de provocation labiale est positive pour les laits de chèvre et de brebis, permettant de retenir le diagnostic d'allergie aux laits de chèvre et de brebis. Une étroite homologie structurale au niveau des caséines présentes dans le lait de chèvre et celles du lait de brebis pourrait expliquer l'apparition d'une allergie isolée aux laits de brebis et de chèvre sans allergie associée au lait de vache.

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[59] - Cases B, García-Ara C, Teresa B, Perez-Gordo M, Pastor C, Vivanco F et al. Allergy to goat's and sheep's milk: identification of allergenic proteins. Allergy 2009;64(Suppl. 90):368

Background: Cow's milk (CM) allergy is frequent in children and some of them react also to goat's and sheep's milk (GSM). There are reports that show IgE mediated allergy to GSM but not to CM. We report 3 children who presented GSM allergy but tolerated CM and its dairy products. Studies were performed in order to characterise the proteins that were involved in the GSM allergy. Methods: Three children (2 males, 1 female), aged 4-5 years, who presented immediate allergic manifestations (urticaria/angioedema, cough, stridor and dysnea) upon contact or after ingestion of minimal doses of GSM products but tolerated CM are studied. Skin prick test (whole milk and casein of CM, SM and GM) and specific IgE (CAP Pharmacia) to whole CM and its proteins, and whole SM and GM were carried out. SDS- PAGE of caseins and milk and inmunoblot assays were performed. Identification of protein bands by MALDI-TOF-MS or MSMS was carried out. Results: Skin prick test and IgE determination confirmed that GSM proteins but not CM proteins were involved in allergic symptoms of these three patients. In the inmunoblotting assays, patients' IgE reacted with several proteins bands (ranging from 15 to 60 kDa) from the caseins and milk of both sheep and goat. The analysis of these proteins turned out to be caseins, corresponding to a-S1 casein, a-S2 casein and .-casein. Conclusion: We report the identification of a-S1 casein, a-S2 casein and .-casein as allergens involved in GSM allergy with good tolerance to CM.

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[60] - Chedevergne F, Paty E, Scheinmann P. Allergie au lait de chèvre et de brebis sans allergie associée au lait de vache. Rev Fr Allergol Immunol Clin 2003;43:94

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[61] - Bidat E, Rancé F, Baranès T, Goulamboussen S. L'allergie au lait de chèvre ou de brebis sans allergie au lait de vache: danger. Rev Fr Allergol Immunol Clin 2003;43:94

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[62] - Bidat E, Rancé F, Baranès T, Goulamhoussen S. Goat's and Sheep's Milk Allergies in Children in the Absence of Cow's Milk Allergy. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°135

RATIONALE: Goat's (GMA) and sheep's (SMA) milk allergies in the absence of cow's milk allergy (CMA) are becoming increasingly frequent. MATERIAL AND METHODS: Nineteen children were diagnosed as having GMA and SMA based on a convincing clinical history and positive skin prick tests and/or specific IgE assays. RESULTS: The children had an average age of 6 at the time of their first allergic reaction (15 months to 16 years). Sixteen reacted on eating goat or sheep cheese, while 9 on consuming cooked food and 3 on consuming cow or buffalo cheese that had been contaminated during manufacture. Symptoms were cutaneous (68%: urticaria, 10 cases; and angioedema 17, including 5 affecting the larynx), respiratory (20%: asthma, 4), and anaphylactic shocks (2). The diagnosis was based on a convincing clinical history and positive skin prick tests with fresh foods or commercial extracts and positive s-IgE (goat: average=17.52 kU/l, 1.09 to >100 kU/l; sheep: average=11.32 kU/l, 3.4 to 31.4 kU/l). Ten children had other food allergies, including 7 with multiple food allergies. Recurrences in 11 patients were caused by consuming unlabeled food (8), a triggering dose (4), a delayed diagnosis (3), or high-risk behavior (1). CONCLUSIONS: The rise in the frequency of GMA and SMA in the absence of CMA is of great concern. Symptoms are more severe than for other food allergies. Minute amounts of GM or SM may trigger severe symptoms.

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[63] - Bidat E, Rancé F, Baranès T, Goulamhoussen S. L'allergie au lait de chèvre ou de brebis chez l'enfant, sans allergie au lait de vache. Rev Fr Allergol Immunol Clin 2003;43:273-277

L'allergie au lait de chèvre et/ou de brebis sans allergie au lait de vache semble augmenter de fréquence. Nous rapportons les caractéristiques cliniques et allergologiques de cette allergie. Patients. - Le diagnostic d'allergie au lait de chèvre et/ou de brebis sans allergie au lait de vache est établi chez 31 enfants sur une « histoire clinique convaincante » et des prick tests et/ou des IgE spécifiques positifs. Résultats. - Les enfants ont un âge moyen de 5,9 ans au moment de leur première réaction allergique (15 mois à 16 ans). Vingt-trois ont réagi en mangeant du fromage de chèvre ou de brebis, 19 en ingérant un plat cuisiné ou du lait « caché » ; dans 4 cas il s'agissait de fromage de vache ou de buffle contaminé lors de la fabrication. Les symptômes sont cutanés (urticaire, 14 cas ; angio-oedème 18, dont 10 laryngés), respiratoire (asthme, 10), et 6 chocs anaphylactiques sont survenus chez 3 patients. Les prick tests sont toujours positifs, sauf chez 2 enfants, les IgE spécifiques sont toujours positives (lait de chèvre médiane 8,2 ; brebis médiane 9,09). Seize enfants ont d'autres allergies alimentaires, et pour 12 il s'agit d'allergies alimentaires multiples. Conclusion. - L'augmentation de fréquence de cette allergie est préoccupante. Les signes sont plus sévères que pour les autres aliments. Des doses infimes d'aliments cachés peuvent déclencher des manifestations sévères.

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[64] - Paty E, Chedevergne F, Scheinmann P, Wal JM, Bernard H. Allergie au lait de chèvre et de brebis sans allergie associée au lait de vache. Rev Fr Allergol Immunol Clin 2003;43:455-462

L'allergie au lait de chèvre et de brebis sans allergie associée au lait de vache est rare et de description récente. Nous rapportons 18 observations d'enfants qui ont présenté des manifestations allergiques sévères après ingestion de fromages de chèvre et de brebis alors que les produits à base de lait de vache sont bien tolérés. L'histoire clinique convaincante, l'enquête allergologique cutanée et biologique permettent de retenir le diagnostic d'allergie aux laits de chèvre et de brebis. Les caséines alpha s1 et bêta du lait de chèvre et de brebis sont impliquées. L'étroite homologie structurale au niveau des caséines présentes dans le lait de chèvre et de brebis peut expliquer l'allergie aux laits de chèvre et de brebis. L'homologie avec le lait de vache est incomplète expliquant la possibilité d'allergie isolée aux laits de chèvre et de brebis sans allergie au lait de vache.

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[65] - Bernard H, Bidat E, Paty E, Scheinmann P, Wal JM. Allergies to goat's and sheep's milk without associated allergies to cow's milk. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°388

Allergies to goat's and sheep's milk without allergies to cow's milk are rare and have only appeared recently. We describe here 29 children (20 boys and 9 girls) with no history of intolerance to cow's milk proteins, but who presented severe allergic symptoms after drinking goat's milk or sheep's milk. Seventeen had a history of atopic dermatitis of varying degrees of severity. Eighteen had a history of asthma and 15 had known allergies to other foods (peanut, nuts, fish, egg, pea, lentil, sesame, apple, kiwi, mustard, shrimp). Symptoms appeared at the age of 5 years on average (12 months-16 years). The food responsible was not always identified immediately. Skin prick tests were carried out with untreated goat's milk and in some cases with goat's cheese and/or sheep's cheese. Skin prick tests were positive for all patients, the diameter of the papule being equal to or greater than that of the positive control. One patient presented a systemic reaction to the test, even though he only had facial urticaria after eating goat's cheese. The radioallergosorbent test (RAST), as measured with the CAP system, was positive in all cases (mean: 32 KU/l; range 1 to >100 KU/l), but not correlated with the severity of the clinical manifestations. No IgE specific to bovine casein or bovine B-lactoglobulin were detected. IgE specific to goat casein and sheep casein were detected by EAST (Enzyme Allergosorbent Tests) (mean: 22 IU/ml; range 0.1 to 200 IU/ml) Allergies to goat's and sheep's milk may cause serious clinical manifestations and need to be more widely recognised. The two allergies are always associated because of the high degree of structural similarity (about 90%) between goat's milk and sheep's milk caseins. However, the fact that these proteins are not identical (incomplete homology) to their counterparts in cow's milk means that it is possible to be allergic to goat's milk and sheep's milk without being allergic to cow's milk.

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[66] - Ah-Leung S, Bernard H, Bidat E, Paty E, Rancé F, Scheinmann P, et al. Allergy to goat and sheep milk without allergy to cow’s milk. Allergy 2006;61:1358-1365

BACKGROUND: Cow's milk (CM) allergy is the most frequent cause of food allergy in infants. Most children who are allergic to CM are also sensitized to whey proteins and/or to the casein fraction and many of them cannot tolerate goat's or sheep's milk (GSM) either. Conversely, the GSM allergies that are not associated with allergic cross-reactivity to CM are rare . METHODS: Twenty-eight children who had severe allergic reactions, including anaphylaxis, after consumption of GSM products but tolerated CM products were recruited in a retrospective study. Whole casein and whey proteins were fractionated from CM and GSM. beta-Lactoglobulin and the different caseins were isolated, purified and used to perform enzyme allergosorbent tests (EAST) and EAST inhibition studies with the sera of the allergic children . RESULTS: Clinical observations, skin prick testing and immunoglobulin (Ig)E-binding studies confirmed the diagnosis of GSM allergy without associated CM allergy. EAST determinations demonstrated that GSM allergy involves the casein fraction and not whey proteins. Cow's milk caseins were not at all or poorly recognized by the patient's IgE, while alphaS(1)-, alphaS(2)- and beta-caseins from GSM were recognized with a high specificity and affinity. In all cases, increasing concentrations of CM caseins failed to inhibit the binding of patient's IgE to sheep or goat milk caseins, whereas this binding was completely inhibited by GSM caseins . CONCLUSIONS: The characteristics of GSM allergy differ from those of the CM allergy because it affects older children and appears later. CM products do not elicit any clinical manifestation in GSM allergic patients, whereas CM allergic patients, usually cross-react to GSM. In all the GSM allergic children, the IgE antibodies recognized the caseins but not the whey proteins. Moreover, IgE specificity and affinity was high to GSM and lower to CM caseins despite their marked sequence homology. Doctors and allergic individuals should be aware that GSM allergy requires a strict avoidance of GSM and milk-derived products because reactions could be severe after ingestion of minimal doses of the offending food.

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[67] - Ah-Leung S, Bernard H, Bidat E, Paty E, Rancé F, Scheinmann P, et al. Allergy to goat and sheep milk without allergy to cow’s milk. Allergy 2006;61:1358-1365

BACKGROUND: Cow's milk (CM) allergy is the most frequent cause of food allergy in infants. Most children who are allergic to CM are also sensitized to whey proteins and/or to the casein fraction and many of them cannot tolerate goat's or sheep's milk (GSM) either. Conversely, the GSM allergies that are not associated with allergic cross-reactivity to CM are rare . METHODS: Twenty-eight children who had severe allergic reactions, including anaphylaxis, after consumption of GSM products but tolerated CM products were recruited in a retrospective study. Whole casein and whey proteins were fractionated from CM and GSM. beta-Lactoglobulin and the different caseins were isolated, purified and used to perform enzyme allergosorbent tests (EAST) and EAST inhibition studies with the sera of the allergic children . RESULTS: Clinical observations, skin prick testing and immunoglobulin (Ig)E-binding studies confirmed the diagnosis of GSM allergy without associated CM allergy. EAST determinations demonstrated that GSM allergy involves the casein fraction and not whey proteins. Cow's milk caseins were not at all or poorly recognized by the patient's IgE, while alphaS(1)-, alphaS(2)- and beta-caseins from GSM were recognized with a high specificity and affinity. In all cases, increasing concentrations of CM caseins failed to inhibit the binding of patient's IgE to sheep or goat milk caseins, whereas this binding was completely inhibited by GSM caseins . CONCLUSIONS: The characteristics of GSM allergy differ from those of the CM allergy because it affects older children and appears later. CM products do not elicit any clinical manifestation in GSM allergic patients, whereas CM allergic patients, usually cross-react to GSM. In all the GSM allergic children, the IgE antibodies recognized the caseins but not the whey proteins. Moreover, IgE specificity and affinity was high to GSM and lower to CM caseins despite their marked sequence homology. Doctors and allergic individuals should be aware that GSM allergy requires a strict avoidance of GSM and milk-derived products because reactions could be severe after ingestion of minimal doses of the offending food.

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[68] - Alonso-Lebrero E, Fuentes V, Zapatero L, Pérez-Bustamante S, Pineda F, Martinez-Molero MI. Goat's milk allergies in children following specific oral tolerance induction to cow's milk. Allergol Immunopathol (Madr) 2008;36:180-181

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[69] - Cases B, García-Ara C, Teresa B, Perez-Gordo M, Pastor C, Vivanco F et al. Allergy to goat's and sheep's milk: identification of allergenic proteins. Allergy 2009;64(Suppl. 90):368

Background: Cow's milk (CM) allergy is frequent in children and some of them react also to goat's and sheep's milk (GSM). There are reports that show IgE mediated allergy to GSM but not to CM. We report 3 children who presented GSM allergy but tolerated CM and its dairy products. Studies were performed in order to characterise the proteins that were involved in the GSM allergy. Methods: Three children (2 males, 1 female), aged 4-5 years, who presented immediate allergic manifestations (urticaria/angioedema, cough, stridor and dysnea) upon contact or after ingestion of minimal doses of GSM products but tolerated CM are studied. Skin prick test (whole milk and casein of CM, SM and GM) and specific IgE (CAP Pharmacia) to whole CM and its proteins, and whole SM and GM were carried out. SDS- PAGE of caseins and milk and inmunoblot assays were performed. Identification of protein bands by MALDI-TOF-MS or MSMS was carried out. Results: Skin prick test and IgE determination confirmed that GSM proteins but not CM proteins were involved in allergic symptoms of these three patients. In the inmunoblotting assays, patients' IgE reacted with several proteins bands (ranging from 15 to 60 kDa) from the caseins and milk of both sheep and goat. The analysis of these proteins turned out to be caseins, corresponding to a-S1 casein, a-S2 casein and .-casein. Conclusion: We report the identification of a-S1 casein, a-S2 casein and .-casein as allergens involved in GSM allergy with good tolerance to CM.

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[70] - Ah-Leung S, Bernard H, Bidat E, Paty E, Rancé F, Scheinmann P, et al. Allergy to goat and sheep milk without allergy to cow’s milk. Allergy 2006;61:1358-1365

BACKGROUND: Cow's milk (CM) allergy is the most frequent cause of food allergy in infants. Most children who are allergic to CM are also sensitized to whey proteins and/or to the casein fraction and many of them cannot tolerate goat's or sheep's milk (GSM) either. Conversely, the GSM allergies that are not associated with allergic cross-reactivity to CM are rare . METHODS: Twenty-eight children who had severe allergic reactions, including anaphylaxis, after consumption of GSM products but tolerated CM products were recruited in a retrospective study. Whole casein and whey proteins were fractionated from CM and GSM. beta-Lactoglobulin and the different caseins were isolated, purified and used to perform enzyme allergosorbent tests (EAST) and EAST inhibition studies with the sera of the allergic children . RESULTS: Clinical observations, skin prick testing and immunoglobulin (Ig)E-binding studies confirmed the diagnosis of GSM allergy without associated CM allergy. EAST determinations demonstrated that GSM allergy involves the casein fraction and not whey proteins. Cow's milk caseins were not at all or poorly recognized by the patient's IgE, while alphaS(1)-, alphaS(2)- and beta-caseins from GSM were recognized with a high specificity and affinity. In all cases, increasing concentrations of CM caseins failed to inhibit the binding of patient's IgE to sheep or goat milk caseins, whereas this binding was completely inhibited by GSM caseins . CONCLUSIONS: The characteristics of GSM allergy differ from those of the CM allergy because it affects older children and appears later. CM products do not elicit any clinical manifestation in GSM allergic patients, whereas CM allergic patients, usually cross-react to GSM. In all the GSM allergic children, the IgE antibodies recognized the caseins but not the whey proteins. Moreover, IgE specificity and affinity was high to GSM and lower to CM caseins despite their marked sequence homology. Doctors and allergic individuals should be aware that GSM allergy requires a strict avoidance of GSM and milk-derived products because reactions could be severe after ingestion of minimal doses of the offending food.

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[71] - Feuillet-Dassonval C, Rossignol B, Baranes T, Sabatié-Garat H, Bidat E. Allergie au lait de chèvre et de brebis: pièges, astuces, conseils. Alim'Inter 2007;12:23-25

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[72] - d'Auria E, Agostoni C, Giovannini M, Riva E, Zetterstrom R, Fortin R, et al. Proteomic evaluation of milk from different mammalian species as a substitute for breast milk. Acta Paediatr 2005;94:1708-1713

As milk represents the main source of nutrition for infants, the question of an effective human milk substitute becomes mandatory when a formula-fed baby is allergic to cows' milk proteins. In this case, formulas containing extensively hydrolysed milk proteins should be preferred, but even such a formula may cause allergic reactions in highly sensitive patients. If there is evidence of cows' milk allergy with IgE-associated symptoms, after 6 mo of age, a soy bean formula may be recommended only when tolerance to soy protein has been established by clinical challenge. In infants with allergic reactions to cows' milk proteins, even after extensive hydrolysation, proteomic techniques coupled to immunological methods may make it possible to select other milk products that do not contain the same allergens as ordinary cow's milk. In this paper, evidence will be presented that proteomic evaluation of proteins from different mammalian species may be a suitable method of testing whether proteins from the milk of diff erent mammalian species may be used as a substitute for untreated bovine milk.Conclusion: Proteomic evaluation of milk from different mammalian species may not only be of help when recommending suitable feeding in cases of cows' milk allergy but also gives new insight into the background to allergic reactions caused by milk proteins

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[73] - Restani P, Gaiaschi A, Plebani A, Beretta B, Cavagni G, Fiocchi A, et al. Cross-reactivity between milk proteins from different animal species. Clin Exp Allergy 1999;29:997-1004

BACKGROUND: Cow's milk allergy is quite frequent in the first years of human life. When breast-feeding is not possible, a cow's milk substitute must be provided for allergic subjects. Different alternatives to cow's milk have been suggested as protein sources (soy, hydrolysed proteins, goat's milk, etc.), but all these dietetic solutions are not without risks for polyallergic or more sensitive subjects. OBJECTIVE: To obtain new information on the suitability of other mammalian milks for allergic children, we evaluated the cross-reactivity between milk proteins from different animal species. METHODS: Milk samples were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). To detect antibody-antigen complexes, immunoblotting was performed by using sera from children allergic to cow's and ewe's milk (RAST class >/= 4) and monoclonal antibodies (MoAb) specific for bovine proteins (caseins and beta-lactoglobulin). RESULTS: IgEs from children allergic to cow's milk are capable of recognizing most part of milk proteins from mammals bred in European countries (ewe, goat, buffalo), while no serum used in this study contains IgEs reacting with camel's milk proteins. Camel's milk was also not recognized from circulating IgEs from a child specifically allergic to ewe's milk. Specific antibovine monoclonal antibodies cross-reacted with proteins from other mammalian species, apart from those of camel. CONCLUSIONS: Homologies in amino acidic composition could justify the cross-reactivity observed between proteins from different animal species. On the other hand, the phylogenetic difference could be responsible for the failed recognition of camel's proteins by circulating IgEs and monoclonal antibodies.

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[74] - Suutari TJ, Valkonen KH, Karttunen TJ, Ehn BM, Ekstrand B, Bengtsson U, et al. IgE Cross Reactivity Between Reindeer and Bovine Milk ß-Lactoglobulins in Cow’s Milk Allergic Patients. J Investig Allergol Clin Immunol 2006;16:296-302

BACKGROUND: Allergic reactions to cow's milk are common in small children. One of the main protein allergens found in cow's milk is beta-lactoglobulin (beta-Lg). Reindeer and bovine milk both contain related beta-Lg proteins, but the allergenicity of reindeer beta-Lg has not previously been studied. The purpose of this study was to analyze the immunological cross-reactivity of IgE antibodies from children with cow's milk allergy to reindeer and bovine beta-Lg . METHODS: Sera from 17 children and a serum pool of 4 patients with elevated cow's milk-specific IgE were investigated. Beta-Lg from bovine and reindeer milk was isolated in native form and an enzyme-linked immunosorbent inhibition assay was developed. Bovine beta-Lg was used as a capturing antigen and the inhibiting effects of reindeer and bovine beta-Lg on the IgE binding were measured . RESULTS: Cross-reactivity patterns of bovine milk beta-Lg specific IgE to reindeer beta-Lg varied among patients. In general, reindeer beta-Lg showed significantly lower inhibition (mean 43%) of IgE binding to the capturing antigen than did bovine beta-Lg (mean 89%). In some patients, even high concentrations of reindeer beta-Lg only partly eliminated the IgE binding to bovine beta-Lg . CONCLUSIONS: The partial cross-reactivity of human anti-bovine IgE with reindeer beta-Lg suggests that it lacks important bovine epitopes and those that are recognized are only weakly bound.

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[75] - Katz Y, Zadik-Mnuchin G. Individuals With Cow's Milk Allergy Are Sensitized to Milk From Deer and Ibex But Not to Milk From Pork. Is There a “Kosher” Epitope? AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°526

Rationale There is almost a complete cross reactivity between cow's milk allergy (CMA) and allergy to milk from goat, ewe and buffalo, all „kosher‰ animals but no cross reactivity to milk from camel and mare, all „non Kosher‰. Here cross reactivity between sensitization to cow's milk and sensitization to milk from deer, ibex („Kosher‰) and pork were tested. Method s : Thirty patients, aged 4 weeks to 30 years, 23 with CMA with typical history and positive skin prick test (SPT) and 7 without CMA were studied. SPT to Cow's milk, and milk from the following: deer, ibex, pork, buffalo, and to histamine and negative control were performed. Result s : Among the 23 patients with CMA all 23 reacted to deer's milk, 21/21 reacted to ibex, 4/21 to pork and 21/21 to buffalo. Among the seven patients without CMA none reacted to any of the SPT except for the histamine. Spearman's rho correlation coefficient between CMA and deer and ibex's milk sensitization were 1 (p=0) and between cow's milk to pork's milk was 0.269, non significant (p=0.162). Conclusions : We assume that milk proteins originated from cow, deer and ibex share a common epitope (or epitopes). If one wishes to draw conclusions regarding clinical allergy an oral challenge has to be carried out.

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[76] - Bernard H, Creminon C, Negroni L, Peltre G, Wal JM. IgE Cross-reactivity with Caseins from Different Species in Humans Allergic to Cow"s Milk. Food Agric Immunol 1999;11:101-111

Fifty-eight sera from humans allergic to cow"s milk proteins were analysed for the specificity of their IgE response to the whole casein fraction of milk from different ruminant and nonruminant species (e.g. cow, sheep, goat, rabbit and rat). IgE-specific responses were determined by an enzyme allergosorbent test using the purified casein fractions as immobilized antigen and an anti-human IgE monoclonal antibody labelled with acetylcholinesterase. Co-and/or cross-sensitizations to caseins of the different ruminant species occurred extensively, though IgE responses to ovine and caprine casein appeared to be lower than that obtained with bovine casein. Cross-reactivity is suggested by the significant reactivity of rat and rabbit casein toward human IgE. In terms of specificity and intensity, the IgE response to caseins demonstrates a great variability. Structural homologies in caseins of such different species, that can share common epitopes for the IgE of some patients, suggest that prevention of cow"s milk allergy cannot be achieved by using milk from other species as substitutes.

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[77] - Bernard H, Wal JM. Comparaison de la composition protéique du lait de différentes espèces et de la structure des principales protéines. Alim'Inter 1998;3(11):14-17

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[78] - Businco L, Giampietro PG, Lucenti P, Lucaroni F, Pini C, Di Felice G, et al. Allergenicity of mare's milk in children with cow's milk allergy. J Allergy Clin Immunol 2000;105:1031-1034

Cow's milk allergy is a common disease of infancy and early childhood. If the baby is not breast-fed, a substitute for cow's milk formula is necessary. OBJECTIVE: The aim of this study was to investigate, in vitro and in vivo, the allergenicity of mare's milk in a population of selected children with severe IgE-mediated cow's milk allergy. METHODS: Twenty-five children (17 male and 8 female) aged 19 to 72 months (median age 34 months) with IgE-mediated cow's milk allergy were selected for this study. All the children underwent skin prick tests with cow's milk and mare's milk and double-blind placebo-controlled oral food challenge (DBPCOFC) with fresh cow's milk, fresh mare's milk, and, as placebo, a soy formula (Isomil, Abbott, Campoverde, Italy). We performed immunoblotting of cow's and mare's milk developed with IgE from allergic children. RESULTS: All the children showed strong positive skin test responses to cow's milk (4+); 2 children had positive skin test responses to mare's milk (2+). All children had positive DBPCOFCs to cow's milk; one child had a positive DBPCOFC to mare's milk. No children reacted to the placebo (Isomil). In the cow's milk, some proteins are able to strongly react with human IgE; when the sera are tested with mare's milk, the bands corresponding to the same proteins are recognized by a lower percentage of sera. CONCLUSION: These data suggest that mare's milk can be regarded as a good substitute of cow's milk in most children with severe IgE-mediated cow's milk allergy. It would be prudent, however, to confirm its tolerability by a supervised titrated oral challenge test.

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[79] - Docena G, Rozenfeld P, Fernández R, Fossati CA. Evaluation of the residual antigenicity and allergenicity of cow's milk substitutes by in vitro tests. Allergy 2002;57:83-91

Background: This study aimed the to investigate presence of residual allergenic cow's milk proteins (CMP) in some milk substitutes employed in the treatment of cow's milk allergy (CMA). These allergens may interfere with the treatment, and elicit allergic reactions in sensitized individuals. Methods: The protein composition of the different extracts was evaluated by Lowry's method and tricine SDS-PAGE. Different immunoenzymatic methods were used (ELISA, EAST and immunoblotting) to quantify total serum IgE and specific serum IgE, as well as to detect the presence of antigenic and allergenic components. Results: The results showed a higher protein content in mammalian milks (cow, sheep, mare, goat, and human) than in hydrolyzed substitutes (partially or extensively hydrolyzed casein or whey proteins). Residual native, processed, or contaminant polypeptides have been identified in the moderate hydrolysates, whereas extensive hydrolysates did not show the presence of residual components by immunoblotting. However, specific antibodies with capacity to bind to peptides have been detected by EAST and ELISA, suggesting that extensive hydrolysates contain residual peptides that preserve immunoreactive epitopes. We were unable to demonstrate either residual antigenicity or allergenicity in an amino-acid-based formula. Conclusions: Immunoenzymatic methods were used to detect the presence of cross-reactive components in mammalian milks. Residual allergenic components from cow's milk could be identified in both the moderate and extensive hydrolysates analyzed. This information may be relevant to the treatment of CMA.

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[80] - Schuller A. Anaphylaxie sévère persistante au lait de vache: le rôle favorisant de l'effort. Alim'Inter 2004;9:88-89

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[81] - Broekaert SM, Darsow U, Ollert M, Ring J, Krause I, Schulmeister U, et al. Anaphylactic shock caused by buffalo's mozzarella cheese. Ann Allergy Asthma Immunol 2008;101:105-107

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[82] - Sheehan WJ, Phipatanakul W. Tolerance to water buffalo milk in a child with cow milk allergy. Ann Allergy Asthma Immunol 2009;102:349

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[83] - Tesse R, D’Alessandro A, Paglialunga C, Braccio S, Martemucci G, Armenio L. Does donkey’s milk may represent an altrnative dietary support in patients with cow’s milk-induced IgE-mediated and non-IgE-mediated disorders ? Case reports. Allergy 2008;63(suppl. 88):183-184

Cow milk is one of the first foods introduced into an infant‚s diet, and cow milk allergy (CMA) is the most prevalent food allergy in the first year of life. Immunoglobulin (Ig)-E mediated allergy accounts for about 60% and non-IgE mediated hypersensitivity accounts for about 40% of adverse reactions to milk, with the majority of IgE mediated reactions involving the skin, and the majority of non-IgE mediated reactions involving the gastrointestinal tract. Avoidance of cow milk (CM) implies the use of alternative dietary supports such as mammalian milks.- Cases: A 3-year-old female child came to our referral Center of Allergy in September 2007. She presented with signs and symptoms of urticaria. Skin prick tests (SPTs) documented a CM proteins sensitisation. We assessed the presence of milk specific serum IgE antibodies, such as casein (1.70 kUA/L), b-lactoglobulin (0.43 kUA/ L), a-lactalbumin (0.45 kUA/L), by Uni- CAP System fluorescent enzyme immunoassay, and found that total IgE levels were high (81 kU/L). A 4-year-old male child, who presented with diarrhea, emesis, abdominal pain, and failure to thrive, also referred in the same period to our Center. SPTs were negative, IgE levels were high (440 kU/L), in the absence of detectable milk-specific serum IgE. Diagnosis of CMA was confirmed in both children by a positive double-blind, placebo-controlled cow‚s milk challenge, following an improvement of signs when the CM proteins were eliminated from the diet of the children. In December 2007, both patients with the IgE mediated and non-IgE mediated CMA, respectively, were enrolled in a longitudinal study which aims to assess the tolerability and the clinical effect of donkey‚s milk in patients with CMA. The ass‚s milk derived by an Apulia donkey‚s race was administered to both children as a single-blind, controlled, donkey‚s milk challenge. Challenges resulted negative in both patients, and after 4 weeks of ass‚s milk dietary intake, neither adverse reactions, nor cutaneous and/or gastrointestinal symptoms were reported by the parents of the children. These two cases highlights the fact that donkey‚s milk seems to be well tolerated in subjects with both forms of CMA, the IgE mediated and the non-IgE mediated. On the basis of these preliminarily results, our purpose is to confirm the safety of donkey‚s milk proteins in a larger group of patients with CMA.

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[84] - Martinez D, Castillo R. Allergy to cheese produced from sheep's and goat's milk in a teenager unaffected by cow's milk protein allergy. Allergy 2007;62(suppl. 83):354

A 15 year old female affected by atopic dermatitis that treated by intermitent pimecrolimus, no affected by rhinitis or Asthma, developed episodes of generalizated urticaria immediately after ingestion of pizza, lasagna and cheese, without clinical manifestation after drinking milk or other products made from cow's milk, she has never drinking another types of milk (sheep, goat, mare∑etc) Skin test were carried out using a battery of commons inhalants (duste mite, mold, pollens, animals) and foods allergens (milk, egg, fruits, dried fruits, types of flour, shellfish) were only positive to cat (8mm) and were also negative to whole milk, a- lactalbumin, ßlactoglobulin and cow's casein. Skin prick by prick test with types or cheese made of cow's, sheep's and goat's milk were positive to goat and sheep. (table 1). Total Ig E level were normal (91.80UI/ml), blood differential count showed 200 eosinophils/mm3 (normal) and specific Ig E (CAP Pharmacia) to cow's casein 1.26 L/l, cheddar cheese 0.96Ku/l, sheep's milk 7.41 Ku/l, and negative to a-lactalbumin, ß- lactaglobulin, cambrie cheese, cow's serum and mare's milk. (table 2) Oral challenge was not performed for ethical reason, the patient actually have and exclusion diet of goat's and sheep's milk without clinical urticaria episodes. Allergy to sheep's or goat's milk cheese has already been described in children affected by cow's milk allergy whereas allergy to sheep or goat without cow's protein is quite rare; the first case report by Johansson in 1995; the hypothesize that sheep's milk may have some antigenic determinant in common with goat's milk but different from that in cow's milk, moreover goat casein seems to be responsible for most of cross reactivity with sheep casein. Goat's and sheep's milk is preferred in the treatment of children affected by cow's milk allergy, is considered more natural and less allergenic; at that time is neccesary to discard that is not sensitized to that proteins before autorized since there are patients allergic to other mammals without clinical to cow's protein or with asintomatic sensitized to cow's milk that tolerated drinking or eating these dairy products but not goat or sheep.

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[85] - Iacono G, Caroccio A, Cavataio F, Montalto G, Soresi M, Balsamo V. Use of ass' milk in multiple food allergy. J Pediatr Gastroenterol Nutr 1992;14:177-181

We report a study of realimentation techniques in 9 unweaned infants with multiple food hypersensitivity. The patients had presented severe symptoms of cow's milk allergy and successive attempts using milk containing soy protein and/or a semielemental formula in their alimentation did not improve their clinical condition, due to the onset of hypersensitivity to these allergens as well. After a short period of parenteral alimentation the infants were refed per os with ass' milk (250 ml/kg/day) + medium chain triglycerides (40 ml/L milk). This food was well tolerated by all patients. No negative clinical reactions were recorded and during hospitalisation average weight increase was 39.8 g/day. The follow-up of the patients showed that ass' milk was tolerated without any problems up to an age ranging from 15 to 20 months, when cow's milk was reintroduced in some patients.

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[86] - Carroccio A, Cavataio F, Iacono G. Cross-reactivity between milk proteins of different animals. Clin Exp Allergy 1999;29:1014-1016

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[87] - Carroccio A, et al Intolerance to hydrolysed cow's milk proteins in infants: clinical characteristics and dietary treatment. Clin Exp Allergy 2000;30:1597-1603

BACKGROUND: Multiple food intolerance in infants, including intolerance to extensively hydrolysed proteins (HP), is often difficult to treat. However, few data have been reported on clinical outcome and dietary treatment of these patients. AIMS: To evaluate the clinical characteristics of patients with HP-intolerance and the long-term outcome of treatment with ass' milk. PATIENTS AND METHODS: This study included 21 HP-intolerant infants (15 males, median age at diagnosis 2 months) treated with an ass' milk-based diet and 70 cow's milk (CM) intolerant infants (40 males, median age at diagnosis 3 months) treated with casein hydrolysate milk-based diet. All patients were followed-up for a median period of 4 years. Both HP-intolerance and intolerance to other foods were diagnosed according to the double-blind placebo-controlled procedure. Formal CM-challenges were conducted at yearly intervals until tolerance was demonstrated. At diagnosis and after one year of the respective diets, the following growth parameters were determined: relative weight for sex and age, relative weight for height and height z-score. RESULTS: During the study period, multiple food intolerance was documented in 21/21 HP-intolerant infants (ass' milk group) and in 20/70 infants with CM-intolerance but tolerating HP (casein hydrolysate group) (P < 0.0001). In the ass' milk group, the more frequent food intolerances were toward soya, oranges, tomatoes and fish; goat's milk intolerance was demonstrated in five out of six patients receiving this food, and sheep's milk derivatives intolerance in four out of seven; these patients tolerated ass' milk. During the study period 3/21 patients in the ass' milk group became ass' milk intolerant; they showed vomiting (one cases) or diarrhoea (two cases). A lower percentage (52%) of patients in the ass' milk group became CM-tolerant during the study period than in the casein hydrolysate group (78%) (P < 0.01) and the age of the children at CM-tolerance was higher in the ass' milk than in the casein hydrolysate-treated children (P < 0.05). At diagnosis, a higher frequency of cases with elevated serum total IgE and specific IgE to CM antigens (P < 0.01) was observed in the ass' milk group. No difference was recorded between the two treatment groups in any of the growth parameters considered either at diagnosis or during the follow-up. CONCLUSIONS: HP-intolerant patients showed a higher frequency of persistent food intolerance and of multiple food intolerance than patients tolerating casein hydrolysate. Ass' milk feeding was confirmed as a safe and valid treatment of the most complicated cases of multiple food intolerance

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[88] - Monti G, Bertino E, Muratore MC, Coscia A, Cresi F, Silvestro L, et al. Efficacy of donkey’s milk in treating highly problematic cow’s milk allergic children: An in vivo and in vitro study. Pediatr Allergy Immunol 2007;18:258-264

Successful therapy in cow's milk protein allergy rests on completely eliminating cow's milk proteins from the child's diet: it is thus necessary to provide a replacement food. This prospective study investigated tolerance of donkey's milk in a population of 46 selected children with cow's milk protein allergy, for whom it was not possible to use any cow's milk substitute. Thirty-eight children (82.6%) liked and tolerated donkey's milk at the challenge and for the entire duration of follow-up. Catch-up growth was observed in all subjects with growth deficit during cow's milk proteins challenge. The degree of cross-reactivity of immunoglobulin E (IgE) with donkey's milk proteins was very weak and aspecific. Donkey's milk was found to be a valid alternative to both IgE-mediated and non-IgE-mediated cow's milk proteins allergy, including in terms of palatability and weight-height gain.

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[89] - Vita D, Passalacqua G, Di Pasquale G, Caminiti L, Crisafulli G, Rulli I, et al. Ass’s milk in children with atopic dermatitis and cow’s milk allergy: Crossover comparison with goat’s milk. Pediatr Allergy Immunol 2007;18:594-598

Cow milk allergy is a common disease of infancy, often associated with atopic dermatitis (AD). Avoidance of cow milk (CM) implies the use of alternative dietary supports such as mammalian milks. In this study, we assessed the tolerability and clinical effect of ass's milk (AM), when compared with the largely used goat's milk (GM) in a single-blind, controlled, randomized crossover. Twenty-eight children with AD and ascertained allergy to CM were enrolled. The children were randomized to AM or GM for 6 months, then switched to the other milk for further 3 months. The SCORAD index (SI) and a visual analog scale (VAS) were evaluated blindly. After termination of the study, food challenges with GM and AM were performed. An SDS-PAGE analysis of different milks was performed. Two children from the GM group dropped out after randomization and 26 completed the study. Ass milk invariantly led to a significant improvement of SI and VAS of symptoms (p < 0.03 vs. baseline and inter-group), whereas GM had no measurable clinical effect. At the end of the study 23 of 26 children had a positive food challenge with GM and one of 26 with AM. Ass's milk had a protein profile closer to human milk than GM. Ass milk is better tolerated and more effective than GM in reducing symptoms of AD. It may represent a better substitute of CM than the currently used GM.

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[90] - Rodriguez Pacheco R, Negro Alvarez J, Peña Arellano M, David Garcia D, Postigo Resa I, Martinez Quesada J. Anaphylaxis due to donkey milk. Allergy 2008;63(suppl. 88):292

Background: We present a clinical case of a patient who presented anaphylaxis after oral exposure to donkey milk. Methods: A 44-year-old male who, since 1985, has presented perennial rhinoconjunctivitis and asthma mainly related to contact with horses. The patient works as horse breeder and is always in contact with them. After several specific immunotherapy (SIT) cycles the patient tolerates contact with horses better, his rhinoconjunctivitis improving considerably and his asthma being adequately controlled with Budesonide/Formoterol 160/4.5, 2 puffs every 12 h and Montelukast 10 mg 1 tablet a day. On September 2004, the patient got milk from a donkey which had delivered a month before, dipped the finger in the glass and took it to his mouth in order to try the milk. Two minutes later he started with the following symptoms: mouth, face and eyelid angioedema. Face and scalp pruritus as well as generalised urticaria. Aphonia and feeling of laryngeal tightening. Wheezing and dyspnea. Vomiting. The clinical picture required emergency care with the administration of parenteral corticosteroids and antihistamines. The patient tolerates cow and other types of milk. Results: Skin prick test for airborne allergens common in the Murcia region: positive to horse epithelium, cat epithelium and grass. Skin prick test for cow milk, casein, chicken, beef and pork: negative. Skin prick test for donkey milk: positive. sIgE DETERMINATION BY UNICAP 100 AGAINST: cat 0.4 KU/L, horse epithelium 14.32 KU/L, grass 2.91 KU/L, donkey milk 34.7 KU/L, donkey milk alpha-lactalbumin 26 KU/L, donkey milk beta-lactalbumin 41.3 KU/L, horse serum proteins e205 4100 KU/L. sIgE DETERMINATION BY SDS-PAGE IMMUNOBLOTTING AGAINST: donkey milk, donkey milk alpha-lactalbumin and donkey milk betalactoglobulin. Bands mainly corresponding to donkey milk alpha-lactalbumin and donkey milk beta-lactoglobulin are detected. Other bands detected correspond to caseins and lactoferrin. Conclusion: We have presented a case of sensitisation to donkey milk proteins. It is possible that the patient‚s sensitisation to donkey milk was secondary to horse epithelium sensitisation. We have found only two similar cases described due to the little consumption there is of this milk by humans.

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[91] - Docena G, Rozenfeld P, Fernández R, Fossati CA. Evaluation of the residual antigenicity and allergenicity of cow's milk substitutes by in vitro tests. Allergy 2002;57:83-91

Background: This study aimed the to investigate presence of residual allergenic cow's milk proteins (CMP) in some milk substitutes employed in the treatment of cow's milk allergy (CMA). These allergens may interfere with the treatment, and elicit allergic reactions in sensitized individuals. Methods: The protein composition of the different extracts was evaluated by Lowry's method and tricine SDS-PAGE. Different immunoenzymatic methods were used (ELISA, EAST and immunoblotting) to quantify total serum IgE and specific serum IgE, as well as to detect the presence of antigenic and allergenic components. Results: The results showed a higher protein content in mammalian milks (cow, sheep, mare, goat, and human) than in hydrolyzed substitutes (partially or extensively hydrolyzed casein or whey proteins). Residual native, processed, or contaminant polypeptides have been identified in the moderate hydrolysates, whereas extensive hydrolysates did not show the presence of residual components by immunoblotting. However, specific antibodies with capacity to bind to peptides have been detected by EAST and ELISA, suggesting that extensive hydrolysates contain residual peptides that preserve immunoreactive epitopes. We were unable to demonstrate either residual antigenicity or allergenicity in an amino-acid-based formula. Conclusions: Immunoenzymatic methods were used to detect the presence of cross-reactive components in mammalian milks. Residual allergenic components from cow's milk could be identified in both the moderate and extensive hydrolysates analyzed. This information may be relevant to the treatment of CMA.

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[92] - Schulmeister U, Swoboda I, Quirce S, de la Hoz B, Ollert M, Pauli G, et al. Sensitization to human milk. Clin Exp Allergy 2008;38:60-68

BACKGROUND: Allergy to milk is one of the earliest manifestations of IgE-mediated allergies and affects about 2.5% of newborn children. Several reports indicate that milk-allergic patients may be sensitized also to human milk proteins . OBJECTIVE: To analyse the specificity and possible biological relevance of IgE reactivity to human milk antigens in milk-allergic patients . METHODS: The specificity of IgE reactivity to cow's milk and human milk antigens was analysed with sera from milk-allergic children and adults by IgE immunoblotting. IgE cross-reactivity between milk antigens was studied by immunoblot inhibition experiments. That IgE reactivity to human milk antigens is not due to alloreactivity or due to the transmission of foreign antigens into mother's milk was demonstrated through the analysis of milk samples from genetically unrelated mothers before and after intake of dietary milk products. The biological relevance of IgE reactivity to human milk was confirmed by skin testing. Results IgE antibodies to human milk were found in more than 80% of the tested milk-allergic patients. Cross-reactive IgE-reactive human antigens such as alpha-lactalbumin and non-cross-reactive human milk antigens were identified. Immediate-type skin reactions could be elicited with human milk samples in patients with IgE reactivity to human milk . CONCLUSION: IgE reactivity to human milk in milk-allergic patients can be due to cross- sensitization and genuine sensitization to human milk and may cause allergic symptoms. IgE-mediated sensitization to human milk is common in milk-allergic patients and may require diagnostic testing and monitoring.

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[93] - Maynard F, Chatel JM, Wal JM. Immunological IgE cross-reactions of bovine and human [alpha]-lactalbumins in cow's milk allergic patients. Food Agric Immunol 1999;11:179-189

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[94] - Cantisani A, Giuffrida MG, Fabris C, Bertino E, Coscia A, Oggero R, et al. Detection of specific IgE to human milk proteins in sera of atopic infants. FEBS Lett 1997;412:515-517

Specific IgE (sIgE) for cow's milk proteins (CMP) have been reported to be present in blood sera of exclusively breast-fed infants. The aim of this study was to find whether the presence of sIgE to human milk proteins in the sera of exclusively breast-fed infants could explain the apparent detection of sIgE to CMP in infants that were never previously in contact with cow's milk. sIgE for human milk whey proteins were found in the blood sera of atopic infants, and these sIgE strongly cross-reacted with the corresponding CMP. In none of the sera examined were sIgE to bovine beta-lactoglobulin detected

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[95] - Bernard H, Negroni L, Chatel JM, Clement G, Adel-Patient K, Peltre G, et al. Molecular basis of IgE cross-reactivity between human beta-casein and bovine beta-casein, a major allergen of milk. Mol Immunol 2000;37:161-167

Twenty patients allergic to cow's milk proteins and with high levels of specific IgE directed against bovine whole casein were selected to evaluate reactivity of their IgE antibodies with human beta-casein. Highly purified human and bovine beta-caseins were prepared by selective precipitations and FPLC separation. Their identity and purity were assessed by HPLC, analysis of amino acid composition, sequencing of the five N-terminal amino acid residues and immunochemical tests. Direct and indirect ELISAs were performed using human and bovine beta-casein coated into microtiter plates and monoclonal anti-human IgE antibody AChE labelled for revelation. Seven sera contained specific IgE directed against human beta-casein. Inhibition studies using native human and bovine beta-caseins as well as bovine beta-casein-derived peptides demonstrated that, depending on the sera, one or several common epitopes located in different parts of the molecule were shared by the two homologous proteins.

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[96] - Cantisani A, Giuffrida MG, Fabris C, Bertino E, Coscia A, Oggero R, et al. Detection of specific IgE to human milk proteins in sera of atopic infants. FEBS Lett 1997;412:515-517

Specific IgE (sIgE) for cow's milk proteins (CMP) have been reported to be present in blood sera of exclusively breast-fed infants. The aim of this study was to find whether the presence of sIgE to human milk proteins in the sera of exclusively breast-fed infants could explain the apparent detection of sIgE to CMP in infants that were never previously in contact with cow's milk. sIgE for human milk whey proteins were found in the blood sera of atopic infants, and these sIgE strongly cross-reacted with the corresponding CMP. In none of the sera examined were sIgE to bovine beta-lactoglobulin detected

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[97] - Jarvinen KM, Sampson HA. Recognition of Human Milk Peptides by IgE Antibodies from Infants with Cow's Milk Allergy. J Allergy Clin Immunol 2008;121:S214

RATIONALE: Occasionally, exclusively breastfed, cow‚s milk-allergic infants continue to be symptomatic despite strict maternal milk avoidance. We sought to determine whether this phenomenon could be due to sensitization against human milk proteins. METHODS: We generated ten peptides (10-12 amino acid in length), representing known bovine milk IgE-binding epitopes of alpha-lactalbumin, beta-, and kappa-casein and the corresponding, highly similar human milk peptides (differing by 1 to 5 amino acids) on the SPOT membranes. The peptides were labeled with sera from 9 breastfed milk-allergic infants (milk-IgE <0.35 to 15.9 kUA/L) who were asymptomatic and 6 infants (milk IgE 100 kUA/L) who were symptomatic during maternal milkelimination diet; aged 3 weeks to 12 months. RESULTS: Human milk peptides were generally less often bound by IgE from milk-allergic infants than the homologous bovine milk peptides. Nine patients had peptide-specific IgE to human alpha-lactalbumin, four to human beta-casein and twelve to human kappa-casein. At least one human milk peptide was strongly bound by IgE from 4/6 symptomatic infants and by 3/9 asymptomatic infants. Interestingly, two infants who were symptomatic while their mothers avoided cow‚s milk had IgE specific to a human beta-casein peptide but none or little to the corresponding bovine counterpart. CONCLUSIONS: These data suggest that human milk proteins are recognized by the IgE from the majority of milk-allergic infants who continue having symptoms despite maternal milk avoidance, although some asymptomatic infants also possess some human milk-specific IgE. The clinical significance of these IgE antibodies requires further investigation in a functional assay.

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[98] - Bernard H, Negroni L, Chatel JM, Clement G, Adel-Patient K, Peltre G, et al. Molecular basis of IgE cross-reactivity between human beta-casein and bovine beta-casein, a major allergen of milk. Mol Immunol 2000;37:161-167

Twenty patients allergic to cow's milk proteins and with high levels of specific IgE directed against bovine whole casein were selected to evaluate reactivity of their IgE antibodies with human beta-casein. Highly purified human and bovine beta-caseins were prepared by selective precipitations and FPLC separation. Their identity and purity were assessed by HPLC, analysis of amino acid composition, sequencing of the five N-terminal amino acid residues and immunochemical tests. Direct and indirect ELISAs were performed using human and bovine beta-casein coated into microtiter plates and monoclonal anti-human IgE antibody AChE labelled for revelation. Seven sera contained specific IgE directed against human beta-casein. Inhibition studies using native human and bovine beta-caseins as well as bovine beta-casein-derived peptides demonstrated that, depending on the sera, one or several common epitopes located in different parts of the molecule were shared by the two homologous proteins.

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[99] - Schulmeister U, Swoboda I, Quirce S, Ollert M, Pauli G, Valenta R, et al. IgE autoreactivity to human milk allergens due to co- and cross-sensitisation to cow’s milk proteins. Allergy Clin Immunol Int 2005;17(Suppl. 1):357

Cow's milk is one of the common causes of IgE-mediated food allergy. Several cow's milk allergens have been characterised and show sequence similarity to human milk proteins. However, IgE reactivity to human milk has not yet been intensively analysed. Out of 18 sera from cow's milk allergic patients 15 showed detectable IgE reactivity to several human milk antigens with molecular weights ranging from 14 to 85 kDa. Some of the IgE-reactive human milk antigens were of comparable size as the IgE-reactive components in cow's milk, whereas for others no IgE-reactive counterpart of similar size was detected in cow's milk. We noted that IgE reactivity was preferentially directed against denatured but not against native human milk proteins. The fact that patients showed identical IgE binding patterns to milk antigens from different mothers, indicated that the observed IgE reactivity was neither due to alloreactivity nor to the presence of bovine milk proteins secreted into human milk. Immunoblot inhibition experiments demonstrated that the reaction with human milk proteins can be due to cross-reaction with cow's milk proteins. One of the cross-reactive allergens was identified as alpha-lactalbumin. In contrast, inhibition experiments also showed that for other patients IgE reactivity to human milk proteins is due to a genuine sensitisation to human milk allergens. We thus conclude that IgE autoreactivity to human milk allergens is due to autosensitisation and/or cross-reactivity with cow's milk proteins.

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[100] - Palma Carlos S, Matos V, Loureiro V, Leiria Pinto P. Breast milk anaphylaxis – a case report. Allergy 2008;63(suppl. 88):124

Background: Cow‚s milk allergy (CMA) is the most common food allergy in children. In rare cases, infants with CMA don‚t tolerate breast feeding. In some studies beta-lactoglobulin (BLG) was detected in breast milk. A great intra and inter-individual breast milk BLG concentration seems to exist, without relation with mother‚s daily cow milk intake. After intense investigation through available documentation, it was not possible to find any reported case about breast milk anaphylaxis or loss of tolerance to it. Case report: A 27 months old girl having CMA with urticaria since 4 months old is being followed in our department since 5 months old. Skin prick test (SPT) were positive to milk, BLG, alpha lactoalbumin (ALA) and casein (CAS). Total IgE was high (115 KU/L) and specific IgE were elevated (milk 17.5 KU/L, BLG 16.9KU/ L, CAS 4.55KU/L and ALA 15.2 KU/L). She was breastfeed since birth until 9 months old. Despite cow‚s milk avoidance diet had been performed, SPT and specific IgE were positive. At 25 months old, her mother had a baby and starts breastfeeding him. One month later, the mother gave her daughter 200 cc of breast milk. The child immediately began coughing followed by urticaria, angioedema, vomiting, shortness of breath and stridor. Medical treatment with epinephrine, b2 agonist and antihistamines was required. Breast milk SPT was positive. Mother‚s cow milk daily intake was 500 cc or equivalent. Discussion: The natural evolution of breast milk tolerance in children with CMA that had tolerated breast feeding in early infancy is unknown. The mechanisms involved in breast milk tolerance in children with CMA, are poorly understood. In this case, a loss of breast milk tolerance in a CMA was observed after an eviction period. This was a serious clinical picture and the clinic shall be aware to advise children‚s parents for this possibility.

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[101] - Schulmeister U, Swoboda I, Quirce S, Ollert M, Pauli G, Valenta R, et al. IgE autoreactivity to human milk allergens due to co- and cross-sensitisation to cow’s milk proteins. Allergy Clin Immunol Int 2005;17(Suppl. 1):357

Cow's milk is one of the common causes of IgE-mediated food allergy. Several cow's milk allergens have been characterised and show sequence similarity to human milk proteins. However, IgE reactivity to human milk has not yet been intensively analysed. Out of 18 sera from cow's milk allergic patients 15 showed detectable IgE reactivity to several human milk antigens with molecular weights ranging from 14 to 85 kDa. Some of the IgE-reactive human milk antigens were of comparable size as the IgE-reactive components in cow's milk, whereas for others no IgE-reactive counterpart of similar size was detected in cow's milk. We noted that IgE reactivity was preferentially directed against denatured but not against native human milk proteins. The fact that patients showed identical IgE binding patterns to milk antigens from different mothers, indicated that the observed IgE reactivity was neither due to alloreactivity nor to the presence of bovine milk proteins secreted into human milk. Immunoblot inhibition experiments demonstrated that the reaction with human milk proteins can be due to cross-reaction with cow's milk proteins. One of the cross-reactive allergens was identified as alpha-lactalbumin. In contrast, inhibition experiments also showed that for other patients IgE reactivity to human milk proteins is due to a genuine sensitisation to human milk allergens. We thus conclude that IgE autoreactivity to human milk allergens is due to autosensitisation and/or cross-reactivity with cow's milk proteins.

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[102] - Wal JM. Cow's milk proteins/allergens. Ann Allergy Asthma Immunol 2002;89(6 Suppl. 1):3-10

OBJECTIVE: The primary objective of this review is to provide updated data on the structure and function of the main cow's milk proteins (CMPs) identified as allergens and on the characterization of their epitopes. DATA SOURCES: The review represents a synthesis of basic literature and most relevant original recent publications on both topics of clinical and epidemiologic aspects of milk allergy and of milk protein's bio- and immunochemistry. STUDY SELECTION: The expert opinion of the author was used to select the relevant data for the review. RESULTS: Most CMPs are potential allergens, even the proteins present at very low concentration. There are both conformational and linear epitopes, widely spread all along the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule which comprise highly conserved sequences responsible for immunoglobulin E cross-reactivity with corresponding milk proteins of other mammals, including human beings. Those sequential epitopes have also been proposed as good markers of persistent allergy to CMPs. CONCLUSIONS: No specific structure nor function is associated with allergenicity of CMPs. Variability and heterogeneity of the human immunoglobulin E response preclude anticipating the allergenic potential of any CMP or fragment thereof, as well as justify the need for being careful before using peptides for desensitization or proposing any milk protein hydrolysate in a diet for highly allergenic children.

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[103] - Duncan JM, Sears MR. Breastfeeding and allergies: time for a change in paradigm ? Curr Opin Allergy Clin Immunol 2008;8:398-405

PURPOSE OF REVIEW: This review examines recent studies of the relationships between breastfeeding and the epidemiology of allergic diseases, especially atopic dermatitis in infants and asthma in early and later childhood. RECENT FINDINGS: Results from observational birth cohort studies, case-control studies, and one cluster randomized intervention trial have generally failed to demonstrate a protective effect of breastfeeding on outcomes of atopic dermatitis, allergic sensitization, wheezing, or asthma. Difficulties in interpretation relate to the absence of nonbreastfed control or reference groups in some studies, meaning outcomes can only be compared between different durations of breastfeeding. Studies with a nonbreastfed control group suggest there is an increased risk for atopy and asthma associated with breastfeeding and that prolonged breastfeeding may eventually reduce this increased risk. The family history, sex of the child, and the presence of other risk factors for allergy and asthma also influence the outcome. SUMMARY: Although breastfeeding is strongly recommended for its multiple benefits on child health, most recent studies do not confirm the 'conventional wisdom' that breastfeeding is protective against allergy and asthma. Early reduction in childhood wheezing may reflect protection from viral infections, but allergies and asthma at later ages may be increased.

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[104] - May CD, Remigio L, Feldman J, Bock SA, Carr RI. A study of serum antibodies to isolated milk proteins and ovalbumin in infants and children. Clin Allergy 1977;7:583-595

Serum antibodies to cow milk proteins and ovalbumin were measured quantitatively. Food hypersensitivity of the immediate type was determined to be present or absent by skin tests and double-blind food challenges. Elevated levels of antibodies to milk proteins in sera characteristic of infants fed cow milk were found to decline with age, so that sera from children who were 6 to 15 years of age (inclusive), not hypersensitive to food, had significantly lower levels than the infants. In contrast, sera from age-matched children, who were shown to have hypersensitivity to some food, were found to have levels of antibodies to milk proteins as elevated as in infancy. Hypersensitivity was not necessarily to milk but often to some other food. This persistence of greater antibody production to milk throughout childhood in those hypersensitive to some food indicates a fundamental difference from those without hypersensitivity to food, either in permeability, in immunological reactivity of the gut or in development of immunological unresponsiveness. Implications for pathogenesis of clinical disorders are discussed.

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[105] - Walsh BJ, Elliott C, Baker RS, Barnett D, Burley RW, Hill DJ, et al. Allergenic cross-reactivity of egg-white and egg-yolk proteins. An in vitro study. Int Arch Allergy Appl Immunol 1987;84:228-232

The radioallergosorbent test (RAST) and RAST inhibition test were used to examine cross-allergenicity amongst the major hen's egg-white and egg-yolk proteins. Using ovalbumin as a reference allergen to compare cross-reactivity, it was apparent that the proteins conalbumin, ovomucoid and lysozyme substantially inhibited binding to ovalbumin discs of IgE in the sera of patients clinically hypersensitive to egg. The converse situation with conalbumin, ovomucoid and lysozyme on the discs and ovalbumin as the inhibitor also resulted in significantly decreased levels of IgE binding to the proteins on the discs. It was also demonstrated that cross-reactions occurred between ovalbumin and the yolk protein, apovitellenin I. Cross-reaction was also observed surprisingly when egg lysozyme was on the disc and the milk protein allergen alpha-lactalbumin was used as the inhibitor. The demonstration of cross-reaction between all of these proteins may signify that there are a number of common allergenic determinants on these egg proteins, thus providing a molecular basis for the phenomenon of cross-reactivity.

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[106] - Moneret-Vautrin DA. A quoi sert le lysozyme dans les fromages ?. Alim'Inter 1999;4(21):13

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[107] - Pérez-Calderón R, Gonzalo-Garijo MA, Lamilla-Yerga A, Mangas-Santos R, Moreno-Gastón I. Recurrent Angioedema Due to Lysozyme Allergy. J Investig Allergol Clin Immunol 2007;17:264-266

A 54-year-old woman suffered an episode of dyspnea and edema affecting her eyelids, tongue, and lips a few minutes after intake of Lizipaina (bacitracin, papain, and lysozyme). She was treated with intravenous drugs and her symptoms improved within 2 hours. She had experienced 3 to 4 bouts of similar symptoms related to the ingestion of cured cheeses or raw egg. Specific serum immunoglobulin (Ig) E against lysozyme was present at a concentration of 0.45 kU/L, and no specific IgE was found against egg white and yolk, ovalbumin, or ovomucoid. Skin prick tests were positive with commercial extracts of egg white and lysozyme but doubtful with yolk, ovalbumin, and ovomucoid. Prick-to-prick tests with raw egg white and yolk gave positive results, but negative results were obtained with cooked egg white and yolk and 5 brands of cheese (3 of them containing lysozyme and the other 2 without lysozyme). Controlled oral administration of papain, bacitracin, and cheeses without lysozyme was well tolerated. We suggest that the presence of lysozyme in a pharmaceutical preparation, cured cheese, and raw egg was responsible for the symptoms suffered by our patient, probably through an IgE-mediated mechanism.

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[108] - Muraro MA, Giampietro PG, Galli E. Soy formulas and nonbovine milk. Ann Allergy Asthma Immunol 2002;89(6 Suppl. 1):97-101

BACKGROUND: Cow's milk allergy is frequently observed during the first year of life when nutritional requirements are critical. In those cases where breast-feeding is not available, a safe and adequate substitute to cow's milk should be offered. OBJECTIVE: The primary aim of this review is to evaluate the clinical use of milk derived from vegetable proteins, such as soy, or from animals such as goat, mare, or donkey, or elemental diet in children with cow's milk allergy. METHODS: MEDLINE searches were conducted with key words such as soy, goat's milk, donkey's milk, mare's milk, and elemental diet. Additional articles were identified from references in books or articles. Original research papers and review articles from peer-reviewed journals were chosen. RESULTS: Soy formulas are nutritionally adequate and can be used in children with immunoglobulin E-mediated nongastrointestinal manifestations of cow's milk allergy. Goat's milk is as allergenic as cow's milk. Mare's milk and donkey's milk may be used in selected cases of cow's milk allergy after appropriate modification to make them suitable for human infants. Elemental diets are usually restricted to the most severe cases of cow's milk allergy (ie, sensitivity to extensively hydrolyzed protein formulas). CONCLUSIONS: Vegetable formulas obtained from soy and milk derived from other mammals, such as mare or donkey, homemade preparations, and elemental diet may represent valid alternatives for children with cow's milk allergy. Extensive clinical trials are needed on the safety profile of any alternative mammal-derived milk. The choice of alternative milk should take into account the clinical profile of the child allergic to cow's milk, particularly as concerns age, severity of symptoms, degree of sensitivity to cow's milk proteins, and any multiple food allergies.

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[109] - Bocquet A, Bresson JL, Briend A, Chouraqui JP, Darmaun D, Dupont C, et al. [Infant formulas and soy protein-based formulas: current data]. Arch Pediatr 2001;8:1226-1233

For many years soy bean-based formulas (SBBF) were the only dietary product used for infants with cow's milk intolerance. At the present time, their place in infant nutrition is reduced as a result of the availability of new dietary products without lactose and/or cow's milk proteins and the recognition of soy bean protein allergy. There is no evidence that SBBF have any efficiency in infant colic. SBBF have no indication in the prevention of allergy, nor in premature infants' nutrition. Their main indication is the feeding of infants of vegetarian parents who do not want to use cow's milk products. Studies have shown that SBBF contain large quantities of phytoestrogens, particularly isoflavone. Because of experimental data suggesting a possible deleterious effect of phytoestrogens on the neuroendocrine maturation, the reduction of their content in SBBF must be considered.

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[110] - Besler M, Eigenmann P, Schwartz RH. Cow's milk (Bos domesticus). Internet Symp Food Allergens 2002;4(1):19-106

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[111] - Klemola T, Vanto T, Juntunen-Backman K, Kalimo K, Korpela R, Varjonen E. Allergy to soy formula and to extensively hydrolyzed whey formula in infants with cow's milk allergy: a prospective, randomized study with a follow-up to the age of 2 years. J Pediatr 2002;140:219-224

OBJECTIVES: We conducted a prospective, randomized study to evaluate the cumulative incidence of allergy or other adverse reactions to soy formula and to extensively hydrolyzed formula up to the age of 2 years in infants with confirmed cow's milk allergy. STUDY DESIGN: Infants (n = 170) with documented cow's milk allergy were randomly assigned to receive either a soy formula or an extensively hydrolyzed formula. If it was suspected that the formula caused symptoms, a double-blind, placebo-controlled challenge (DBPCFC) with the formula was performed. The children were followed to the age of 2 years, and soy-specific immunoglobulin E antibodies were measured at the time of diagnosis and at the ages of 1 and 2 years. RESULTS: An adverse reaction to the formula was confirmed by challenge in 8 patients (10%; 95% confidence interval, 4.4%-18.8%) randomly assigned to soy formula and in 2 patients (2.2%; 95% confidence interval, 0.3% to 7.8%) randomly assigned to extensively hydrolyzed formula. Adverse reactions to soy were similar in IgE-associated and non-IgE-associated cow's milk allergy (11% and 9%, respectively). IgE to soy was detected in only 2 infants with an adverse reaction to soy. Adverse reactions to soy formula were more common in younger (<6 months) than in older (6 to 12 months) infants (5 of 20 vs 3 of 60, respectively, P =.01). CONCLUSIONS: Soy formula was well tolerated by most infants with IgE-associated and non-IgE-associated cow's milk allergy. Development of IgE-associated allergy to soy was rare. Soy formula can be recommended as a first-choice alternative for infants >or=6 months of age with cow's milk allergy.

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[112] - Ahn K, Nam S, Han Y, Lee S. Prevalence of Soy Protein Hypersensitivity in Korean Children Sensitized to Cow Milk Protein. J Korean Med Sci 2003;18:473-477

RATIONALE: Soy protein formula (SPF) has been in limited use in cow milk allergy (CMA) patients, because some of CMA patients are allergic to soy protein as well. The purpose of this study is to evaluate the prevalence of soy protein hypersensitivity in cow milk protein-sensitive children in Korea. METHODS: A total of 1363 patients with atopic disease were recruited in this study. Serum IgE antibodies to either cow milk or soy was measured by CAP assay, and specific IgE levels higher than 0.7 kU/L were considered as positive. CMA patients who were confirmed by history, elimination or open challenge of cow milk protein were evaluated for soy allergy by open challenge test. RESULTS: Patients with high soy-specific IgE accounted for 14.7% of 224 children who are sensitized to cow milk. The prevalence of sensitization to soy decreased with age (28.9% in the first year of life, 14.5% in the second year, 14.0% in the third year, 8.8% thereafter). Of 22 CMA patients, 40.9% (n=9) were determined to have soy allergy. The mean age of patients experiencing positive challenge was 9.5 months, whereas those with negative challenge was 15.0 months. CONCLUSIONS: Our results suggest that SPF should be carefully used as a substitute for cow milk in CMA patients, especially in the first year of life.

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[113] - Ahn K, Nam S, Han Y, Lee S. Prevalence of Soy Protein Hypersensitivity in Korean Children Sensitized to Cow Milk Protein. J Korean Med Sci 2003;18:473-477

RATIONALE: Soy protein formula (SPF) has been in limited use in cow milk allergy (CMA) patients, because some of CMA patients are allergic to soy protein as well. The purpose of this study is to evaluate the prevalence of soy protein hypersensitivity in cow milk protein-sensitive children in Korea. METHODS: A total of 1363 patients with atopic disease were recruited in this study. Serum IgE antibodies to either cow milk or soy was measured by CAP assay, and specific IgE levels higher than 0.7 kU/L were considered as positive. CMA patients who were confirmed by history, elimination or open challenge of cow milk protein were evaluated for soy allergy by open challenge test. RESULTS: Patients with high soy-specific IgE accounted for 14.7% of 224 children who are sensitized to cow milk. The prevalence of sensitization to soy decreased with age (28.9% in the first year of life, 14.5% in the second year, 14.0% in the third year, 8.8% thereafter). Of 22 CMA patients, 40.9% (n=9) were determined to have soy allergy. The mean age of patients experiencing positive challenge was 9.5 months, whereas those with negative challenge was 15.0 months. CONCLUSIONS: Our results suggest that SPF should be carefully used as a substitute for cow milk in CMA patients, especially in the first year of life.

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[114] - Bardare M, Magnolfi C, Zani G. Soy sensitivity: personal observation on 71 children with food intolerance. Allerg Immunol (Paris) 1988;20:63-66

The controversies on the use of soy milk as a substitute in cow's milk intolerance prompted us to study: the incidence of soy sensitivity in a pediatric population (71 children, mean age 5.9 years, 45 boys and 26 girls) with food intolerance: the influence of a prior soy milk feeding on soy sensitivity: the relationship between soy, cow's milk and seed allergy. The patients were subdivided in two groups, one of atopic patients (50 subjects, 28 of which previously fed soy) and the other of non atopic patients (21 subjects, 12 of which previously fed soy). In the atopic group prick and RAST gave positive results to soy in 46% of case, with no difference between subjects fed soy and not. There was a relationship between any and peanut RAST in 82%; between soy and pea in 70%; between soy and cow's milk in 27% of cases. Soy milk challenge was positive in 10 out of 58 children (6 atopic and 4 non atopic); 4 our of 21 atopic patients with a cow's milk intolerance had a positive soy milk challenge: 3 of 10 non atopic patients with cow's milk intolerance were reactive to soy too. 77% of atopic and 90% of non atopic children were responsive both to seeds and soy. It can be concluded that soy sensitivity is rather rare in patients with food intolerance (17.2% of cases) and is not correlated with cow's milk intolerance while is significantly correlated with seeds allergy; there is no difference between atopic and non atopic subjects and between patients previously fed soy and never fed soy.

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[115] - Zeiger RS, Sampson HA, Bock SA, Burks AW Jr, Harden K, Noone S, et al. Soy allergy in infants and children with IgE-associated cow's milk allergy. J Pediatr 1999;134:614-622

OBJECTIVES: To determine the prevalence of soy allergy in IgE-associated cow's milk allergy (CMA). STUDY DESIGN: Children <3.5 years with documented IgE-associated CMA (n = 93) were evaluated for soy allergy by double-blind, placebo-controlled food challenge, open challenge, or convincing previous history of an anaphylactic reaction to soy. Children tolerant to soy at entry received soy formula and were followed up for 1 year. RESULTS: Of this IgE-associated CMA cohort (ages 3 to 41 months), 14% (95% CI = 7. 7%-22.7%) were determined to have soy allergy, 12 definitely at entry and 1 possibly after 1 year of soy ingestion. The latter child experienced severe failure to thrive at enrollment and exhibited improved growth while receiving soy during follow-up but was diagnosed with eosinophilic esophagitis at study completion. Improved growth (P <.05) occurred in the non-soy-allergic cohort ingesting soy formula (579 31 mL/d) during the year of follow-up. CONCLUSIONS: Soy allergy occurs in only a small minority of young children with IgE-associated CMA. As such, soy formula may provide a safe and growth-promoting alternative for the majority of children with IgE-associated CMA shown to be soy tolerant at the time of introduction of soy formula.

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[116] - Klemola T, Kalimo K, Poussa T, Juntunen-Backman K, Korpela R, Valovirta E, et al. Feeding soy formula to cow's milk allergic children: the development of IgE-mediated allergy to soy and peanuts. Pediatr Allergy Immunol 2005;16:641-646

Background: Peanut allergy has been associated with the intake of soy milk or soy formula. We studied the development of IgE antibodies specific for soy and peanut and of allergic reactions caused by peanuts in children with confirmed cow's milk allergy fed with either a soy formula or an extensively hydrolyzed formula. Methods: Infants (n = 170) with documented cow's milk allergy were randomly assigned to receive either a soy formula or an extensively hydrolyzed formula (EHF). The children were followed to the age of 4 years. Peanut-specific IgE was measured at the age of 4 years. Detailed history of the occurrence allergic reactions caused by peanut was recorded by parents. Soy-specific IgE antibodies were measured at the time of diagnosis and at the ages of 1, 2 and 4 years. Results: IgE antibodies to soy (> 0.35 kU/l) were found in 22/70 children fed with soy formula, and in 14/70 of children fed with EHF (p = 0.082). In an open challenge with soy at the age of 4, no immediate reactions to soy were observed. One out of 72 children from the soy group had a delayed reaction to soy. IgE antibodies to peanut (> 0.35 kU/l) were found in 21/70 children fed with soy formula and 17/69 infants in fed with EHF (p= 0.717). The frequency of reported peanut allergy in the soy group was 2/72 (3%) and 4/76 (5%) in the EHF group (p = 0.68) Conclusions: Development of IgE-associated allergy to soy and peanut was rare in our study group of milk allergic children. The use of soy formula during the first two years of life did not increase the risk of development of peanut-specific IgE antibodies or clinical peanut allergy.

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[117] - Zeiger RS, Sampson HA, Bock SA, Burks AW Jr, Harden K, Noone S, et al. Soy allergy in infants and children with IgE-associated cow's milk allergy. J Pediatr 1999;134:614-622

OBJECTIVES: To determine the prevalence of soy allergy in IgE-associated cow's milk allergy (CMA). STUDY DESIGN: Children <3.5 years with documented IgE-associated CMA (n = 93) were evaluated for soy allergy by double-blind, placebo-controlled food challenge, open challenge, or convincing previous history of an anaphylactic reaction to soy. Children tolerant to soy at entry received soy formula and were followed up for 1 year. RESULTS: Of this IgE-associated CMA cohort (ages 3 to 41 months), 14% (95% CI = 7. 7%-22.7%) were determined to have soy allergy, 12 definitely at entry and 1 possibly after 1 year of soy ingestion. The latter child experienced severe failure to thrive at enrollment and exhibited improved growth while receiving soy during follow-up but was diagnosed with eosinophilic esophagitis at study completion. Improved growth (P <.05) occurred in the non-soy-allergic cohort ingesting soy formula (579 31 mL/d) during the year of follow-up. CONCLUSIONS: Soy allergy occurs in only a small minority of young children with IgE-associated CMA. As such, soy formula may provide a safe and growth-promoting alternative for the majority of children with IgE-associated CMA shown to be soy tolerant at the time of introduction of soy formula.

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[118] - Klemola T, Kalimo K, Poussa T, Juntunen-Backman K, Korpela R, Valovirta E, et al. Feeding soy formula to cow's milk allergic children: the development of IgE-mediated allergy to soy and peanuts. Pediatr Allergy Immunol 2005;16:641-646

Background: Peanut allergy has been associated with the intake of soy milk or soy formula. We studied the development of IgE antibodies specific for soy and peanut and of allergic reactions caused by peanuts in children with confirmed cow's milk allergy fed with either a soy formula or an extensively hydrolyzed formula. Methods: Infants (n = 170) with documented cow's milk allergy were randomly assigned to receive either a soy formula or an extensively hydrolyzed formula (EHF). The children were followed to the age of 4 years. Peanut-specific IgE was measured at the age of 4 years. Detailed history of the occurrence allergic reactions caused by peanut was recorded by parents. Soy-specific IgE antibodies were measured at the time of diagnosis and at the ages of 1, 2 and 4 years. Results: IgE antibodies to soy (> 0.35 kU/l) were found in 22/70 children fed with soy formula, and in 14/70 of children fed with EHF (p = 0.082). In an open challenge with soy at the age of 4, no immediate reactions to soy were observed. One out of 72 children from the soy group had a delayed reaction to soy. IgE antibodies to peanut (> 0.35 kU/l) were found in 21/70 children fed with soy formula and 17/69 infants in fed with EHF (p= 0.717). The frequency of reported peanut allergy in the soy group was 2/72 (3%) and 4/76 (5%) in the EHF group (p = 0.68) Conclusions: Development of IgE-associated allergy to soy and peanut was rare in our study group of milk allergic children. The use of soy formula during the first two years of life did not increase the risk of development of peanut-specific IgE antibodies or clinical peanut allergy.

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[119] - Bruno G, Giampietro PG, Del Guercio MJ, Gallia P, Giovannini L, Lovati C, et al. Soy allergy is not common in atopic children: a multicenter study. Pediatr Allergy Immunol 1997;8:190-193

The aim of the present study was to evaluate the prevalence of soy allergy (positive skin test and positive challenge test) in a large cohort of atopic children, many of them soy fed early in life for several months. In order to investigate the prevalence of soy allergy, two groups of children were enrolled into the study. The first group comprised a cohort of 505 children with personal history suggestive of food allergy. The second group included 243 children born of atopic parents, who had been soy protein formula fed for the first six months of life for the prevention of cow's milk allergy and who had been prospectively followed up, from birth to 5 years. As regards the prevalence of soy allergy in the cohort of children suffering from allergic disease: 31/505 children (6%) had positive skin prick test to soy, however only six of the 31 children with positive skin prick test to soy had positive challenge test to soy. With regard to the prevalence of soy allergy in the children who had been soy protein formula fed in the first six months of life (second group): 14/243 children (6%) had positive skin prick test to soy, but the double blind placebo control oral food challenge to soy was positive in only one of these 14 children. In conclusion documented soy allergy is not common in atopic children

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[120] - Bardare M, Magnolfi C, Zani G. Soy sensitivity: personal observation on 71 children with food intolerance. Allerg Immunol (Paris) 1988;20:63-66

The controversies on the use of soy milk as a substitute in cow's milk intolerance prompted us to study: the incidence of soy sensitivity in a pediatric population (71 children, mean age 5.9 years, 45 boys and 26 girls) with food intolerance: the influence of a prior soy milk feeding on soy sensitivity: the relationship between soy, cow's milk and seed allergy. The patients were subdivided in two groups, one of atopic patients (50 subjects, 28 of which previously fed soy) and the other of non atopic patients (21 subjects, 12 of which previously fed soy). In the atopic group prick and RAST gave positive results to soy in 46% of case, with no difference between subjects fed soy and not. There was a relationship between any and peanut RAST in 82%; between soy and pea in 70%; between soy and cow's milk in 27% of cases. Soy milk challenge was positive in 10 out of 58 children (6 atopic and 4 non atopic); 4 our of 21 atopic patients with a cow's milk intolerance had a positive soy milk challenge: 3 of 10 non atopic patients with cow's milk intolerance were reactive to soy too. 77% of atopic and 90% of non atopic children were responsive both to seeds and soy. It can be concluded that soy sensitivity is rather rare in patients with food intolerance (17.2% of cases) and is not correlated with cow's milk intolerance while is significantly correlated with seeds allergy; there is no difference between atopic and non atopic subjects and between patients previously fed soy and never fed soy.

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[121] - Klemola T, Kalimo K, Poussa T, Juntunen-Backman K, Korpela R, Valovirta E, et al. Feeding soy formula to cow's milk allergic children: the development of IgE-mediated allergy to soy and peanuts. Pediatr Allergy Immunol 2005;16:641-646

Background: Peanut allergy has been associated with the intake of soy milk or soy formula. We studied the development of IgE antibodies specific for soy and peanut and of allergic reactions caused by peanuts in children with confirmed cow's milk allergy fed with either a soy formula or an extensively hydrolyzed formula. Methods: Infants (n = 170) with documented cow's milk allergy were randomly assigned to receive either a soy formula or an extensively hydrolyzed formula (EHF). The children were followed to the age of 4 years. Peanut-specific IgE was measured at the age of 4 years. Detailed history of the occurrence allergic reactions caused by peanut was recorded by parents. Soy-specific IgE antibodies were measured at the time of diagnosis and at the ages of 1, 2 and 4 years. Results: IgE antibodies to soy (> 0.35 kU/l) were found in 22/70 children fed with soy formula, and in 14/70 of children fed with EHF (p = 0.082). In an open challenge with soy at the age of 4, no immediate reactions to soy were observed. One out of 72 children from the soy group had a delayed reaction to soy. IgE antibodies to peanut (> 0.35 kU/l) were found in 21/70 children fed with soy formula and 17/69 infants in fed with EHF (p= 0.717). The frequency of reported peanut allergy in the soy group was 2/72 (3%) and 4/76 (5%) in the EHF group (p = 0.68) Conclusions: Development of IgE-associated allergy to soy and peanut was rare in our study group of milk allergic children. The use of soy formula during the first two years of life did not increase the risk of development of peanut-specific IgE antibodies or clinical peanut allergy.

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[122] - Klemola T, Kalimo K, Poussa T, Juntunen-Backman K, Korpela R, Valovirta E, et al. Feeding soy formula to cow's milk allergic children: the development of IgE-mediated allergy to soy and peanuts. Pediatr Allergy Immunol 2005;16:641-646

Background: Peanut allergy has been associated with the intake of soy milk or soy formula. We studied the development of IgE antibodies specific for soy and peanut and of allergic reactions caused by peanuts in children with confirmed cow's milk allergy fed with either a soy formula or an extensively hydrolyzed formula. Methods: Infants (n = 170) with documented cow's milk allergy were randomly assigned to receive either a soy formula or an extensively hydrolyzed formula (EHF). The children were followed to the age of 4 years. Peanut-specific IgE was measured at the age of 4 years. Detailed history of the occurrence allergic reactions caused by peanut was recorded by parents. Soy-specific IgE antibodies were measured at the time of diagnosis and at the ages of 1, 2 and 4 years. Results: IgE antibodies to soy (> 0.35 kU/l) were found in 22/70 children fed with soy formula, and in 14/70 of children fed with EHF (p = 0.082). In an open challenge with soy at the age of 4, no immediate reactions to soy were observed. One out of 72 children from the soy group had a delayed reaction to soy. IgE antibodies to peanut (> 0.35 kU/l) were found in 21/70 children fed with soy formula and 17/69 infants in fed with EHF (p= 0.717). The frequency of reported peanut allergy in the soy group was 2/72 (3%) and 4/76 (5%) in the EHF group (p = 0.68) Conclusions: Development of IgE-associated allergy to soy and peanut was rare in our study group of milk allergic children. The use of soy formula during the first two years of life did not increase the risk of development of peanut-specific IgE antibodies or clinical peanut allergy.

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[123] - Morisset M, Lee T, Frémont S, Errahali Y, Codreanu F, Guénard L, et al. Exceptionnels cas d'allergie à un substitut du lait à base d'acides aminés: une allergie à l'huile de soja incriminée. Rev Fr Allergol Immunol Clin 2006;46:373

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[124] - Moneret-Vautrin DA, Morisset M, Flabbee J, Kanny G, Kirch F, Parisot L. Unusual soy oil allergy. Allergy 2002;57:266-267

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[125] - Rozenfeld P, Docena GH, Añon MC, Fossati CA. Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk. Clin Exp Immunol 2002;130:49-58

Soy-based formulas are the most employed cow's milk substitutes in the treatment of cow's milk allergy in our country. Since adverse reactions have been reported in allergic patients as a consequence of exposure to soy proteins, we have investigated the possible cross-reactivity between components from soybean and cow's milk. A cow's milk specific polyclonal antiserum and casein specific monoclonal antibodies were used in immunoblotting and competitive ELISA studies to identify a 30-kD component from soybean that cross-reacts with cow's milk caseins. Its IgE binding capacity was tested by EAST, employing sera from cow's milk allergic patients, not previously exposed to soy proteins. The 30 kD protein was isolated and partially sequenced. It is constituted by two polypeptides (A5 and B3) linked by a disulphide bond. The protein's capacity to bind to the different antibodies relies on the B3 poly-peptide. These results indicate that soy-based formula, which contains the A5-B3 glycinin molecule, could be involved in allergic reactions observed in cow's milk allergic patients exposed to soy-containing foods.

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[126] - Curciarello R, Lareu JF, Fossati CA, Docena GH, Petruccelli S. Immunochemical characterization of Glycine max L. Merr. var Raiden, as a possible hypoallergenic substitute for cow’s milk-allergic patients. Clin Exp Allergy 2008;38:1559-1565

BACKGROUND: Cows' milk allergy (CMA) is the most common cause of food allergy in infancy. The only proven treatment is the complete elimination of cows' milk proteins (CMPs) from the diet by means of hypoallergenic formulas. Soybean-based formulae are widely used although intolerance to soy has been reported to occur in 15-40% of infants with CMA . OBJECTIVE: The aim of this work was to analyse the in vitro reactivity of the soybean cultivar Raiden, which naturally lacks glycinin A(4)A(5)B(3), to evaluate whether this genotype could be a safe CMP substitute for CMA patients . METHODS: The reactivity of conventional soybean (CS) and Raiden soybean (RS) genotypes and also recombinant glycinin A(4)A(5)B(3) and alphabeta-conglycinin with casein-specific monoclonal antibodies and CMP-specific polyclonal serum was evaluated by immunoblotting and ELISA. A sequential competitive ELISA with the polyclonal antiserum and different soluble inhibitors was performed. In addition, an indirect ELISA with sera of atopic children with CMA was carried out to analyse the IgE-binding capacity of the different soybean components . RESULTS: We have shown that CS contains four components that cross-react with CMP, while RS has only one. The remaining cross-reactive component in RS was identified as alpha-subunit beta-conglycinin. By means of inhibitory ELISA, we demonstrated that CS, RS and the alpha-subunit beta-conglycinin extracts inhibited the binding of CMP-specific antibodies to the CMP-coated solid phase. Finally, we showed that CS, RS and the recombinant proteins were recognized by human CMP-specific IgE antibodies . CONCLUSION: This work shows that although Raiden has fewer cross-reactive components than conventional soybean, it still has a residual cross-reactive component: the alpha-subunit beta-conglycinin. This reactivity might make this genotype unsuitable to treat CMA and also explains adverse reactions to soybean in CMA infants.

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[127] - Fiocchi A, Bouygue GR, Sarratud T, Terracciano L, Martelli A, Restani P. Clinical tolerance of processed foods. Ann Allergy Asthma Immunol 2004;93(5 Suppl. 3):S38-S46

OBJECTIVE: To review the effects of technological processing on selected foods of relevance to childhood allergy from the viewpoints of reduced allergenicity, contamination of processed foods by allergens introduced during processing, and ad hoc technologies to produce reduced hypoallergenic products. DATA SOURCES: We searched the literature (PubMed/MEDLINE) for articles published between January 1994 and April 2004 using the following keywords: food allergy AND process* OR heat* OR cooking OR toleran*. STUDY SELECTION: We drew on our collective clinical and biological experience to restrict retrieved studies to those of more frequent relevance to a hospital allergy practice. RESULTS: Comparatively few clinical studies address the modification of allergenicity of food through cooking or processing. Dairy foods are largely unaffected by processing and may be contaminated by, or themselves become, hidden allergens. Hypoallergenic formulas based on milk, soy, or rice and homogenized beef are successful applications of allergenicity reduction via technological processing. Egg, fish, condiments, and vegetables all carry heat-resistant allergens and should also be considered contaminants. Cereals and bakery products are generally well tolerated, but their allergenicity may be enhanced by processing; the case of rice is still open. Peanut allergens are stable, and the evidence is scant that thermal processing affects the allergenicity of soybean and soy hydrolysates. The debate is ongoing about the tolerance of vegetable oils. CONCLUSIONS: It is too early to systematize clinical studies based on single procedures. Processing affects antigenicity, but this does not always translate into safety recommendations. Industrial processing is liable to contamination, and monitoring and labeling are industry priorities. Clinicians should evaluate foods by as complete a workup as possible before recommending processed foods.

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[128] - Moneret-Vautrin DA, Hatahet R, Kanny G. Hydrolysats de protéines: laits hypoallergéniques et formules extensivement hydrolysées. Bases immuno-allergologiques de leur utilisation dans la prévention et le traitement de l'allergie au lait. Arch Pediatr 2001;8:1348-1357

Allergy to milk has an estimated incidence of 2.5%. Hypoallergenic milks are partial hydrolysates of proteins with lactose. Extensive hydrolysates of casein are mainly small peptides and do not include lactose in their formula. A primary allergenicity as well as a cross-sensitivity are shown for all partial hydrolysates. Even extensive hydrolysates have a weak potential of cross-allergy. The prevention of cow's milk allergy in infants at peculiar risk, born from atopic parents, is based on breast-feeding and/or partial or extensive hydrolysates. There is no conclusive evidence for the usefulness of eviction of dairy products during pregnancy or during the period of breast-feeding. The treatment needs exclusively extensive casein hydrolysates ensuring a 90% protection with 95% confidence intervals. Predictive prick tests in certain cases may help the choice of another hydrolysate. Severe cases with failure to thrive or multiple food intolerance require an amino-acid based formula (Neocate). A rational diversification of the diet is advised in order to prevent the onset of a multiple food intolerance syndrome which could avert from the recovery of cow milk allergy.

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[129] - Fanta C, Ebner C. Allergy to mare's milk. Allergy 1998;53:539-540

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[130] - Gall H, Kalveram CM, Sick H, Sterry W. Allergy to the heat- labile proteins alpha-lactalbumin and beta-lactoglobulin in mare's milk. J Allergy Clin Immunol 1996;97:1304-1307

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[131] - Robles S, Torres MJ, Mayorga C, Rodríguez-Bada JL, Fernández TD, Blanca M, et al. Anaphylaxis to Mare's milk. Ann Allergy Asthma Immunol 2007;98:600-602

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[132] - Fanta C, Ebner C. Allergy to mare's milk. Allergy 1998;53:539-540

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[133] - Robles S, Torres MJ, Mayorga C, Rodríguez-Bada JL, Fernández TD, Blanca M, et al. Anaphylaxis to Mare's milk. Ann Allergy Asthma Immunol 2007;98:600-602

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[134] - Szépfalusi Z, Ebner C, Urbanek R, Ebner H, Scheiner O, Boltz-Nitulescu G, et al. Detection of IgE antibodies specific for allergens in cow milk and cow dander. Int Arch Allergy Immunol 1993;102:288-294

Sera from patients with cow milk protein allergy (n = 6) and cow dander allergy (n = 5) were analyzed for reactivity of IgE antibodies specific for allergens derived from milk and cow dander. The cow milk- and cow dander-allergic patients exhibited elevated specific IgE levels to milk and to cow dander, respectively, as determined by RAST. IgE immunoblot analysis revealed that cow milk-allergic patients exhibited IgE binding to the milk allergens casein, beta-lactoglobulin, alpha-lactalbumin and bovine serum albumin. Four of six cow milk-reactive patients also exhibited IgE binding to cow dander proteins with molecular weights of 20, 22, 36, 50 and > 200 kD. IgE from the sera of cow dander-allergic patients reacted with the major allergens of cow dander (20 and 22 kD) and with other proteins with molecular weights of 24, 36, 42, 50 and 70 kD. Only one out of five of these sera displayed IgE reactivity with cow milk proteins with molecular weights of 69, 92 and > 200 kD. Inhibition studies revealed the cross-reactive nature of the IgE antibodies. Preincubation of cow milk-positive sera with cow milk and subsequent immunoblotting led to complete blocking of IgE binding to cow dander proteins and to cow milk proteins. Preincubation of cow milk-positive sera with cow dander extract led to blocking of IgE binding only to defined cow milk proteins: these could be identified in 2 cases as casein and in 1 case as beta-lactoglobulin.

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[135] - Osvath P, Endre L, Peter F. [Cross reaction of specific IgE antibodies directed against cow's milk and bovine hair]. Allerg Immunol (Leipz) 1979;25:203-206

Sera of 25 children living in town were examined by means of RAST on the presence of IgE antibody against milk and cow's hair antigens. 18 cases had IgE antibodies reacting with milk and 9 with cow's hair. There was correlation between the levels of these antibodies with the exception of two cases. The results give evidence that half of the asthmatic children allergic to milk will react with bronchospasm in the cases if they ar exposed to inhalation of cow's hair.

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[136] - Hoppé A, Nicolie B, Beauvillain C, Drouet M. Sensibilisation croisée entre lait de vache et chien. Rev Fr Allergol 2009;49:305

Introduction.ˆ L‚allergie croisée dans la famille des mammifères a été décrite par plusieurs équipes (porc et chat, cheval et hamster, boeuf et chien). Nous nous proposons d‚étudier la relation entre lait de vache et chien. Méthodes.ˆ Huit enfants présentant une anaphylaxie au lait de vache (prick-test et IgE spécifiques [IgE sp. LV] positifs) ont bénéficié d‚un dosage des IgE spécifiques pour le chien (IgE sp. chien) et d‚une inhibition des IgE sp. chien par les IgE sp. LV. Résultats.ˆ Patient maximum IgEsp LV (kUI/L) IgEsp chien (kUI/L) Inhibition (%) 1 6,2 0,4 2,9 2 69,6 42,8 20,6 3 9,6 1,5 71,7 4 > 100 3,7 100 42,8 2,2 83,8 5 0,9 0,7 75,6 6 52 3,7 100 6,1 1,4 49,8 7 10,8 2 42,3 8 9 9,7 24,8 Conclusion.ˆ Une réaction croisée entre lait de vache et épithélia de chien est confirmée chez quatre patients. Deux patients ont bénéficié des mêmes examens à quelques années d‚intervalle avec une diminution franche des IgE sp. LVet de l‚inhibition du chien par le lait de vache dans le même temps.

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[137] - Drouet M, Nicolie B, Bonneau JC, Leclère JM, Le Sellin J. Cow's milk anaphylaxy - cross reacitivity with dog. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°602

Background: In the mammalian family we previously described the Pork-Cat syndrom (patients allergic to pork meat and Cat hair) and in this syndrom we noticed a great poly sensitization with other mammalian hair and other mammalian foods including cow's milk(CM). That's why we decide to investigate the CM allergy to look after other mammalian allergy that could suggest a cross reactivity. Methods:/ Subjects: 9 children have been selected with a well documented story of CM anaphylaxy (from acute urticaria to anaphylactic shock) and both positive Prick test and specific IgE (sIgE) to CM. sIgE measurements: sIgE to CM, casein, alpha-lactalbumin, beta-lactoglobulin are measured in each case, completed by sIgE to mite (d1), grass pollen (g6) cat (e1) and dog (e5). sIgE are measured by using the CAP FEIA system (Pharmacia, Uppsala, Sweden). Patients are considered as sensitized if the levels were 0.75 KUI/L or greater. sIgE inhibition were realized between CM and dog. Results: The 9 patients have sIgE to CM (0.9-75 KUI/L) and casein is in all the cases the most relevant allergen. For the aeroallergens, among the 9 patients we find 3 positive sIgE to mite (27-93KUI/L), 2 positive sIgE to Grass pollen (5-41 KUI/L), 5 positive sIgE to cat (1.2-7KUI/l). and 9 positive sIgE to dog (1.5-22.3KUI/l). The association between dog and CM is highly significant. sIgE inhibition : In 2 patients we completely inhibit sIgE to dog by serum pre incubated with CM. Conclusion: In this group of 9 children with anaphylactic reaction to CM, we find sIgE to dog in all the cases. This high frequency suggests a crossed sensitivity and this hypothesis is increased by other arguments : 1) all the children are sensitized to dog whereas only 3 are exposed, 2) sIgE inhibition is complete for dog by CM and impossible for CM by dog, 3) For 8 children sIgE to CM are more elevated than sIgE to dog. The 2 last arguments suggest that CM is probably the inducing allergen in the cross sensitivity.

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[138] - Szépfalusi Z, Ebner C, Urbanek R, Ebner H, Scheiner O, Boltz-Nitulescu G, et al. Detection of IgE antibodies specific for allergens in cow milk and cow dander. Int Arch Allergy Immunol 1993;102:288-294

Sera from patients with cow milk protein allergy (n = 6) and cow dander allergy (n = 5) were analyzed for reactivity of IgE antibodies specific for allergens derived from milk and cow dander. The cow milk- and cow dander-allergic patients exhibited elevated specific IgE levels to milk and to cow dander, respectively, as determined by RAST. IgE immunoblot analysis revealed that cow milk-allergic patients exhibited IgE binding to the milk allergens casein, beta-lactoglobulin, alpha-lactalbumin and bovine serum albumin. Four of six cow milk-reactive patients also exhibited IgE binding to cow dander proteins with molecular weights of 20, 22, 36, 50 and > 200 kD. IgE from the sera of cow dander-allergic patients reacted with the major allergens of cow dander (20 and 22 kD) and with other proteins with molecular weights of 24, 36, 42, 50 and 70 kD. Only one out of five of these sera displayed IgE reactivity with cow milk proteins with molecular weights of 69, 92 and > 200 kD. Inhibition studies revealed the cross-reactive nature of the IgE antibodies. Preincubation of cow milk-positive sera with cow milk and subsequent immunoblotting led to complete blocking of IgE binding to cow dander proteins and to cow milk proteins. Preincubation of cow milk-positive sera with cow dander extract led to blocking of IgE binding only to defined cow milk proteins: these could be identified in 2 cases as casein and in 1 case as beta-lactoglobulin.

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[139] - Vicente-Serrano J, Caballero ML, Rodriguez-Pérez R, Carretero P, Pérez R, Blanco JG, et al. Sensitization to serum albumins in children allergic to cow’s milk and epithelia. Pediatr Allergy Immunol 2007;18:503-507

Patients with persistent milk allergy and specific immunoglobulin E (IgE) to bovine serum albumin (BSA) have a greater risk of rhinoconjunctivitis and asthma because of animal dander. To prove the cross-reactivity between serum albumin (SA) of different mammals in milk, meat, and epithelia and determine if heat treatment of meats decrease the allergenicity of albumins. The study was performed using SDS-PAGE and IgE-immunoblotting using sera from eight patients sensitized to milk, BSA, and animal danders. Sera from non-allergic and only animal dander allergic subjects served as a control. With one exception, all patients' sera recognized SA in different meats (beef, lamb, deer, and pork), epithelia (dog, cat, and cow), and cow's milk. Some patients even were only sensitized to SA in meat and epithelia. Danders' allergic only recognized other proteins in epithelia but not SA. No patients reacted to SA from heated meat extracts. Serum albumin is an important allergen involved in milk, meat, and epithelia allergy. The first contact with SA was through cow's milk and patients developed sensitization to epithelia SA even without direct contact with animals. Patients with both BSA and cow's milk allergy must avoid raw meats and furry pets.

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[140] - Bencharitiwong R, Butler J, Vereda A, Wegrzyn AH. Effect of Heating on Allergenicity of Cow (CM), Goat (GM) and Sheep (SM) Milk Proteins. J Allergy Clin Immunol 2009;123:S27

RATIONALE: The majority of children allergic to CM tolerate extensively heated CM. We sought to compare the effects of heating on the allergenicity of CM, GM, and SM proteins. METHODS: Milk samples were heated in a water bath at 95-100°C for up to 90 minutes, defatted and clarified; 8 mg/ml proteins were loaded on a SDS-PAGE gel and separated electrophoretically. Sera were obtained from 5 subjects with documented CM, orGMand SM allergy, and with detectable CM, GM, and/or SM specific-IgE antibody levels by UniCAP (Phadia). IgE-antibody binding (diluted 1:5-1:40, based on the milk IgE levels) to unheated and heated proteins was analyzed by immunoblot assay. RESULTS:We observed no significant consistent changes following boiling in the appearance of protein bands on a SDS-PAGE gel in GM; whereas in CM and SM appeared less intense following >= 10 minutes of boiling. However, immunolabeling of CMsamples boiled >= 10 minutes was weaker compared with unheated milk. In contrast, immunolabeling of GMand SM boiled for >= 10 minutes was significantly stronger to caseins, a band between 49 and 62 KDa in GM and SM, and serum albumin band only in SM compared with unheated milk. CONCLUSION: Boiling decreases binding of anti-cow milk IgE to CM proteins, whereas it increases binding of anti-goat and anti-sheep milk IgE to caseins, serum albumin, and the band between 49 and 62 KDa in GM and SM. This observation is surprising in view of high protein sequence homology between these mammalian milks.

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[141] - Sanchez C, Frémont S. Conséquences des traitements thermiques et de la formulation sur la structure et l'allergénicité des protéines alimentaires. Rev Fr Allergol Immunol Clin 2003;43:13-20

Une augmentation importante du nombre de cas d'allergies alimentaires a été constatée au cours des cinq dernières années. Parmi les causes possibles, les procédés alimentaires et les interactions entre ingrédients et additifs sont fortement suspectés. Cette revue présente l'état des connaissances sur la relation entre chauffage, formulation, structure et allergénicité des protéines. Les procédés de fabrication alimentaire comprennent un ensemble d'opérations unitaires thermiques et mécaniques dont l'objet est de structurer, texturer et permettre une conservation satisfaisante de l'aliment. Les opérations de chauffage entraînent dans la plupart des cas une dénaturation irréversible de la conformation de la protéine pouvant conduire à son agrégation. Ces changements structuraux ne sont pas corrélés avec une diminution du potentiel allergénique des protéines. Selon les cas, un chauffage peut n'avoir aucun effet, diminuer ou augmenter ce potentiel. L'existence d'épitopes séquentiels et conformationnels, le démasquage de nouveaux épitopes ou la modification d'épitopes par réaction de Maillard peut expliquer partiellement les résultats reportés dans la littérature. Des interactions entre protéines allergènes et autres molécules présentes dans les aliments peuvent entraîner des modifications conformationnelles des protéines, même sans chauffage, et affecter leur stabilité thermique. En particulier, une augmentation ou une diminution de l'agrégation thermique des protéines peuvent être observées. L'effet de ces interactions sur le potentiel allergénique des protéines est aujourd'hui pratiquement inconnu.

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[142] - Mills ENC, Jenkins JA, Alcocer MJ, Shewry PR. Structural, biological, and evolutionary relationships of plant food allergens sensitizing via the gastrointestinal tract. Crit Rev Food Sci Nutr 2004;44:379-407

The recently completed genome sequence of the model plant species Arabidopsis has been estimated to encode over 25,000 proteins, which, on the basis of their function, can be classified into structural and metabolic (the vast majority of plant proteins), protective proteins, which defend a plant against invasion by pathogens or feeding by pests, and storage proteins, which proved a nutrient store to support germination in seeds. It is now clear that almost all plant food allergens are either protective or storage proteins. It is also becoming evident that those proteins that trigger the development of an allergic response through the gastrointestinal tract belong primarily to two large protein superfamilies: (1) The cereal prolamin superfamily, comprising three major groups of plant food allergens, the 2S albumins, lipid transfer proteins, and cereal alpha-amylase/trypsin inhibitors, which have related structures, and are stable to thermal processing and proteolysis. They include major allergens from Brazil nut, peanuts, fruits, such as peaches, and cereals, such as rice and wheat; (2) The cupin superfamily, comprising the major globulin storage proteins from a number of plant species. The globulins have been found to be allergens in plant foods, such as peanuts, soya bean, and walnut; (3) The cyteine protease C1 family, comprising the papain-like proteases from microbes, plants, and animals. This family contains two notable allergens that sensitize via the GI tract, namely actinidin from kiwi fruit and the soybean allergen, Gly m Bd 30k/P34. This study describes the properties, structures, and evolutionary relationships of these protein families, the allergens that belong to them, and discusses them in relation to the role protein structure may play in determining protein allergenicity.

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[143] - Wijesinha-Bettoni R, Gao C, Jenkins JA, Mackie AR, Wilde PJ, Mills EN, et al. Heat Treatment of Bovine alpha-Lactalbumin Results in Partially Folded, Disulfide Bond Shuffled States with Enhanced Surface Activity. Biochemistry 2007;46:9774-9784

Prolonged heating of holo bovine alpha-lactalbumin (BLA) at 80 degrees C in pH 7 phosphate buffer in the absence of a thiol initiator improves the surface activity of the protein at the air:water interface, as determined by surface tension measurements. Samples after 30, 60, and 120 min of heating were analyzed on cooling to room temperature. Size-exclusion chromatography shows sample heterogeneity that increases with the length of heating. After 120 min of heating monomeric, dimeric, and oligomeric forms of BLA are present, with aggregates formed from disulfide bond linked hydrolyzed protein fragments. NMR characterization at pH 7 in the presence of Ca2+ of the monomer species isolated from the sample heated for 120 min showed that it consisted of a mixture of refolded native protein and partially folded protein and that the partially folded protein species had spectral characteristics similar to those of the pH 2 molten globule state of the protein. Circular dichroism spectroscopy showed that the non-native species had approximately 40% of the alpha-helical content of the native state, but lacked persistent tertiary interactions. Proteomic analysis using thermolysin digestion of three predominant non-native monomeric forms isolated by high-pressure liquid chromatography indicated the presence of disulfide shuffled isomers, containing the non-native 61-73 disulfide bond. These partially folded, disulfide shuffled species are largely responsible for the pronounced improvement in surface activity of the protein on heating K

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[144] - Roth-Walter F, Berin MC, Arnaboldi P, Escalante CR, Dahan S, Rauch J, et al. Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches. Allergy 2008;63:882-890

BACKGROUND: The underlying mechanisms responsible for allergic sensitization to food proteins remain elusive. To investigate the intrinsic properties (as well as the effect of pasteurization) of the milk proteins alpha-lactalbumin, beta-lactoglobulin and casein that promote the induction of milk allergy . METHODS: Alteration of structure and immune-reactivity of native and pasteurized proteins was assessed by gel filtration and ELISA. Uptake of these proteins was compared in vitro and in vivo. The biological effect was assessed by orally sensitizing C3H/HeJ mice with milk proteins followed by a graded oral challenge. Required dose to induce anaphylaxis, symptoms and mean body temperature was recorded. Antigen-specific antibodies and cytokine production by splenocytes were analyzed . RESULTS: Soluble beta-lactoglobulin and alpha-lactalbumin but not insoluble casein were readily transcytosed through enterocytes in vitro and in vivo. Pasteurization caused aggregation of beta-lactoglobulin and alpha-lactalbumin inhibiting uptake by intestinal epithelial cells in vitro and in vivo. Furthermore, aggregation redirected uptake to Peyer's patches, which promoted significantly higher Th2-associated antibody and cytokine production in mice than their native counterparts. Despite this only the soluble forms of beta-lactoglobulin and alpha-lactalbumin elicited anaphylaxis (following priming) when allergens were administered orally. Aggregated beta-lactoglobulin and alpha-lactalbumin as well as casein required systemic administration to induce anaphylaxis . CONCLUSIONS: These results indicate that triggering of an anaphylactic response requires two phases (1) sensitization by aggregates through Peyer's patches and (2) efficient transfer of soluble protein across the epithelial barrier. As the majority of common food allergens tend to form aggregates, this may be of clinical importance.

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[145] - Sanchez C, Frémont S. Conséquences des traitements thermiques et de la formulation sur la structure et l'allergénicité des protéines alimentaires. Rev Fr Allergol Immunol Clin 2003;43:13-20

Une augmentation importante du nombre de cas d'allergies alimentaires a été constatée au cours des cinq dernières années. Parmi les causes possibles, les procédés alimentaires et les interactions entre ingrédients et additifs sont fortement suspectés. Cette revue présente l'état des connaissances sur la relation entre chauffage, formulation, structure et allergénicité des protéines. Les procédés de fabrication alimentaire comprennent un ensemble d'opérations unitaires thermiques et mécaniques dont l'objet est de structurer, texturer et permettre une conservation satisfaisante de l'aliment. Les opérations de chauffage entraînent dans la plupart des cas une dénaturation irréversible de la conformation de la protéine pouvant conduire à son agrégation. Ces changements structuraux ne sont pas corrélés avec une diminution du potentiel allergénique des protéines. Selon les cas, un chauffage peut n'avoir aucun effet, diminuer ou augmenter ce potentiel. L'existence d'épitopes séquentiels et conformationnels, le démasquage de nouveaux épitopes ou la modification d'épitopes par réaction de Maillard peut expliquer partiellement les résultats reportés dans la littérature. Des interactions entre protéines allergènes et autres molécules présentes dans les aliments peuvent entraîner des modifications conformationnelles des protéines, même sans chauffage, et affecter leur stabilité thermique. En particulier, une augmentation ou une diminution de l'agrégation thermique des protéines peuvent être observées. L'effet de ces interactions sur le potentiel allergénique des protéines est aujourd'hui pratiquement inconnu.

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[146] - Anema SG, Lee SK, Klostermeyer H. Effect of Protein, Nonprotein-Soluble Components, and Lactose Concentrations on the Irreversible Thermal Denaturation of beta-Lactoglobulin and alpha-Lactalbumin in Skim Milk. J Agric Food Chem 2006;54:7339-7348

The effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible denaturation of beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) in reconstituted skim milk samples was studied over a wide temperature range (75-100 degrees C). The irreversible thermal denaturation of beta-LG had a reaction order of 1.5 and that of alpha-LA had a reaction order of 1.0 in all systems and under all conditions. The rates of irreversible denaturation of beta-LG and alpha-LA were markedly dependent upon the composition of the milk. At all temperatures, the irreversible denaturations of beta-LG and alpha-LA were enhanced at a higher protein concentration and were retarded when the nonprotein-soluble components and lactose concentrations were increased. The effects of increasing the concentrations of lactose and nonprotein-soluble components were interpreted using the preferential hydration theory and allowed for the interpretation of the changes in the denaturations of beta-LG and alpha-LA when the milk total solids concentration was increased.

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[147] - Wehbi Z, Perez MD, Sanchez L, Pocovi C, Barbana C, Calvo M. Effect of heat treatment on denaturation of bovine alpha-lactalbumin: determination of kinetic and thermodynamic parameters. J Agric Food Chem 2005;53:9730-9736

The effect of heat treatment on the denaturation of alpha-lactalbumin was studied, under different conditions, over a temperature range of 78-94 degrees C. The concentration of the residual immunoreactive protein after different treatments was determined by kinetic analysis, obtaining D and Z values. Thermodynamic parameters were also calculated. Denaturation of alpha-lactalbumin, measured by the loss of immunoreactivity, could be described as an order of reaction of n = 1.5. Results obtained indicated that alpha-lactalbumin was more heat-sensitive when treated in milk than in phosphate buffer. The protein was also denatured more rapidly in the apo form than in the calcium-saturated form. Besides, the thermal stability of apo-alpha-lactalbumin decreased with the binding of oleic acid.

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[148] - Zhang X, Keiderling TA. Lipid-Induced Conformational Transitions of beta-Lactoglobulin. Biochemistry 2006;45:8444-8452

Bovine beta-lactoglobulin (betaLG) provides an excellent model protein system for beta-to-alpha conformational change, but its behavior varies when the change is induced by alcohols, surfactants, or lipid vesicles. Here the interaction and orientation of betaLG in association with various artificial lipid vesicles at neutral and acidic pH have been studied by use of several complementary spectroscopic techniques. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra demonstrated that betaLG acquires a non-native alpha-helical structure upon binding with anionic lipids, while zwitterionic lipids do not have a significant effect on its conformation. The degree of induced alpha-helix depends on the lipid concentration and is strongly affected by the charge of the protein and lipids as well as the ionic strength of the solution. Near-UV CD and Trp emission spectra revealed that the tertiary structure of lipid-bound betaLG is highly expanded but not completely disrupted. Fluorescence quenching toge ther with a Trp emission blue shift showed that the Trp residues remain largely shielded from the solvent when interacting with DMPG, which would be consistent with at least some portions of betaLG having been inserted into the lipid membrane. The orientations of the alpha-helix and beta-sheet axes in membrane-bound betaLG were found to be parallel and perpendicular, respectively, to the membrane film normal, as determined by use of polarized attenuated total reflection (ATR) FTIR spectra. Our findings reveal that the lipid-induced beta-to-alpha transition in betaLG, accompanied by a substantial disruption in tertiary structure, is mainly driven by strong electrostatic interactions. Once the tightly packed betaLG is disrupted, hydrophobic residues become exposed and available for insertion into the lipid bilayer, where hydrophobic interaction with the lipids may play a role in stabilizing the helical components

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[149] - Davis PJ, Williams SC. Protein modification by thermal processing. Allergy 1998;53(Suppl. 46):102-105

This paper is a review concerning the way in which heat treatment can modify the allergenicity of food proteins. Any food protein may be allergenic if it can be absorbed intact, or as substantial fragments, through the gut mucosa and then evoke an immune (allergic) response. The intrinsic properties of the protein, the overall composition of the food, and the past processing history (especially thermal processing) all have an effect on the allergic potential. When a protein is denatured by heat, most of the original tertiary structure is lost, so that many of the sites recognized by antibodies on the native molecule are destroyed. There are many examples of allergenicity being reduced, but not eliminated, by heating. But heat-denatured proteins can also present new antigenic sites, uncovered by the unfolding process or created by new chemical reactions with other molecules present in the food (e.g., beta-lactoglobulin associating with alpha-lactalbumin in milk). We have found that heat-denatured beta-lactoglobulin has at least one new epitope, not found in the native state. Therefore, thermal processing can be part of a procedure for making hypoallergenic food, but will rarely be sufficient on its own. Increased understanding will help in evaluating novel proteins and processes.

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[150] - Bertrand-Harb C, Baday A, Dalgalarrondo M, Chobert JM, Haertlé T. Thermal modifications of structure and co-denaturation of alpha-lactalbumin and beta-lactoglobulin induce changes of solubility and susceptibility to proteases. Nahrung 2002;46:283-289

Study of heat denaturation of major whey proteins (beta-lactoglobulin or alpha-lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other codenaturation partner, and of existence of a previous thermal pretreatment (industrial whey). alpha-Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at > or = 85 degrees C. After its denaturation, alpha-lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys-X and Arg-X bonds). Heating over 85 degrees C of beta-lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co-denaturation of alpha-lactalbumin with beta-lactoglobulin increased their aggregation and resulted in complete exposure of beta-lactoglobulin peptic cleavage sites and partial unveiling of alpha-lactalbumin tryptic cleavage sites. The exposure of alpha-lactalbumin tryptic cleavage sites was slightly enhanced when the alpha-lactalbumin/beta-lactoglobulin mixture was heated at pH 7.5. Co-denaturation of fresh whey by heating at 95 degrees C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by beta-mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.

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[151] - Meltretter J, Schmidt A, Humeny A, Becker CM, Pischetsrieder M. Analysis of the Peptide Profile of Milk and Its Changes during Thermal Treatment and Storage. J Agric Food Chem 2008;56:2899-2906

In this study a new method was developed for analysis of the low molecular weight protein fraction of milk, allowing a simple and fast overview of the peptide profile of various milk samples. For this purpose, immobilized metal affinity chromatography (IMAC) was coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). By this technique, two major peptides in milk could be identified as fragments of alpha-s1-casein. During heat treatment of raw milk, five new peptides were generated, the origin of which could be assigned to the casein fraction. Storage experiments with extended shelf life milk at 4 degrees C did not show any changes in the peptide profile, whereas in ultra high temperature milk stored at room temperature, one peptide increased significantly, which was identified as the N-terminus of alpha-s1-casein. The peptide was assumed to be formed in an enzymatic reaction, which was confirmed in a storage experiment with sterilized milk. Analyses of different commercially available milk samples confirmed the results obtained with the heated and stored milk. Furthermore, differences in the peptide profiles of the samples, probably due to different cow breeds or lactation stages, were observed. These results establish IMAC prior to MALDI-TOF-MS as a valid tool for the rapid analysis of the peptide profile of milk.

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[152] - Wal JM. Cow's milk proteins/allergens. Ann Allergy Asthma Immunol 2002;89(6 Suppl. 1):3-10

OBJECTIVE: The primary objective of this review is to provide updated data on the structure and function of the main cow's milk proteins (CMPs) identified as allergens and on the characterization of their epitopes. DATA SOURCES: The review represents a synthesis of basic literature and most relevant original recent publications on both topics of clinical and epidemiologic aspects of milk allergy and of milk protein's bio- and immunochemistry. STUDY SELECTION: The expert opinion of the author was used to select the relevant data for the review. RESULTS: Most CMPs are potential allergens, even the proteins present at very low concentration. There are both conformational and linear epitopes, widely spread all along the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule which comprise highly conserved sequences responsible for immunoglobulin E cross-reactivity with corresponding milk proteins of other mammals, including human beings. Those sequential epitopes have also been proposed as good markers of persistent allergy to CMPs. CONCLUSIONS: No specific structure nor function is associated with allergenicity of CMPs. Variability and heterogeneity of the human immunoglobulin E response preclude anticipating the allergenic potential of any CMP or fragment thereof, as well as justify the need for being careful before using peptides for desensitization or proposing any milk protein hydrolysate in a diet for highly allergenic children.

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[153] - Bernard H, Wal JM. Comparaison de la composition protéique du lait de différentes espèces et de la structure des principales protéines. Alim'Inter 1998;3(11):14-17

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[154] - Morgan PE, Treweek TM, Lindner RA, Price WE, Carver JA. Casein Proteins as Molecular Chaperones. J Agric Food Chem 2005;53:2670-2683

Under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. We have investigated the chaperone activity of the major milk proteins alpha(S)-, beta-, and kappa-casein with reduced insulin and the milk whey proteins, alpha-lactalbumin and beta-lactoglobulin, and compared it with that of the mammalian small heat shock protein (sHsp), alpha-crystallin, and clusterin. alpha(S)-Casein exhibited different chaperone behavior under reduction and heat stresses, i.e., chaperone activity increased with increasing temperature (as observed with alpha-crystallin), but under reduction stress, its chaperone activity increased at lower temperatures. beta- and kappa-casein had comparable chaperone ability with each other but were less effective than alpha(S)-casein. Under molecular crowding conditions, precipitation of stressed protein was accelerated, and alpha(S)-casein was a poorer chaperone. Furthermore, at slightly alkaline pH values, alpha(S)-casein was a less effective chaperone than at neutral pH. Detailed fluorescence, size exclusion chromatography, and real-time NMR studies studies indicated that the casein proteins underwent conformational changes and stabilized the partially unfolded whey proteins prior to formation of high molecular weight soluble complexes. These results are consistent with casein proteins acting as molecular chaperones in a manner similar to sHsps and clusterin.

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[155] - Wal JM. Bovine milk allergenicity. Ann Allergy Asthma Immunol 2004;93(5 suppl.3):S2-S11

OBJECTIVE: To provide updated data on the characteristics (eg, structure, function, stability) of the main milk proteins identified as allergens and on the characterization of their epitopes. DATA SOURCES: Basic literature and the most relevant original recent publications on clinical and epidemiologic aspects of milk allergy and the biochemistry and immunochemistry of milk proteins. STUDY SELECTION: The expert opinion of the author was used to select the relevant data for the review . RESULTS: Most milk proteins, even proteins present at low concentrations, are potential allergens. Epitopes on milk proteins are both conformational and linear epitopes, widely spread throughout the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule, that comprise highly conserved sequences responsible for IgE cross-reactivity with corresponding milk proteins of other mammals, including humans. Those sequential epitopes have also been proposed as good markers of persistent allergy to milk proteins and may be of particular clinical significance . CONCLUSIONS: No specific structure or function is associated with allergenicity of milk proteins. Due to the great variability and heterogeneity of the human IgE response, no single allergen or particular structure can account for a major part of milk allergenicity. Furthermore, the available evidence is not sufficient to establish an intake threshold below which allergic reactions are not triggered or to predict reliably the effect of food processing on allergenic potential of milk proteins.

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[156] - Yong YH, Foegeding EA. Effects of Caseins on Thermal Stability of Bovine beta-Lactoglobulin. J Agric Food Chem 2008;56:10352-10358

Casein fractions have been shown to act as molecular chaperones and inhibit aggregation of whey proteins in dilute solutions (/=75 degrees C. Moreover, prolonged heating (90 min) of BLG with 2% (w/v) BCN (pH 6.0) at 90 degrees C produced a clear solution while BLG-ACN solutions formed translucent gels after heating for 15 min. The weight-averaged molar mass and root-mean-square (rms) radius of soluble aggregates were determined by size exclusion chromatography in conjunction with multiangle laser light scattering (SEC-MALS). SEC-MALS confirmed the turbidity results by showing that the BLG-BCN mixture (8% w/v protein) produced aggregates with lower molar mass and smaller rms radius (majority 20-40 nm). These results showed that BCN is a feasible component to stabilize higher concentrations of whey proteins in beverages.

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[157] - Lowe EK, Anema SG, Bienvenue A, Boland MJ, Creamer LK, Jimenez-Flores R. Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine beta-lactoglobulin and kappa-casein. J Agric Food Chem 2004;52:7669-7680

Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.

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[158] - Patel HA, Singh H, Anema SG, Creamer LK. Effects of Heat and High Hydrostatic Pressure Treatments on Disulfide Bonding Interchanges among the Proteins in Skim Milk. J Agric Food Chem 2006;54:3409-3420

Traditionally, milk has been heat treated to control microorganisms and to alter its functionality, for example, to increase its heat stability. Pressure treatment has been considered as a possible alternative for microorganism control, but some of the functionality-related milk protein interactions have not been explored. The present study used two novel two-dimensional polyacrylamide gel electrophoresis (2D PAGE) methods to explore the differences in the irreversible disulfide bond changes among the milk proteins after four common heat treatments and after 30-min pressure treatments of milk at 200, 400, 600, and 800 MPa at ambient temperature (22 degrees C). The pasteurizing heat treatment (72 degrees C for 15 s) denatured and aggregated only a few minor whey proteins, but the high heat treatments (100 degrees C for 120 s, 120 degrees C for 120 s, and 140 degrees C for 5 s) formed disulfide-bonded aggregates that included a high proportion of all of the whey proteins and kappa-casein (kappa-CN) and a proportion of the alpha(s2)-CN. Pressure treatment of milk at 200 MPa caused beta-lactoglobulin (beta-LG) to form disulfide-bonded dimers and incorporated beta-LG into aggregates, probably disulfide-bonded to kappa-CN. The other whey proteins appeared to be less affected at 200 MPa for 30 min. In contrast, pressure treatment at 800 MPa incorporated beta-LG and most of the minor whey proteins, as well as kappa-CN and much of the alpha(s2)-CN, into aggregates. The accessibility of alpha(s2)-CN and formation of complexes involving alpha(s2)-CN, kappa-CN, and whey proteins in the pressure treated milk is an important novel finding. However, only some of the alpha-lactalbumin was denatured or incorporated into the large aggregates. These and other results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that give the differences among the resultant aggregates, the sizes of the aggregates, and the product functionalities.

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[159] - Kohno Y, Honma K, Saito K, Shimojo N, Tsunoo H, Kaminogawa S, et al. Preferential recognition of primary protein structures of alpha-casein by IgG and IgE antibodies of patients with milk allergy. Ann Allergy 1994;73:419-422

We studied the binding activities of IgE and IgG antibodies in patients with allergy to cow milk proteins, against different alpha-casein preparations: alpha-casein treated with urea, hydrochloric acid, sodium hydroxide, or sodium dodecyl sulfate (SDS); or heat-denatured alpha- casein. The binding activities of IgE and IgG antibodies to these denatured alpha-casein preparations were compared with those to native alpha-casein. The binding activities of IgE and IgG antibodies to these denatured alpha-casein preparations were similar to those to native alpha-casein although the binding activities of IgG antibodies to these denatured alpha-casein preparations were relatively heterogeneous compared with those of IgE antibodies. Since modifications of alpha- casein did not alter the ability of alpha-casein to react with these antibodies, IgE and IgG antibodies to alpha-casein in sera from patients with allergy preferentially bind to the antigenic determinants associated with primary protein structures

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[160] - Taheri-Kafrani A, Gaudin JC, Rabesona H, Nioi C, Agarwal D, Drouet M et al. Effects of heating and glycation of beta-lactoglobulin on its recognition by IgE of sera from cow milk allergy patients. J Agric Food Chem 2009;57:4974-4982

beta-Lactoglobulin (beta-LG) is one of the cow's major milk proteins and the most abundant whey protein. This globular protein of about 18 kDa is folded, forming a beta-barrel (or calyx) structure. This structure is stabilized by two disulfide bonds and can be altered by heating above 65 degrees C. beta-LG is also one of the major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. During heating in the presence of reducing sugars, beta-LG is also submitted to the Maillard reaction, which at the first stage consists of the covalent fixation of sugars on the epsilon-amino groups of lysyl residues. The following steps are condensation and polymerization reactions leading to the formation of melanoidins (brown pigments). Despite the frequency of use of heating during milk transformation, the effects of heat-induced denaturation and of glycation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) patients are not fully understood. The objectives of our work were to evaluate the effect of heat-induced denaturation of bovine beta-LG on binding of IgE from CMA patients and to determine the effect of moderate glycation on the degree of recognition by IgE. We showed that heat-induced denaturation (loss of tertiary and secondary structures) of beta-LG is associated with weaker binding of IgE from CMA patients. It was also shown that moderate glycation of beta-LG in early stages of Maillard reaction has only a small effect on its recognition by IgE, whereas a high degree of glycation has a clear "masking" effect on the recognition of epitopes. This demonstrates the importance of epsilon-amino groups of lysines in the definition of epitopes recognized by IgE.

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[161] - Host A, Samuelsson EG. Allergic reactions to raw, pasteurized, and homogenized/pasteurized cow milk: a comparison. A double- blind placebo- controlled study in milk allergic children. Allergy 1988;43:113-118

Five children aged 12-40 months with IgE-mediated adverse reactions to cow milk (immediate onset clinical pattern of cow milk allergy) were orally challenged double-blind in random order with three different milk preparations processed from the same batch of milk 1) raw untreated cow milk, 2) pasteurized cow milk, 3) homogenized and pasteurized cow milk, and 4) Nutramigen (a commercial hypoallergenic infant formula based on hydrolysed casein) as placebo. Skin prick tests with the same preparations were also performed. On oral challenge the three different processed milk types provoked significant and similar allergic reactions in each child, and no adverse reactions followed the challenge with placebo (Nutramigen). Skin prick test with the same milk products were positive in all children and comparable to the results with an extract of purified raw cow milk protein (Soluprick), whereas Nutramigen did not elicit any skin reactions. A tendency towards a lower threshold of reaction and larger skin reactions induced by the processed milk preparations might indicate an increased ability of pasteurized and homogenized/pasteurized milk to evoke allergic reactions in patients allergic to milk.

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[162] - Paschke A, Besler M. Stability of bovine allergens during food processing. Ann Allergy Asthma Immunol 2002;89(6 Suppl. 1):16-20

OBJECTIVE: The primary objective of this review was to summarize reported findings about the influence of various food manufacturing processes on the potential alteration of bovine allergens in cow's milk, beef, and related food products. DATA SOURCES: This review was based on literature research in two German databases. STUDY SELECTION: The expert opinion of the authors was used to select the relevant data for the review. RESULTS: Changes in allergenic activity during food processing are attributable to inactivation or destruction of epitope structures, formation of new epitopes, or improved access of previously hidden epitopes. The allergenic potency of food could be altered by several food manufacturing procedures--such as mechanical, purification, thermal, biochemical, and chemical processes. The main processing steps studied by investigators were heating (dry heating, boiling, or cooking) and enzymatic digestion. A review of the available literature on the alteration of bovine allergens in cow's milk, meat, and related food products revealed reduction (but not elimination) of allergenicity by heating of cow's milk for 10 minutes. Although homogenization did not change the allergenic potency of cow's milk, it decreased the allergenicity of beef, as did freeze-drying. Digestion studies showed varied results. CONCLUSIONS: The allergenicity of some food products decreased during certain processing steps, but the results of other investigations differed. Therefore, more systematic research on the influence of food processing on allergenicity should be undertaken.

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[163] - Besler M, Steinhart H, Paschke A. Stability of food allergens and allergenicity of processed foods. J Chromatogr B Biomed Appl 2001;756:207-228

The allergenicity of food could be altered by several processing procedures. For various foods of animal and plant origin the available literature on this alteration is described. Investigations on hidden allergens in food products are also dealt with. [References: 160

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[164] - Morisawa Y, Matsumoto K, Akasawa A, Saito H, Wakiguchi H. Influence of Heating and Digestion on the B-cell Epitopes of Cow's Milk Allergens. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°731

RATIONALE: Regarding cow's milk allergy, it is reported that the responsible B-cell epitopes varied with patients, and that such differences appeared to be associated with clinical outgrow. In the present study, the influence of heat-treatment and pepsin-digestion of the milk protein on B-cell epitopes was investigated using passive histamine release test (HRT) from cultured human basophils. METHODS: Sera from 13 patients with positive IgE-RAST against at least one of the milk proteins, b-lactoglobulin or aS1casein were examined. All patients showed immediate type hypersensitivity upon ingestion of milk. b-lactoglobulin and aS1casein were untreated (UT), heat-treated (HT) or HT and pepsin-digested (PD). Cultured human basophils were derived from umbilical cord blood mononuclear cells in the presence of IL-3. After sensitization with 10% serum for 48 hours, cells were challenged with UT, HT or PD protein for 1 hour. The concentration of histamine in the supernatant was measured by RIA. RESULTS: b-lactoglobulin-induced histamine release was remarkably reduced by HT compared to UT when the cells were sensitized with 4 out of 7 patients' sera, whereas aS1casein-induced histamine release was not reduced by HT in all cases (with 6 patients). However, PD markedly reduced the histamine release by both b-lactoglobulin (6/7) and aS1casein (4/6). CONCLUSIONS: The susceptibility of B-cell epitopes to HT and PD varied with patients. b-lactoglobulin seems to be more sensitive to HT than aS1casein. Such differences might participate in the molecular mechanisms how different B-cell epitopes might associate with sensitization, onset of symptoms or clinical outgrow.

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[165] - Morisawa Y, Kitamura A, Ujihara T, Zushi N, Kuzume K, Shimanouchi Y et al. Effect of heat treatment and enzymatic digestion on the B cell epitopes of cow’s milk proteins. Clin Exp Allergy 2009;39:918-925

BACKGROUND: Processing milk leads to changes in clinical allergenicity. However, the mechanism by which heat treatment affects the allergenicity of milk proteins is not fully understood . OBJECTIVE: We investigated the effect of heat treatment and enzymatic digestion on the allergenicity of B cell epitopes of milk proteins using a histamine release assay . METHODS: Human basophils were passively sensitized using sera from 10 patients with allergies to cow's milk. All the patients experienced symptoms immediately after ingesting milk. The human basophils were obtained from umbilical cord blood mononuclear cells after culturing the mononuclear cells for 3-4 weeks in the presence of IL-3. After sensitization with 10% patient sera for 48 h, the cells were stimulated with untreated, heat-treated, or heat-treated and pepsin-and-trypsin-digested beta-lactoglobulin or alpha-casein for 1 h. The histamine concentrations in the supernatants were then measured by radioimmunoassay . RESULTS: Heat treatment alone did not alter the molecular weight of beta-lactoglobulin or alpha-casein. Heat treatment of beta-lactoglobulin significantly increased its susceptibility to enzymatic digestion in a time- and temperature-dependent manner and reduced its ability to induce histamine release from sensitized basophils. Similar findings were not observed for alpha-casein. The combination of heat treatment and enzymatic digestion reduced the abilities of both beta-lactoglobulin and alpha-casein to induce histamine release from passively sensitized basophils . CONCLUSIONS: Heat treatment reduced the allergenicity of beta-lactoglobulin by inducing conformational changes and by increasing its susceptibility to enzymatic digestion, both of which disrupted B cell epitopes, whereas heat treatment alone did not alter the allergenicity of alpha-casein.

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[166] - Rytkonen J, Valkonen KH, Virtanen V, Foxwell RA, Kyd JM, Cripps AW, et al. Enterocyte and M-Cell Transport of Native and Heat-Denatured Bovine beta-Lactoglobulin: Significance of Heat Denaturation. J Agric Food Chem 2006;54:1500-1507

The three-dimensional structure, digestibility, and immunological properties of bovine beta-lactoglobulin (beta-lg) are modified by heat treatments used in processing of liquid milk products. Because it is not known if such treatments also modify the intestinal transport properties of beta-lg, the transport of native and heat-denatured bovine beta-lg was investigated in experimental cell models using Caco-2 cells and M cells. Transport of beta-lg labeled with a fluorescent marker was followed with fluorometric measurements, electrophoretic analyses, and fluorescence microscopy. The data show that both cell types transported native beta-lg more efficiently than they did heat-denatured beta-lg. In addition, M cells transported native beta-lg more than Caco-2 cells. Transport of native and heat-denatured beta-lg was transcellular. The electrophoretic data also suggest that heat-denatured beta-lg may have degraded more than native beta-lg during the transport.

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[167] - Roth-Walter F, Berin MC, Arnaboldi P, Escalante CR, Dahan S, Rauch J, et al. Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches. Allergy 2008;63:882-890

BACKGROUND: The underlying mechanisms responsible for allergic sensitization to food proteins remain elusive. To investigate the intrinsic properties (as well as the effect of pasteurization) of the milk proteins alpha-lactalbumin, beta-lactoglobulin and casein that promote the induction of milk allergy . METHODS: Alteration of structure and immune-reactivity of native and pasteurized proteins was assessed by gel filtration and ELISA. Uptake of these proteins was compared in vitro and in vivo. The biological effect was assessed by orally sensitizing C3H/HeJ mice with milk proteins followed by a graded oral challenge. Required dose to induce anaphylaxis, symptoms and mean body temperature was recorded. Antigen-specific antibodies and cytokine production by splenocytes were analyzed . RESULTS: Soluble beta-lactoglobulin and alpha-lactalbumin but not insoluble casein were readily transcytosed through enterocytes in vitro and in vivo. Pasteurization caused aggregation of beta-lactoglobulin and alpha-lactalbumin inhibiting uptake by intestinal epithelial cells in vitro and in vivo. Furthermore, aggregation redirected uptake to Peyer's patches, which promoted significantly higher Th2-associated antibody and cytokine production in mice than their native counterparts. Despite this only the soluble forms of beta-lactoglobulin and alpha-lactalbumin elicited anaphylaxis (following priming) when allergens were administered orally. Aggregated beta-lactoglobulin and alpha-lactalbumin as well as casein required systemic administration to induce anaphylaxis . CONCLUSIONS: These results indicate that triggering of an anaphylactic response requires two phases (1) sensitization by aggregates through Peyer's patches and (2) efficient transfer of soluble protein across the epithelial barrier. As the majority of common food allergens tend to form aggregates, this may be of clinical importance.

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[168] - Fu TJ, Abbott UR, Hatzos C. Digestibility of food allergens and nonallergenic proteins in simulated gastric fluid and simulated intestinal fluid-a comparative study. J Agric Food Chem 2002;50:7154-7160

Information on the comparative digestibility of food allergens and nonallergenic proteins is crucial when stability to digestion is to be used as a criterion to assess the allergenic potential of novel proteins. In this work, we compared the digestive stability of a number of food allergens and proteins of unproven allergenicity and examined whether allergens possess a higher stability than nonallergenic proteins of similar cellular functions, and whether there is a correlation between protein digestibility and allergenicity. The stability of groups of storage proteins, plant lectins, contractile proteins, and enzymes, both allergens and proteins with unproven allergenicity, in a standard simulated gastric fluid and a standard simulated intestinal fluid was measured. Food allergens were not necessarily more resistant to digestion than nonallergenic proteins. There was not a clear relationship between digestibility measured in vitro and protein allergenicity.

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[169] - Thomas K, Aalbers M, Bannon GA, Bartels M, Dearman RJ, Esdaile DJ, et al. A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Regul Toxicol Pharmacol 2004;39:87-98

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

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[170] - Untersmayr E, Jensen-Jarolim E. The effect of gastric digestion on food allergy. Curr Opin Allergy Clin Immunol 2006;6:214-219

PURPOSE OF REVIEW: The role of the stomach as the primary location of protein digestion is very well recognized, leading to classification of proteins as digestion-resistant or digestion-labile. This review analyses the role of gastric digestion in food allergy. RECENT FINDINGS: Hindrance of gastric digestion by elevation of the gastric pH, the therapeutic goal of anti-ulcer medication, was recently shown to trigger food allergy via oral sensitization in a murine food allergy model. The relevance in humans was assessed in an observational study of 152 gastroenterological patients who were medicated with anti-ulcer drugs due to dyspeptic disorders. Twenty-five percent of all patients developed a boost or de-novo IgE formation towards regular constituents of the daily diet. The clinical relevance of the induced antibodies was confirmed by positive skin and oral-provocation tests. Moreover, the importance of gastric digestion was also proven for food-allergic patients, as the allergenicity of allergens were reduced up to a 10,000-fold by gastric digestion. SUMMARY: These recent studies indicate for the first time the important gate-keeping function of gastric digestion, both in the sensitization and the effector phases of food allergy.

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[171] - Goodman RE, Taylor SL, Yamamura J, Kobayashi T, Kawakami H, Kruger CL, et al. Assessment of the potential allergenicity of a Milk Basic Protein fraction. Food Chem Toxicol 2007;45:1787-1794

A specific basic fraction of bovine milk, termed Milk Basic Protein (MBP), has the potential to provide nutritionally important benefits if used as a food ingredient. Although derived from milk, MBP is intended for use as an ingredient in other foods. Although the proteins in MBP are not identified as milk allergens, food products containing MBP will be labelled as containing milk as a caution to milk allergic consumers under food labelling guidelines in the US and the European Union as MBP has not been demonstrated to be free of milk allergens. This study evaluated the potential allergenicity of the five identified proteins in MBP. While extensive studies have not demonstrated allergenicity of lactoferrin, the four other proteins are less studied. The four were tested here by sequence identity comparison to known allergens, and for stability of these proteins in acidic pepsin as a characteristic common to many food allergens. No significant sequence similarities were identified for the MBP proteins compared to known allergens. All but one of the protein components of MBP were digested relatively quickly by pepsin. Therefore, based on molecular characteristics and expected exposure, the protein components in MBP are unlikely to present any increased risk of allergy for milk allergic subjects or of cross-reactivity for other allergic subjects. However, since the proteins are derived from milk, products containing MBP will need to be labelled as containing milk proteins to warn milk allergic subjects of the potential risk of allergic reactions

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[172] - Besler M, Steinhart H, Paschke A. Stability of food allergens and allergenicity of processed foods. J Chromatogr B Biomed Appl 2001;756:207-228

The allergenicity of food could be altered by several processing procedures. For various foods of animal and plant origin the available literature on this alteration is described. Investigations on hidden allergens in food products are also dealt with. [References: 160

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[173] - Schmidt DG, Meijer RJ, Slangen CJ, van Beresteijn EC. Raising the pH of the pepsin-catalysed hydrolysis of bovine whey proteins increases the antigenicity of the hydrolysates. Clin Exp Allergy 1995;25:1007-1017

BACKGROUND: Hypersensitivity to cow milk protein is frequently observed in infancy. Since the pH in the infant's stomach is relatively high (pH 3-4) compared with adults (pH 2), an incomplete digestion of the milk proteins is expected to occur. OBJECTIVE: The determination of the degree of hydrolysis by pepsin of the four main proteins of bovine whey, i.e. alpha-lactalbumin (alpha La), beta-lactoglobulin (beta Lg), bovine serum albumin (BSA) and bovine immunoglobulin G (B-IgG), in the pH range 2.0-4.0 and of the antigenic properties of the resulting hydrolysates. METHODS: Whey proteins were successively hydrolysed with pepsin at pH values ranging from 2.0 to 4.0 and with pancreatic enzymes at pH 7.5 using a pH-stat. The resulting hydrolysates were characterized by their degree of hydrolysis, and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, gel permeation chromatography and immunologically by competitive enzyme-linked immunosorbent assay. RESULTS: In general, the degree of hydrolysis, the gel electrophoretic patterns, the contents of peptides of molecular mass > 5 kDa and the residual human-immunoglobulin E and G antigenicities of the hydrolysates did not differ much whether the pepsin incubation was done at pH 2.0 or 3.0. Pepsin incubation at pH 4.0, however, resulted in a decreased hydrolysis and enhanced residual antigenicity of alpha La, BSA and B-IgG, but not of beta Lg. CONCLUSION: The poor and slow degradation of the antigenic epitopes of whey proteins when pepsin digestion occurs under conditions that prevail in the stomach of infants could be of much importance for the development of cow milk hypersensitivity. The immature gastrointestinal mucosal barrier of infants allows large antigenic fragments of these proteins to pass into the systemic circulation

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[174] - Sakai K, Yoshino K, Satter MA, Ota F, Nii Y, Fukuta K, et al. Effects of pH variation and NaCl on in vitro digestibility of cow's milk proteins in commercially available infant formulas. J Nutr Sci Vitaminol (Tokyo) 2000;46:325-328

Allergenicity and antigenicity of food proteins are generally dependent on their heat-stability and resistance to digestive enzymes. Using the methods of SDS-PAGE and immunoblots, we assessed peptic-digestibility of major milk proteins (casein, beta-lactoglobulin and alpha-lactalbumin) in commercially available infant formulas in acidic pH range (pH 1.5 to 4.0), and we also investigated the effect of NaCl on peptic-digestibility of cow's milk proteins at pH 2.0. The profile of peptic-digestion of casein was similar at pH range from 1.5 to 3.5 where the intact protein of casein rapidly disappeared, whereas slow digestibility of the protein was found at pH 4.0. Beta-lactoglobulin and alpha-lactalbumin were digested at pH range from 1.5 to 2.5 and were almost entirely resistant to peptic digestion at over pH 3.0. In the presence of NaCl at pH 2.0, casein showed a good digestibility by pepsin similar to that in the absence of NaCl, in contrast to beta-lactoglobulin and alpha-lactalbumin both having their peptic-d igestibility decreased in the presence of 0.2 M NaCl. As the state of the stomach in new born infants shows low amounts of secretary pepsin and out of optimum pH of peptic activity, low digestibility of beta-lactoglobulin and alpha-lactalbumin in cow's milk based infant formulas at over pH 3.0 is supposed to be responsible for their allergenicity

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[175] - Mazon A, Solera E, Alentado N, Oliver F, Pamies R, Caballero L, et al. Frequent IgE sensitization to latex, cow’s milk, and egg in children with short bowel syndrome. Pediatr Allergy Immunol 2008;19:180-183

Children with short bowel syndrome (SBS) undergo frequent operations, so they are at risk for sensitizing to latex. There have been isolated reports of sensitization to food in these children. In a cross-sectional study, we assessed sensitization to latex, cow's milk, and egg with skin prick tests (SPT) and serum-specific immunoglobulin E (IgE) in 14 children with SBS. Data were collected about the number of operations with latex devices, serum total IgE, and history of feeding with milk formula. Ten children were sensitized to latex (specific IgE median: 6.7 kU/l, range: 0.5-33). Compared with those non-sensitized, sensitized children had significantly (p < 0.05) higher levels of serum total IgE in z-units (mean rank 3.25 vs. 9.2, respectively), and more operations with latex devices (mean rank 3.75 vs. 9). Eight children were sensitized to cow's milk, one with only positive SPT, the other seven with serum-specific IgE (median: 3.5, range: 0.5-21.1 kU/l), and five to egg (specific IgE median: 0.68, range: 0.58-2.17 kU/l). Except for some isolated days with cow's milk formula, the children had been initially fed with a diet without intact cow's milk proteins. Sensitization to latex, cow's milk, and egg is very frequent in children with SBS. They should be treated in a latex-free environment since the very early stages of the disease, and should be routinely studied regarding food sensitization, as this might contribute as an added factor in the chronic diarrhea of these patients.

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[176] - Benlounes N, Dupont C, Candalh C, Blaton MA, Darmon N, Desjeux JF, et al. The threshold for immune cell reactivity to milk antigens decreases in cow's milk allergy with intestinal symptoms. J Allergy Clin Immunol 1996;98:781-789

BACKGROUND: In cow's milk allergy (CMA) with intestinal symptoms, peripheral blood mononuclear cells (PBMCs) secrete tumor necrosis factor-alpha (TNF-alpha), altering intestinal function. However, the type of cow's milk protein (CMP) that triggers symptoms (intact or intestinally processed) is not known, and neither is the minimal amount required. METHODS: PBMCs were isolated from infants with active CMA or cured infants just before a new challenge and stimulated with intact or intestinally processed CMP. Supernatants were tested for cytokine content and for their ability to perturb intestinal barrier capacity, measured in Using chambers in HT29-19A intestinal cells. RESULTS: PBMCs from infants with active CMA secreted more TNF-alpha, when they were stimulated with intact rather than intestinally processed CMPs, and more TNF-alpha than PBMCs from cured infants. Accordingly, supernatants from PBMCs stimulated with intact but not intestinally processed CMPs significantly increased intestinal permeability. The C MP concentration required to trigger TNF-alpha secretion capable of altering intestinal function was very small in infants with active CMA (approximately 2 micrograms/ml), but about 300 times higher in cured infants. CONCLUSION: Intact rather than intestinally processed proteins stimulate PBMCs to release TNF-alpha and alter intestinal barrier capacity. The threshold for PBMC reactivity to milk antigens drops considerably during active CMA with intestinal symptoms

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[177] - Burnett GR, Wickham M, Fillery-Travis A, Robertson JA, Belton PS, Gilbert SM, et al. Interaction between protein allergens and model gastric emulsions. Biochem Soc Trans 2002;30:916-918

The observed resistance to pepsinolysis of known food allergens has been suggested as a predictor of their allergenic risk. Consequently, resistance to pepsinolysis has become incorporated into decision tree assessment for potential allergenic risk posed by novel foods. However, existing methods take little account of the interaction between food structure and physiological conditions existing during digestion in vivo. Here we show that a range of protein allergens can adsorb to model stomach emulsions, providing a further means of resisting digestion. We also show that raising the pH and the addition of bile salts to a model stomach emulsion, thereby mimicking the duodenal environment, has the effect of desorbing the adsorbed protein.

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[178] - Papageorgiou PS, Mondoulet L, Rigby NR, Bossios A, Papadopoulos NG, Bernard H, et al. Effect of in vitro gastric digestion on the allergenicity of purified B-lactoglobulin. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1026

Introduction: Digestibility of milk allergens has been related to their allergenicity but direct information is limited. The purpose of the study was to assess the effect of in vitro gastric digestion on the allergenicity of purified B-lactoglobulin (Blg). Methodology: Digesta were prepared by digestion of Blg (Sigma Chemical Co.) using pepsin with or without phosphatidylcholine (PC). Blg concentration in digesta and controls was measured by a sandwich ELISA using 2 different monoclonal antibodies. Assessment of the allergenicity of digesta was done in vitro by specific basophil activation(Basotest-Becton Dickinson) and lymphocyte proliferation (PCNA-flow cytometry) using cells from patients allergic toBlg, and in vivo by skin prick testing (SPT) of these patients following Ethics Committee and parent approval. Results: Blg was partially digested by pepsin. Residual concentrations of Blg in digesta were 28% (28mg/g digesta) when digestion was performed without PC but 60% (600mg/g digesta) in the presence of PC during digestion. No statistically significant difference was found in the CD63 expression (activation) of basophils or lymphocyte proliferation to Blg digesta compared to native Blg regardless of the presence of PC. Regarding skin reactivity there was a tendency towards reduction in mean diameter of SPT in the presence of pepsin alone but a 3-6 fold increase in mean diameter was found when PC was added to the digestion. Conclusion: Our results showed that pepsin partially digests Blg with a significant inhibition of this process by PC. Presence of PC during digestion results in an increased allergenicity of Blg as assessed by SPT reactivity of patients allergic to Blg.

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[179] - Moreno FJ, Mackie AR, Mills EN. Phospholipid Interactions Protect the Milk Allergen alpha-Lactalbumin from Proteolysis during in Vitro Digestion. J Agric Food Chem 2005;53:9810-9816

Interactions with food components may alter the resistance of food proteins to digestion, a property thought to play an important role in determining allergenic properties. The kinetics of breakdown of the bovine milk allergen alpha-lactalbumin during in vitro gastrointestinal digestion was found to be altered by interactions with physiologically relevant levels of phosphatidylcholine (PC), a surfactant that is abundant both in milk and is actively secreted by the stomach. Breakdown during gastric digestion was slowed in the presence of PC and accompanied by small alterations in the profile of resulting peptides, with little effect being observed during subsequent duodenal digestion. alpha-Lactalbumin was found to unfold at gastric (acid) pH, giving a CD spectrum similar to that obtained for the partially folded state it is known to adopt at pH values below its isoelectric point. Fluorescence polarization studies performed at low pH indicated that this partially unfolded form of the protein was able to penetrate into the PC vesicles. These interactions are probably responsible for the slowing of gastric digestion by reducing the accessibility of the protein to pepsin. These findings show that interactions with other food components, such as lipids, may alter the rate of breakdown of food proteins in the gastrointestinal tract. It underlines the importance of the food matrix in affecting patterns of food allergen digestion and hence presentation to the immune system and that in vitro digestion systems used for assessing digestibility of allergens must take account of surfactants.

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[180] - Sakai K, Yoshino K, Satter MA, Ota F, Nii Y, Fukuta K, et al. Effects of pH variation and NaCl on in vitro digestibility of cow's milk proteins in commercially available infant formulas. J Nutr Sci Vitaminol (Tokyo) 2000;46:325-328

Allergenicity and antigenicity of food proteins are generally dependent on their heat-stability and resistance to digestive enzymes. Using the methods of SDS-PAGE and immunoblots, we assessed peptic-digestibility of major milk proteins (casein, beta-lactoglobulin and alpha-lactalbumin) in commercially available infant formulas in acidic pH range (pH 1.5 to 4.0), and we also investigated the effect of NaCl on peptic-digestibility of cow's milk proteins at pH 2.0. The profile of peptic-digestion of casein was similar at pH range from 1.5 to 3.5 where the intact protein of casein rapidly disappeared, whereas slow digestibility of the protein was found at pH 4.0. Beta-lactoglobulin and alpha-lactalbumin were digested at pH range from 1.5 to 2.5 and were almost entirely resistant to peptic digestion at over pH 3.0. In the presence of NaCl at pH 2.0, casein showed a good digestibility by pepsin similar to that in the absence of NaCl, in contrast to beta-lactoglobulin and alpha-lactalbumin both having their peptic-d igestibility decreased in the presence of 0.2 M NaCl. As the state of the stomach in new born infants shows low amounts of secretary pepsin and out of optimum pH of peptic activity, low digestibility of beta-lactoglobulin and alpha-lactalbumin in cow's milk based infant formulas at over pH 3.0 is supposed to be responsible for their allergenicity

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[181] - Spuergin P, Mueller H, Walter M, Schiltz E, Forster J. Allergenic epitopes of bovine alpha S1-casein recognized by human IgE and IgG. Allergy 1996;51:306-312

B-cell epitopes of bovine alpha S1-casein, one of the major allergens of cow's milk, were identified by a screening method based on synthetic peptides. According to the known amino acid sequence of alpha S1-casein, a set of 188 overlapping sequential decapeptides shifted by one amino acid was manually synthesized on polyethylene pins by the 9-fluorenyl-methoxycarbonyl (Fmoc) method. Peptides were screened by an enzyme-linked immunosorbent assay (ELISA) specific for human IgE and IgG. Bound antibodies were detected by successive incubation with up to three polyclonal antibodies, the last one conjugated to horseradish peroxidase. Tested sera were from 15 patients with acute clinical reactions to cow's milk and IgE-specific reactions to bovine alpha-casein in the ELISA and immunoblot. Sera from 10 healthy subjects without remarkable reactions to cow's milk proteins were used as controls. All sera from allergic subjects showed reactions with three regions of alpha S1-casein, corresponding to amino acids 19-30, 93-98, and 141-150. Furthermore, individual sera showed reactions with other parts of the protein. No essential differences in the epitope specificity of IgE and IgG were found. Inhibition of IgE binding to alpha S1-casein with soluble synthetic peptides confirmed the results and revealed peptide CN-2 as the most inhibiting one.

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[182] - Sélo I, Clément G, Bernard H, Chatel J, Créminon C, Peltre G, et al. Allergy to bovine beta-lactoglobulin: specificity of human IgE to tryptic peptides. Clin Exp Allergy 1999;29:1055-1063

BACKGROUND: Bovine beta-lactoglobulin (Blg) is a major cow's milk allergen. It is the main whey protein, without any counterpart in human milk. Blg chemical hydrolysates appeared to retain most of the immunoreactivity of the native protein. Allergenicity of Blg has already been shown to be associated with the four peptides derived from cyanogen bromide cleavage of Blg. OBJECTIVES: To map the major allergenic epitopes (e.g. regions of the molecule able to bind IgE) on Blg using specific IgE from sera of 46 milk-allergic patients as a probe. METHODS: Direct and competitive inhibition enzyme immunoassays involving immobilized native protein or purified peptides derived from Blg tryptic cleavage. RESULTS: Several peptides capable of specifically binding human IgEs were identified and were classified according to the intensity and frequency of the responses. The major epitopes appeared to be fragments (41-60), (102-124) and (149-162) recognized by 92, 97 and 89% of sera, respectively, whilst a second group which contained the fragments (1-8) and (25-40) was recognized by 58 and 72% of the population. A third group, comprising peptides (9-14), (84-91) and (92-100), was still detected by more than 40% of sera. CONCLUSION: Three peptides were identified as major epitopes, recognized by a large majority of human IgE antibodies. Numerous other epitopes are scattered all along the Blg sequence.

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[183] - Wal JM. Cow's milk proteins/allergens. Ann Allergy Asthma Immunol 2002;89(6 Suppl. 1):3-10

OBJECTIVE: The primary objective of this review is to provide updated data on the structure and function of the main cow's milk proteins (CMPs) identified as allergens and on the characterization of their epitopes. DATA SOURCES: The review represents a synthesis of basic literature and most relevant original recent publications on both topics of clinical and epidemiologic aspects of milk allergy and of milk protein's bio- and immunochemistry. STUDY SELECTION: The expert opinion of the author was used to select the relevant data for the review. RESULTS: Most CMPs are potential allergens, even the proteins present at very low concentration. There are both conformational and linear epitopes, widely spread all along the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule which comprise highly conserved sequences responsible for immunoglobulin E cross-reactivity with corresponding milk proteins of other mammals, including human beings. Those sequential epitopes have also been proposed as good markers of persistent allergy to CMPs. CONCLUSIONS: No specific structure nor function is associated with allergenicity of CMPs. Variability and heterogeneity of the human immunoglobulin E response preclude anticipating the allergenic potential of any CMP or fragment thereof, as well as justify the need for being careful before using peptides for desensitization or proposing any milk protein hydrolysate in a diet for highly allergenic children.

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[184] - Kleber N, Krause I, Illgner S, Hinrichs J. The antigenic response of beta-lactoglobulin is modulated by thermally induced aggregation. Eur Food Res Technol 2004;219:105-110

Abstract The antigenic response of thermally denatured and aggregated -lactoglobulin was determined by an indirect competitive enzyme-linked immunosorbent assay using polyclonal antibodies from the egg yolk of chicken immunized with heat-denatured -lactoglobulin as a measure for the potential antigenicity/allergenicity. The heat denaturation and the aggregation of heated whey protein isolate solutions were followed by reversed-phase high-performance liquid chromatography and photon correlation spectroscopy. Thermally modified whey proteins showed a remarkable increase of antigenicity when heated to 90 °C, possibly as a consequence of the exposure or formerly hidden epitopes. Above 90 °C, the antigenic response decreased owing to the loss of conformational epitopes and masking of sequential epitopes in the course of aggregation to particles. When large and compact particles were formed, the antigenicity was reduced remarkably. Depending the heating conditions applied, the structure and the size of whey protein particles and thus the potential allergenicity may be modulated in a wide range.

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[185] - Morisawa Y, Matsumoto K, Akasawa A, Saito H, Wakiguchi H. Influence of Heating and Digestion on the B-cell Epitopes of Cow's Milk Allergens. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°731

RATIONALE: Regarding cow's milk allergy, it is reported that the responsible B-cell epitopes varied with patients, and that such differences appeared to be associated with clinical outgrow. In the present study, the influence of heat-treatment and pepsin-digestion of the milk protein on B-cell epitopes was investigated using passive histamine release test (HRT) from cultured human basophils. METHODS: Sera from 13 patients with positive IgE-RAST against at least one of the milk proteins, b-lactoglobulin or aS1casein were examined. All patients showed immediate type hypersensitivity upon ingestion of milk. b-lactoglobulin and aS1casein were untreated (UT), heat-treated (HT) or HT and pepsin-digested (PD). Cultured human basophils were derived from umbilical cord blood mononuclear cells in the presence of IL-3. After sensitization with 10% serum for 48 hours, cells were challenged with UT, HT or PD protein for 1 hour. The concentration of histamine in the supernatant was measured by RIA. RESULTS: b-lactoglobulin-induced histamine release was remarkably reduced by HT compared to UT when the cells were sensitized with 4 out of 7 patients' sera, whereas aS1casein-induced histamine release was not reduced by HT in all cases (with 6 patients). However, PD markedly reduced the histamine release by both b-lactoglobulin (6/7) and aS1casein (4/6). CONCLUSIONS: The susceptibility of B-cell epitopes to HT and PD varied with patients. b-lactoglobulin seems to be more sensitive to HT than aS1casein. Such differences might participate in the molecular mechanisms how different B-cell epitopes might associate with sensitization, onset of symptoms or clinical outgrow.

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[186] - Maynard F, Jost R, Wal JM. Human IgE binding capacity of tryptic peptides from bovine alpha-lactalbumin. Int Arch Allergy Immunol 1997;113:478-488

The specific IgE binding capacity of native bovine alpha-lactalbumin (alpha-La), a globular whey protein, and tryptic peptides was investigated using 19 sera from patients with cow's milk protein allergy. The specific anti-bovine alpha-La IgE titers ranged from 0.6 to 125 IU/ml. Highly purified tryptic peptides from native and disulfide-bond-reduced alpha-La were obtained by reverse phase chromatography. By ELISA technique using immobilized native protein or peptides, 11 of the 19 sera reacted exclusively with intact protein while 8 of them also presented a specific IgE response to different tryptic peptides. Polyclonal IgE population specificity was not related to anti-bovine alpha-La IgE levels. Sequence (17G-K58) and larger peptides sharing this sequence were most strongly and frequently recognized. Competitive ELISA inhibition tests confirmed this IgE-specific response and gave also clear evidence for IgE binding to smaller peptides corresponding to sequences (6C-R10):S-S:(115L-L123) and (109A-L123). IgE binding to native alpha-La and large peptides confirmed the importance of conformational epitope(s). However, in some sera reduced and S-alkylated peptide (59I-K94) exhibited a similar or higher IgE binding capacity than the native corresponding fragment, suggesting the existence of sequential epitope(s) exposed through protein denaturation. Moreover, IgE binding sequences were also located within hydrophobic regions of alpha-La and/or within parts with high sequence homology to human alpha-La.

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[187] - Sélo I, Clément G, Bernard H, Chatel J, Créminon C, Peltre G, et al. Allergy to bovine beta-lactoglobulin: specificity of human IgE to tryptic peptides. Clin Exp Allergy 1999;29:1055-1063

BACKGROUND: Bovine beta-lactoglobulin (Blg) is a major cow's milk allergen. It is the main whey protein, without any counterpart in human milk. Blg chemical hydrolysates appeared to retain most of the immunoreactivity of the native protein. Allergenicity of Blg has already been shown to be associated with the four peptides derived from cyanogen bromide cleavage of Blg. OBJECTIVES: To map the major allergenic epitopes (e.g. regions of the molecule able to bind IgE) on Blg using specific IgE from sera of 46 milk-allergic patients as a probe. METHODS: Direct and competitive inhibition enzyme immunoassays involving immobilized native protein or purified peptides derived from Blg tryptic cleavage. RESULTS: Several peptides capable of specifically binding human IgEs were identified and were classified according to the intensity and frequency of the responses. The major epitopes appeared to be fragments (41-60), (102-124) and (149-162) recognized by 92, 97 and 89% of sera, respectively, whilst a second group which contained the fragments (1-8) and (25-40) was recognized by 58 and 72% of the population. A third group, comprising peptides (9-14), (84-91) and (92-100), was still detected by more than 40% of sera. CONCLUSION: Three peptides were identified as major epitopes, recognized by a large majority of human IgE antibodies. Numerous other epitopes are scattered all along the Blg sequence.

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[188] - Papageorgiou PS, Mondoulet L, Rigby NR, Bossios A, Papadopoulos NG, Bernard H, et al. Effect of in vitro gastric digestion on the allergenicity of purified B-lactoglobulin. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1026

Introduction: Digestibility of milk allergens has been related to their allergenicity but direct information is limited. The purpose of the study was to assess the effect of in vitro gastric digestion on the allergenicity of purified B-lactoglobulin (Blg). Methodology: Digesta were prepared by digestion of Blg (Sigma Chemical Co.) using pepsin with or without phosphatidylcholine (PC). Blg concentration in digesta and controls was measured by a sandwich ELISA using 2 different monoclonal antibodies. Assessment of the allergenicity of digesta was done in vitro by specific basophil activation(Basotest-Becton Dickinson) and lymphocyte proliferation (PCNA-flow cytometry) using cells from patients allergic toBlg, and in vivo by skin prick testing (SPT) of these patients following Ethics Committee and parent approval. Results: Blg was partially digested by pepsin. Residual concentrations of Blg in digesta were 28% (28mg/g digesta) when digestion was performed without PC but 60% (600mg/g digesta) in the presence of PC during digestion. No statistically significant difference was found in the CD63 expression (activation) of basophils or lymphocyte proliferation to Blg digesta compared to native Blg regardless of the presence of PC. Regarding skin reactivity there was a tendency towards reduction in mean diameter of SPT in the presence of pepsin alone but a 3-6 fold increase in mean diameter was found when PC was added to the digestion. Conclusion: Our results showed that pepsin partially digests Blg with a significant inhibition of this process by PC. Presence of PC during digestion results in an increased allergenicity of Blg as assessed by SPT reactivity of patients allergic to Blg.

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[189] - Peyron S, Mouecoucou J, Fremont S, Sanchez C, Gontard N. Effects of Heat Treatment and Pectin Addition on beta-Lactoglobulin Allergenicity. J Agric Food Chem 2006;54:5643-5650

The specific effects of heat treatment and/or addition of low/high-methylated pectin (LMP/HMP) on the allergenicity of beta-lactoglobulin (beta-Lg) and its hydrolysis products were investigated through a two-step in vitro digestion approach. beta-Lg was first hydrolyzed by pepsin and then by a trypsin/chymotrypsin (T/C) mixture done in a dialysis bag with a molecular weight cutoff of 1000. The protein digestion was followed by SDS-PAGE electrophoresis performed on each digestion product, and their in vitro allergenicity was analyzed by immunoblotting. Such procedure was applied on beta-Lg samples mixed with the two kinds of pectin before or after heating (80 degrees C, 25 min) to determine the respective impact of heat treatment and pectin addition. Heat denaturation improved significantly the susceptibility of beta-Lg against the pepsin and the T/C. This effect, which was coupled to a reduction in immunoreactivity of the digested beta-Lg, appeared to be distinctively modulated by LMP and HMP. Through nonspecific interaction with the beta-Lg, pectin could reduce the accessibility of cleavage sites and/or epitope sequences. This mechanism of action is discussed in relation to the intra- and intermolecular interactions between beta-Lg and pectin initiated under the experimental conditions.

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[190] - Khwaldia K, Perez C, Banon S, Desobry S, Hardy J. Milk proteins for edible films and coatings. Crit Rev Food Sci Nutr 2004;44:239-251

Due to the recent increase in ecological consciousness, research has turned toward finding edible materials. Viable edible films and coatings have been produced using milk proteins. These films and coatings may retard moisture loss, are good oxygen barriers, show good tensile strength and moderate elongation, are flexible, and generally have no flavor or taste. Incorporation of lipids in protein films, either in an emulsion or as a coating, improve their properties as barriers to moisture vapor. Interactions between chemical, structural properties, as well as film-forming conditions and functional properties of edible milk films are elucidated. Some potential uses of milk protein packaging, which are hinged on film properties, are described with examples.

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[191] - Ikeuchi T, Aoki T, Yoshida T, Takahashi K, Hattori M. Functional improvements to beta-lactoglobulin by preparing an edible conjugate with cationic saccharide using microbial mransglutaminase (MTGase). Biosci Biotechnol Biochem 2008;72:1227-1234

Bovine beta-lactoglobulin (BLG) was conjugated with cationic saccharides to improve its functions. We used a polylysine-dextran conjugate (PL-Dex) as the cationic saccharide which had been prepared by the Maillard reaction. The molar ratio of PL:Dex was 1:1. The emulsifying property of PL in the acidic pH range was improved by conjugating with Dex. BLG and PL-Dex were conjugated by using microbial transglutaminase (MTGase), the effective conjugation being confirmed by SDS-PAGE. The molar ratio of BLG:PL-Dex was 1:1. Structural analyses by a fluorescence study, ELISA with monoclonal antibodies and measurement of the retinol-binding activity indicated that the conjugates had almost retained the native structure of BLG. The emulsifying property of BLG in the acidic pH range and in the presence of NaCl was improved by conjugating with PL-Dex. The immunogenicity of BLG was reduced by this conjugation, while the antigenicity of the BLG-PL-Dex conjugate was similar to that of BLG in BALB/c mice.

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[192] - Oliver CM, Melton LD, Stanley RA. Creating Proteins with Novel Functionality via the Maillard Reaction: A Review. Crit Rev Food Sci Nutr 2006;46:337-350

Proteins are widely utilized to add functional properties, such as gelling and emulsification to foods. These attributes depend on a number of factors such as molecular structure of the protein, the pH, and the composition of its chemical environment. There is substantial evidence to suggest that the functional properties of food proteins can be further improved by derivatization. Covalent bonding of proteins to polysaccharides and smaller reducing sugars via the Maillard reaction has been shown to alter the functionality of proteins without requiring the addition of chemical reagents. Establishment of a technologically feasible method for preparing the conjugates and optimization of the processing conditions, however, is needed to promote their development as functional food ingredients. This paper provides a state-of-the-art contribution to the impact of the Maillard reaction on protein functionality. It presents a deeper understanding of the influence of processing conditions and reactant formulation on improving desirable properties of proteins. In particular attention is given to how potential improvements could be achieved in the emulsifying, textural, and solubility properties of proteins to add value to commodity food ingredients. Elements that are considered to be critical to the design of functional Maillard conjugates are highlighted and suggestions proposed to facilitate progress in this area.

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[193] - Graveland-Bikker JF, de Kruif CG. Unique milk protein based nanotubes: Food and nanotechnology meet. Trends Food Sci Technol 2006;17:196-203

Partial hydrolysis of the milk protein a-lactalbumin by a protease from Bacillus licheniformis results in building blocks, which self-assemble into nanometer-sized tubular structures at appropriate conditions. These nanostructures promise various applications in food, nanomedicine and nanotechnology. Important aspects for application of a-lactalbumin nanotubes are the formation conditions and nanotube stability. Therefore, this paper reviews the stability and possible applications of a-lactalbumin nanotubes. The stability can be controlled by solvent conditions as well as by cross-linking the nanotubes with for example transglutaminase. This controllable stability considerably widens the scope of applications.

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[194] - Meltretter J, Becker CM, Pischetsrieder M. Identification and Site-Specific Relative Quantification of beta-Lactoglobulin Modifications in Heated Milk and Dairy Products. J Agric Food Chem 2008;56:5165-5171

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.

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[195] - Monaci L, Hengel AJ. Effect of Heat Treatment on the Detection of Intact Bovine beta-Lactoglobulins by LC Mass Spectrometry. J Agric Food Chem 2007;55:2985-2992

Lactoglobulin (LG) is the most abundant protein of the whey fraction of cow's milk, and due to its high nutritional value as well as its technological properties it is widely used as an ingredient in food preparation. As a consequence of heat treatment, milk proteins may undergo structural changes such as protein unfolding and aggregation, in addition to chemical modifications. This, in turn can change the allergenic potential of LG. In this study, the potential of mass spectrometry has been exploited to investigate LG protein modification and stability as a consequence of thermal treatments applied to both standard solutions and milk samples. An investigation into the charge-state distribution in ESI-MS source revealed that, in standard solutions, a higher degree of protonation accompanies increases in the severity of the heat treatment applied. In contrast, the analysis of milk samples revealed a higher stability of the charge-state distribution of LG. However, we observed modification of LG spectra after heating of standard solutions as well as milk samples caused by lactosylation. The degree of LG lactosylation has been investigated in raw milk samples by LC-MS and provides a potential marker to trace heat treatments

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[196] - Meltretter J, Becker CM, Pischetsrieder M. Identification and Site-Specific Relative Quantification of beta-Lactoglobulin Modifications in Heated Milk and Dairy Products. J Agric Food Chem 2008;56:5165-5171

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.

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[197] - Taheri-Kafrani A, Gaudin JC, Rabesona H, Nioi C, Agarwal D, Drouet M et al. Effects of heating and glycation of beta-lactoglobulin on its recognition by IgE of sera from cow milk allergy patients. J Agric Food Chem 2009;57:4974-4982

beta-Lactoglobulin (beta-LG) is one of the cow's major milk proteins and the most abundant whey protein. This globular protein of about 18 kDa is folded, forming a beta-barrel (or calyx) structure. This structure is stabilized by two disulfide bonds and can be altered by heating above 65 degrees C. beta-LG is also one of the major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. During heating in the presence of reducing sugars, beta-LG is also submitted to the Maillard reaction, which at the first stage consists of the covalent fixation of sugars on the epsilon-amino groups of lysyl residues. The following steps are condensation and polymerization reactions leading to the formation of melanoidins (brown pigments). Despite the frequency of use of heating during milk transformation, the effects of heat-induced denaturation and of glycation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) patients are not fully understood. The objectives of our work were to evaluate the effect of heat-induced denaturation of bovine beta-LG on binding of IgE from CMA patients and to determine the effect of moderate glycation on the degree of recognition by IgE. We showed that heat-induced denaturation (loss of tertiary and secondary structures) of beta-LG is associated with weaker binding of IgE from CMA patients. It was also shown that moderate glycation of beta-LG in early stages of Maillard reaction has only a small effect on its recognition by IgE, whereas a high degree of glycation has a clear "masking" effect on the recognition of epitopes. This demonstrates the importance of epsilon-amino groups of lysines in the definition of epitopes recognized by IgE.

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[198] - Chevalier F, Chobert JM, Dalgalarrondo M, Choiset Y, Haertlé T. Maillard glycation of beta-lactoglobulin induces conformation changes. Nahrung 2002;46:58-63

Glycation by the Maillard reaction is an ubiquitous reaction of condensation of a reducing sugar with amino groups of proteins, which products could improve the functional and/or biological properties for food and non-food uses. It can induce structural modifications in proteins, modifying their properties. The aim of this work was to investigate the association behavior and the conformational changes of beta-lactoglobulin (BLG) after its glycation by the Maillard reaction with several alimentary sugars (arabinose, galactose, glucose, lactose, rhamnose and ribose). Protein samples were heated in the presence or in the absence (heated control) of different sugars during 3 days at 60 degrees C. Glycation induced oligomerization of BLG monomers. Depending on the reactivity of the sugar, the population of produced oligomers showed smaller or greater heterogeneity in molecular masses. Analysis of modified BLG by circular dichroism and by its susceptibility to pepsinolysis showed that the conditions of heating used did not significantly alter the conformation of BLG. Heating of BLG in presence of sugars induced only minor structural modification, when using the less reactive sugars such as lactose and rhamnose. It was, however, at the origin of major three-dimensional destructuring in the case of the more reactive sugars such as arabinose and ribose. Pepsinolysis of glycated BLG did not affect about 62 and 35% of the protein molecules modified with lactose or rhamnose, and arabinose or ribose, respectively. The increase of susceptibility of glycated BLG to pepsinolysis could be related to the alteration of the conformation of the protein when glycation was performed with highly reactive sugars, as observed by circular dichroism and calorimetry analysis.

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[199] - Meltretter J, Becker CM, Pischetsrieder M. Identification and Site-Specific Relative Quantification of beta-Lactoglobulin Modifications in Heated Milk and Dairy Products. J Agric Food Chem 2008;56:5165-5171

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.

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[200] - Ehn BM, Ekstrand B, Bengtsson U, Ahlstedt S. Modification of IgE Binding during Heat Processing of the Cow's Milk Allergen beta-Lactoglobulin. J Agric Food Chem 2004;52:1398-1403

The effect of heat treatment on the IgE binding ability of beta-lactoglobulin, as pure protein or in whole milk, was studied by inhibition of IgE antibody binding using FEIA-CAP inhibition. A slight but significant decreased IgE binding was seen between unheated and heat-treated beta-lactoglobulin solution at 74 degrees C (IC(50) = 2.03 and 3.59 microg/mL, respectively, p = 0.032). A more pronounced decrease was found at 90 degrees C with an IC(50) of 8.45 microg/mL (p = 0.014). The inhibition of IgE binding of milk after heat treatment at 90 degrees C was also significantly decreased (p = 0.007). However, at all heat treatments, a similar total amount of IgE antibodies could be inhibited at a sufficiently high concentration of beta-lactoglobulin. The inhibiting ability of beta-lactoglobulin was significantly impaired in some fermented acidified milk products such as yogurt as compared to that in nonfermented milk (p < 0.001). There was only a small difference of IgE binding between the native forms of genetic variants A and B.

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[201] - Ehn BM, Allmere T, Telemo E, Bengtsson U, Ekstrand B. Modification of IgE Binding to beta-Lactoglobulin by Fermentation and Proteolysis of Cow's Milk. J Agric Food Chem 2005;53:3743-3748

The effect of fermentation by Lactobacilli and of proteolytic hydrolysis of whole milk on the IgE binding ability of beta-lactoglobulin was studied using an ELISA inhibition assay. Sera from nine adult milk allergic patients were tested. The individual sera showed a similar inhibition pattern in the changes during fermentation and proteolysis. The degradation of beta-lactoglobulin was studied with liquid chromatography. In general, fermentation with Lactobacilli gave little effect on IgE binding, even though chromatography data showed a gradual degradation of beta-lactoglobulin. Proteolysis with trypsin, however, gave extensive degradation of beta-lactoglobulin and strongly decreased IgE binding. In addition, we measured the inhibition pattern of beta-lactoglobulin in various selected commercially available fermented milk products. These showed an IgE binding capacity similar to that of nonfermented high pasteurized milk.

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[202] - Papageorgiou PS, Douladiris N, Tassios I, Manoussakis E, Kolakowski P, Szpendowska E. Reduced skin reactivity to yoghurt and kefir in children with cows' milk allergy. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1028

Introduction: Processing can affect the allergenicity of various foods. The purpose of the study was to assess the skin prick test (SPT) response of patients with cows‚ milk (CM) allergy to commercially available fermented milk products. Methodology: Prick to prick SPT was performed using 3 types of yoghurt (1,5% fat) varied in microflora composition, viscosity, exopolisaccharides content and protein degradation produced on following cultures: TM-111, MYE95, MYBio6 and kefir. Kefir was produced using freeze-dried kefit grains. Semi-skimmed non- fermented milk reconstituted in water from powder (prepared in the same way like the yoghurt and kefir) and fresh full fat milk was used as controls. The mean diameter was measured and compared to fresh full fat CM. 30 patients, 7 mos to 7 yrs (mean 2.4 yrs), 9 F and 21 M with clinical allergy to CM and serum IgE to CM > Class 3 (FEIA, Pharmacia CAP System) participated in the study following Ethics Committee and parent/patient approval. Results: A tendency towards reduction of SPT reactivity with all processed samples including semi skimmed milk compared to fresh full fat CM was noted. Significant reduction of SPT mean diameter in comparison to CM (8.3 mm) was found for Kefir (mean diameter 5.7 mm) and the 3 yoghurt preparations (TM-111: 6.3 mm, MYE95: 6.1 mm and MYBio6: 5.9 mm) with P value 0.007, 0.033, 0.011 and 0.003 respectively. Conclusion: Our data show that milk fermentation reduces its allergenicity as assessed by skin prick testing of patients clinically allergic to CM. Oral provocation is necessary to evaluate the clinical significance of these findings.

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[203] - Besler M, Steinhart H, Paschke A. Stability of food allergens and allergenicity of processed foods. J Chromatogr B Biomed Appl 2001;756:207-228

The allergenicity of food could be altered by several processing procedures. For various foods of animal and plant origin the available literature on this alteration is described. Investigations on hidden allergens in food products are also dealt with. [References: 160

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[204] - Jedrychowski L, Wroblewska B. Reduction of the Antigenicity of Whey Proteins by Lactic Acid Fermentation. Food Agric Immunol 1999;11:91-99

Lactic acid bacteria (399 meso- and thermophilic strains or mixed cultures) were screened for their ability to reduce the antigenicity of bovine alpha -lactalbumin ( alpha -la) and beta -lactoglobulin ( beta -lg). Residual antigenicity of these proteins in whey from the fermented milks was determined by indirect ELISA and competitive ELISA for alpha -la and beta -lg, using rabbit polyclonal antibodies. The antigenicity was related to allergenicity, as determined by skintesting with selected samples of whey from the fermented milks. The antigenicity of whey proteins was found to be reduced by over 99% as compared to raw milk after the lactic acid fermentation of sterilized cow"s milk. The allergenicity of alpha -la and beta -lg was not, however, eliminated, being only slightly attenuated.

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[205] - Fiocchi A, Bouygue GR, Sarratud T, Terracciano L, Martelli A, Restani P. Clinical tolerance of processed foods. Ann Allergy Asthma Immunol 2004;93(5 Suppl. 3):S38-S46

OBJECTIVE: To review the effects of technological processing on selected foods of relevance to childhood allergy from the viewpoints of reduced allergenicity, contamination of processed foods by allergens introduced during processing, and ad hoc technologies to produce reduced hypoallergenic products. DATA SOURCES: We searched the literature (PubMed/MEDLINE) for articles published between January 1994 and April 2004 using the following keywords: food allergy AND process* OR heat* OR cooking OR toleran*. STUDY SELECTION: We drew on our collective clinical and biological experience to restrict retrieved studies to those of more frequent relevance to a hospital allergy practice. RESULTS: Comparatively few clinical studies address the modification of allergenicity of food through cooking or processing. Dairy foods are largely unaffected by processing and may be contaminated by, or themselves become, hidden allergens. Hypoallergenic formulas based on milk, soy, or rice and homogenized beef are successful applications of allergenicity reduction via technological processing. Egg, fish, condiments, and vegetables all carry heat-resistant allergens and should also be considered contaminants. Cereals and bakery products are generally well tolerated, but their allergenicity may be enhanced by processing; the case of rice is still open. Peanut allergens are stable, and the evidence is scant that thermal processing affects the allergenicity of soybean and soy hydrolysates. The debate is ongoing about the tolerance of vegetable oils. CONCLUSIONS: It is too early to systematize clinical studies based on single procedures. Processing affects antigenicity, but this does not always translate into safety recommendations. Industrial processing is liable to contamination, and monitoring and labeling are industry priorities. Clinicians should evaluate foods by as complete a workup as possible before recommending processed foods.

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[206] - Enomoto H, Li CP, Morizane K, Ibrahim HR, Sugimoto Y, Ohki S, et al. Glycation and Phosphorylation of beta-Lactoglobulin by Dry-Heating: Effect on Protein Structure and Some Properties. J Agric Food Chem 2007;55:2392-2398

beta-Lactoglobulin (beta-Lg) was glycated with maltopentaose and subsequently phosphorylated by dry-heating in the presence of pyrophosphate to investigate the structural and functional properties of phosphorylated beta-Lg. The circular dichroism spectra showed that the change of the secondary structure in the beta-Lg molecule by glycation and subsequent phosphorylation was small. The differential scanning calorimetry thermograms of beta-Lg showed that the denaturation temperature of the most stable domain was only slightly affected, whereas the retinol-binding activity of beta-Lg was somewhat reduced by glycation and subsequent phosphorylation. These results indicated that the conformational changes of the beta-Lg molecule by glycation and subsequent phosphorylation were mild. The anti-beta-Lg antibody response was somewhat reduced by glycation, but significant changes were not observed by phosphorylation. Although the stability of beta-Lg against heat-induced insolubility was improved by glycation alone, it was further enhanced by phosphorylation. The calcium phosphate solubilizing ability of beta-Lg was enhanced by phosphorylation following glycation. Keywords: beta-Lactoglobulin; dry-heating; Maillard reaction; phosphorylation; structural properties; functional properties.

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[207] - Devoisins JM. Allergie isolée à certains fromages: néoallergènes issus des caséines ? Alim'Inter 2004;9:84-87

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[208] - Kilara A, Panyam D. Peptides from milk proteins and their properties. Crit Rev Food Sci Nutr 2003;43:607-633

This review has attempted to study the literature pertaining to peptides derived from milk proteins. Hydrolysis of milk proteins to generate peptides has been practiced for a long time and it was recognized early on in this process that the taste of hydrolyzates might hinder use of these products in food formulations. Modification of protein is necessary to form a more acceptable or utilizable product, to form a product that is less susceptible to deteriorative reactions and to form a product that is of higher nutritionall quality. Modifications may be achieved by a number of chemical and enzymatic means. This review has considered only enzymatic modification of dairy proteins. Modified proteins contain peptides and some of these peptides have been purified and their functionalities have been compared with unmodified proteins. This paper has examined the literature pertaining to improvement in functionality of enzyme-modified proteins. Improvements in solubility, emulsification, foaming and gelation were examined. There is limited information available on the sequence of the peptides necessary to improve the functional characteristics of proteins. Knowing the sequences of desirable functional peptides can lead to genetic alteration of proteins to improve functionality. Addition of synthetic peptides to intact proteins may be another way in which the functionality of proteins can be augmented. Some of the peptides in milk proteins are capable of affecting biological functions of an organism. These effects can be antimicrobial and probiotic, i.e., prevent the growth and proliferation of undesirable and pathogenic organisms, or they may promote the growth of desirable bacteria in the digestive tract of humans and animals. Peptides derived from milk protein have been shown to exert digestive and metabolic effects as well. They may also influence the immune system. These biological effects may play an important role in the development of medical foods that treat or mitigate the effects of diseases. Proteins are allergens and therefore it is possible that products derived from modification of proteins may also be allergens. The known literature about the allergenicity of peptides derived from milk proteins has been examined in this article. Last, but not the least, the taste attributes of peptides is also considered. Bitterness of hydrolyzates is a common occurrence and the origins of these bitter peptides and possible ways of mitigating this sensory defect has been discussed. Many of the peptides that enhance functionality and exert biological activity are likely to be bitter. Therefore, the bitter taste of hydrolysis products has to be dealt with in boosting the functional or nutraceutical aspects of foods containing these peptides. Analytical techniques for sequencing peptides have become more accessible and purification of peptides is commercially feasible. Computer based modeling techniques have aided the prediction of structures in these peptides. These advances, coupled with the advances in biotechnology, promise to revolutionize the future of nutraceutical and functional foods.

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[209] - Malandain H. Transglutaminases : a meeting point for wheat allergy, celiac disease, and food safety. Eur Ann Allergy Clin Immunol 2005;37:397-403

Wheat is the staple cereal in many countries and its uses in manufactured foods are ever growing due to the technological qualities of gluten proteins. Transglutaminases (TG) are ubiquitous enzymes with many functions. They are able to transform proteins by deamidation and/or transamidation. This last reaction can cross-link proteins together. Intestinal tissue TG has been shown to play an important role in two kinds of immune reactions to wheat: celiac disease and wheat-dependent exercise-induced anaphylaxis. In addition, new epitopes have been suspected in cases of anaphylaxis to wheat isolates, a food ingredient consisting mainly of deamidated gluten proteins. As a microbial TG is included in many food technological processes, its safe use should be checked. This assessment must cover not only the safety of the TG itself but also that of the deamidated/cross-linked proteins generated by this enzyme. This article aims at discussing the possible consequences of using TG in food industry in the light of today knowledge about immune reactions to wheat.

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[210] - Walker MK, Farkas DF, Anderson SR, Meunier-Goddik L. Effects of high-pressure processing at low temperature on the molecular structure and surface properties of beta-lactoglobulin. J Agric Food Chem 2004;52:8230-8235

High-pressure processing (HPP) was utilized to induce unfolding of beta-lactoglobulin (beta-LG). beta-Lactoglobulin solutions at concentrations of 0.5 mg/mL, in pH 7.5 phosphate buffer, were pressure treated at 510 MPa for 10 min at either 8 or 24 degrees C. The secondary structure, as determined by circular dichroism (CD), of beta-LG processed at 8 degrees C appeared to be unchanged, whereas beta-LG processed at 24 degrees C lost alpha-helix structure. Tertiary structures for beta-LG, as determined by near-UV CD, intrinsic protein fluorescence spectroscopy, hydrophobic fluorescent probe binding, and thiol group reactivity, were changed following processing at either temperature. The largest changes to tertiary structure were observed for the samples processed at 24 degrees C. Model solutions containing the pressure-treated beta-LG showed significant decreases in surface tension at liquid-air interfaces with values of 54.00 and 51.69 mN/m for the samples treated at 24 and 8 degrees C, respectively. In comparison, the surface tension for model solutions containing the untreated control was 60.60 mN/m. Changes in protein structure during frozen and freeze-dried storage were also monitored, and some renaturation was observed for both storage conditions. Significantly, the sample pressure-treated at 8 degrees C continued to display the lowest surface tension.

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[211] - Lopez-Fandino R. Functional improvement of milk whey proteins induced by high hydrostatic pressure. Crit Rev Food Sci Nutr 2006;46:351-363

High pressure is emerging as a new processing technology that produces particular changes in the molecular structure of proteins and thus gives rise to new properties inaccessible via conventional methods of protein modification. This review deals with the main effects of high hydrostatic pressure on the physicochemical characteristics of milk whey proteins and how modifications in their structural properties contribute to functionality. In this paper the mechanism underlying pressure-induced changes in ss-lactoglobulin, a-lactabumin, and bovine serum albumin is explained, and related to functional properties such as gel-forming ability, emulsifying activity, or foaming capacity. The possibility of using high pressures to favor chemical reactions of proteins with other food components, such as carbohydrates, to produce novel molecules with new food uses is also considered

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[212] - Patel HA, Singh H, Anema SG, Creamer LK. Effects of Heat and High Hydrostatic Pressure Treatments on Disulfide Bonding Interchanges among the Proteins in Skim Milk. J Agric Food Chem 2006;54:3409-3420

Traditionally, milk has been heat treated to control microorganisms and to alter its functionality, for example, to increase its heat stability. Pressure treatment has been considered as a possible alternative for microorganism control, but some of the functionality-related milk protein interactions have not been explored. The present study used two novel two-dimensional polyacrylamide gel electrophoresis (2D PAGE) methods to explore the differences in the irreversible disulfide bond changes among the milk proteins after four common heat treatments and after 30-min pressure treatments of milk at 200, 400, 600, and 800 MPa at ambient temperature (22 degrees C). The pasteurizing heat treatment (72 degrees C for 15 s) denatured and aggregated only a few minor whey proteins, but the high heat treatments (100 degrees C for 120 s, 120 degrees C for 120 s, and 140 degrees C for 5 s) formed disulfide-bonded aggregates that included a high proportion of all of the whey proteins and kappa-casein (kappa-CN) and a proportion of the alpha(s2)-CN. Pressure treatment of milk at 200 MPa caused beta-lactoglobulin (beta-LG) to form disulfide-bonded dimers and incorporated beta-LG into aggregates, probably disulfide-bonded to kappa-CN. The other whey proteins appeared to be less affected at 200 MPa for 30 min. In contrast, pressure treatment at 800 MPa incorporated beta-LG and most of the minor whey proteins, as well as kappa-CN and much of the alpha(s2)-CN, into aggregates. The accessibility of alpha(s2)-CN and formation of complexes involving alpha(s2)-CN, kappa-CN, and whey proteins in the pressure treated milk is an important novel finding. However, only some of the alpha-lactalbumin was denatured or incorporated into the large aggregates. These and other results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that give the differences among the resultant aggregates, the sizes of the aggregates, and the product functionalities.

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[213] - Patel HA, Singh H, Havea P, Considine T, Creamer LK. Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions. J Agric Food Chem 2005;53:9590-9601

Whey protein concentrate solutions (12% w/v, pH 6.65 +/- 0.05) were pressure treated at 800 MPa for 20-120 min and then examined using size exclusion chromatography (SEC), small deformation rheology, transmission electron microscopy, and various types of one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). The pressure-treated samples showed a time-dependent loss of native whey proteins by SEC and 1D PAGE and a corresponding increase in non-native proteins and protein aggregates of different sizes. These aggregates altered the viscosity and opacity of the samples and were shown to be cross-linked by intermolecular disulfide bonds and by noncovalent interactions using 1D PAGE [alkaline (or native), sodium dodecyl sulfate (SDS), and SDS of reduced samples (SDS(R))] and 2D PAGE (native:SDS and SDS:SDS(R)). The sensitivity of the major whey proteins to pressure was in the order beta-lactoglobulin B (beta-LG B) > beta-LG A > bovine serum albumin (BSA) > alpha-lactalbumin (alpha-LA), and the large internal hydrophobic cavity of beta-LG may have been partially responsible for its sensitivity to high-pressure treatments. It seemed likely that, at 800 MPa, the formation of a beta-LG disulfide-bonded network preceded the formation of disulfide bonds between alpha-LA or BSA and beta-LG to form multiprotein aggregates, possibly because the disulfide bonds of alpha-LA and BSA are less exposed than those of beta-LG either during or after pressure treatment. It may be possible that intermolecular disulfide bond formation occurred at high pressure and that hydrophobic association became important after the high-pressure treatment.

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[214] - Chicón R, Belloque J, Alonso E, Martín-Alvarez PJ, López-Fandiño R. Hydrolysis under high hydrostatic pressure as a means to reduce the binding of beta-lactoglobulin to immunoglobulin E from human sera. J Food Prot 2008;71:1453-1459

.Cows' milk allergy is the most frequent food allergy in children, and beta-lactoglobulin (beta-Lg) is a major allergen. Milk-based hypoallergenic ingredients are manufactured by enzymatic hydrolysis, a process that could be improved by the application of high-pressure treatments. This study showed that the treatment of beta-Lg dissolved in buffer with chymotrypsin and trypsin under high pressure for relatively short times accelerated proteolysis by leading to a rapid removal of the intact protein. The rapid proteolysis of the beta-Lg substrate under pressure led to the production, in 20 min, of hydrolysates with lower immunoglobulin (Ig) G binding than those produced in 8 h (chymotrypsin) or 48 h (trypsin) at atmospheric pressure. However, those hydrolysates retained some residual IgE-binding properties that could be traced to the preferential release, during the initial stages of proteolysis, of peptides containing IgE epitopes, such as (Val-41-Lys-60), (Leu-149-Ile-162), and (Ser-21-Arg-40). The formation of these fragments was favored when proteolysis was conducted under high pressure due to the preferential hydrolysis of Arg-40 and Arg-148 by trypsin, and Tyr-42 and Leu-149 by chymotrypsin, all located at the dimer interface of beta-Lg or very close to it. Although our results do not support that trypsin and chymotrypsin under high pressure selectively address the allergenic regions of beta-Lg, it is possible to select the conditions that quickly produce hydrolysates with reduced potential allergenicity that could be used in hypoallergenic foods.

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[215] - Lopez-Fandino R. Functional improvement of milk whey proteins induced by high hydrostatic pressure. Crit Rev Food Sci Nutr 2006;46:351-363

High pressure is emerging as a new processing technology that produces particular changes in the molecular structure of proteins and thus gives rise to new properties inaccessible via conventional methods of protein modification. This review deals with the main effects of high hydrostatic pressure on the physicochemical characteristics of milk whey proteins and how modifications in their structural properties contribute to functionality. In this paper the mechanism underlying pressure-induced changes in ss-lactoglobulin, a-lactabumin, and bovine serum albumin is explained, and related to functional properties such as gel-forming ability, emulsifying activity, or foaming capacity. The possibility of using high pressures to favor chemical reactions of proteins with other food components, such as carbohydrates, to produce novel molecules with new food uses is also considered

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[216] - Koppelman SJ, Wensing M, de Jong GA, Knulst AC. Anaphylaxis caused by the unexpected presence of casein in salmon. Lancet 1999;354:2136

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[217] - Novembre E, Caparrelli C, Mori F, Calogero C, Vierucci A, De Martino M. Casein As An "Hidden" Allergen In Manufactured Foods. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°166

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[218] - Morisset M, Moneret-Vautrin DA, Commun N, Schuller A, Kanny G. Allergy to Cow Milk Proteins Contaminating Lactose, Common Excipient of Dry Powder Inhalers for Asthma. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°369

RATIONALE: Lactose intolerance and cow milk allergy are two distinct diseases, the first is due to a congenital or acquired lactase deficiency and the second is due to an immunologic disorder. Although disputed, the risk of anaphylaxis to cow's milk (CM) after ingestion or lactose inhalation really exists, as that is shown in the following clinical case METHODS: A woman allergic to milk presented several atopic dermatitis and asthma exacerbations after respiratory exposure to CM proteins traces. The symptoms began when bronchodilator (formoterol) was delivered with a dry powder inhaler, instead of a spray. The prick-tests and challenges were carried out with natural food and pharmaceutical lactose RESULTS: A sensitization to CM proteins was shown by positive skin prick-test and specific IgE to cow's milk, respectively 5 mm and 15.4 KU/l. The oral and bronchial challenges with CM were positive as well as the oral challenge with lactose. A bronchial challenge with lactose induced bronchospasm, rhinitis and an exacerbation of eczema. Stop of exposure to this excipient and diet avoidance, made it possible to note a clear improvement of eczema and asthma CONCLUSIONS: This clinical case is the second published observation (1st case in the adult) showing the risk of anaphylaxis in case of severe allergy to CM, at the time of the use of dry powder inhalers containing lactose (1) REFERENCES: (1)Nowak-Wegrzyn A, Shapiro GG, Beyer K, Bardina L, Sampson HA. Contamination of dry powder inhalers for asthma with milk proteins containing lactose. J Allergy Clin Immunol 2004; 113(3): 558-60 Funding: Nancy University Hospital

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[219] - Lee TT, Morisset M, Astier C, Moneret-Vautrin DA, Cordebar V, Beaudouin E, et al. Contamination of probiotic preparations with milk allergens can cause anaphylaxis in children with cow's milk allergy. J Allergy Clin Immunol 2007;119:746-747

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[220] - Morisset M, Moneret-Vautrin DA, Kanny G, Guénard L, Beaudouin E, Flabbée J, et al. Thresholds of clinical reactivity to milk, egg, peanut and sesame in IgE-dependent allergies: evaluation by double-blind or single-blind placebo-controlled oral challenges. Clin Exp Allergy 2003;33:1046-1051

BACKGROUND: The prevalence of food anaphylaxis due to masked allergens has increased within the last 10 years. Contamination of manufactured products by food allergens is a key concern for food industries . OBJECTIVE: To determine quantities eliciting reactions in patients who have an IgE-dependent food allergy, thanks to standardized oral provocation tests. To evaluate the subsequent levels of sensitivity required for the detection tests of allergens for egg, peanut, milk and sesame . METHODS: Prick-in-prick tests, Cap system RAST, and single or double-blind placebo-controlled food challenges (SBPCFC or DBPCFC) were performed. The doses of natural food were gradually increased from 5 to 5000 mg for solid food and from 1 to 30 mL for peanut oil, sunflower oil, soy oil and sesame oil . RESULTS: Data from 125 positive oral challenges to egg, 103 to peanut, 59 to milk and 12 to sesame seeds were analysed. Haemodynamic modifications were observed in 2%, 3%, 1.7%, and 8% of the oral challenges (OCs) to egg, peanut, milk and sesame, respectively. Respiratory symptoms were observed in 12%, 20%, 10% and 42% of egg, peanut milk and sesame allergies, respectively. A cumulative reactive dose inferior or equal to 65 mg of solid food or 0.8 mL of milk characterized 16%, 18%, 5% and 8% of egg, peanut, milk and sesame allergies, respectively. 0.8% of egg allergies, 3.9% of peanut allergies, and 1.7% of milk allergies reacted to 10 mg or less of solid food or to 0.1 mL for milk. The lowest reactive threshold has been observed at less than 2 mg of egg; 5 mg of peanut, 0.1 mL of milk and 30 mg of sesame seed. Ten out of 29 OC with peanut oil, two out of two OC with soy oil and three out of six OC with sunflower oil were positive. Five out six OC with sesame oil were positive: 1 and 5 mL induced an anaphylactic shock . CONCLUSION: The risk of asthma and anaphylactic shock to sesame and peanut is confirmed. Minimal reactive quantities show that, in order to guarantee a 95% safety for patients who are allergic to egg, peanut and milk, and on the basis of consumption of 100 g of food, the detection tests should ensure a sensitivity of 10 p.p.m. for egg, 24 p.p.m. for peanut and 30 p.p.m. for milk proteins. Oil allergies being considered, the limit of sensitivity should fall to 5 p.p.m.

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[221] - Tabar AI, Alvarez MJ, Echechipia S, Acero S, Garcia BE, Olaguibel JM. Anaphylaxis from cow's milk casein. Allergy 1996;51:343-345

The case of a 35-year-old woman who suffered anaphylaxis after the ingestion of milk or milk-containing products is presented. Symptoms also appeared after the patient used a cosmetic which contained casein. Skin prick tests, specific IgE measurement, and immunoblotting were carried out with cow's milk extract, and its purified proteins: casein, beta-lactoglobulin, and alpha-lactalbumin. Specific IgE to cow's milk casein was detected with the three tests. Immunoblotting revealed one band of 24 kDa approximately corresponding to the beta-fraction of casein. Negative results to beta-lactoglobulin and alpha-lactalbumin were obtained with the three methods. Symptoms were controlled by rigorous dietary and behavior measures

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[222] - Bonadonna P, Crivellaro M, Dama A, Guarnieri G, Schiappoli M, Senna G. [Occupational asthma induced by casein inhalation]. G Ital Med Lav Ergon 2003;25(Suppl. 3):192-193

Reported cases of occupational asthma due to inhalation of milk proteins are rare. Our report is about the first evidence of occupational asthma in a health care worker, caused by an hidden exposure to casein, contained in a commercial dermatological powder widely used in geriatric patients treatment. The diagnosis is based on the case-history and on the positive skin prick test to a commercial and extemporary extract of the occupational allergen.

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[223] - Bernaola G, Echechipia S, Urrutia I, Fernandez E, Audicana M, Fernandez de Corres L. Occupational asthma and rhinoconjunctivitis from inhalation of dried cow's milk caused by sensitization to alpha-lactalbumin. Allergy 1994;49:189-191

A chocolate candy worker was diagnosed as having occupational asthma and rhinoconjunctivitis on the basis of clinical record and methacholine challenge. Positive conjunctival and bronchial challenge tests with lactalbumin showed that this protein was the pathogenetic agent. Type I hypersensitivity mechanism is demonstrated by means of skin prick test and RAST

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[224] - Olaguibel JM, Hernandez D, Morales P, Peris A, Basomba A. Occupational asthma caused by inhalation of casein. Allergy 1990;45:306-308

Solutions of casein are usually sprayed over the leather in the final stage of tanning. We present the result of a thorough investigation of an atopic tannery worker with occupational asthma. Data from clinical record and metacholine challenges support the occupational source of the patient's symptoms, and positive bronchial challenge with casein clearly defines it as the specific aetiological agent. The presence of specific IgE suggests a hypersensitivity type I mechanism. In the tanning process chromium salts, paraphenylendiamine and formaldehyde have all been included as specific aetiological agents of asthma, but occupational asthma induced by inhalation of casein has not previously been reported

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[225] - Rossi GL, Corsico A, Moscato G. Occupational asthma caused by milk proteins: report on a case. J Allergy Clin Immunol 1994;93:799-801

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[226] - Mäkinen-Kiljunen S, Mussalo-Rauhamaa H. Casein, an important house dust allergen. Allergy 2002;57:1084-1085

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[227] - Rossi GL, Corsico A, Moscato G. Occupational asthma caused by milk proteins: report on a case. J Allergy Clin Immunol 1994;93:799-801

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[228] - Vargiu A, Vargiu G, Locci F, Del Giacco S, Del Giacco GS. Hypersensitivity reactions from inhalation of milk proteins. Allergy 1994;49:386-387

We describe the case of a young man with IGE-mediated hypersensitivity to milk, casein, and lactoglobulin, who went into respiratory crisis every time he milked his sheep

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[229] - Bernaola G, Echechipia S, Urrutia I, Fernandez E, Audicana M, Fernandez de Corres L. Occupational asthma and rhinoconjunctivitis from inhalation of dried cow's milk caused by sensitization to alpha-lactalbumin. Allergy 1994;49:189-191

A chocolate candy worker was diagnosed as having occupational asthma and rhinoconjunctivitis on the basis of clinical record and methacholine challenge. Positive conjunctival and bronchial challenge tests with lactalbumin showed that this protein was the pathogenetic agent. Type I hypersensitivity mechanism is demonstrated by means of skin prick test and RAST

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[230] - Vargiu A, Vargiu G, Locci F, Del Giacco S, Del Giacco GS. Hypersensitivity reactions from inhalation of milk proteins. Allergy 1994;49:386-387

We describe the case of a young man with IGE-mediated hypersensitivity to milk, casein, and lactoglobulin, who went into respiratory crisis every time he milked his sheep

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[231] - Fernández Rivas M. Food Allergy in Alergológica-2005. J Investig Allergol Clin Immunol 2009;19(Suppl. 2):37-44

BACKGROUND: Alergológica-2005 is a cross-sectional study carried out in Spain in 4991 new patients prospectively recruited in allergy clinics . OBJECTIVE: To describe the characteristics of food allergic patients included in Alergológica-2005 . RESULTS: Food allergy was diagnosed in 369 subjects (7.4%, 95% CI, 6.7%-8.1%). The most common offending foods were fruits (33.3% of cases), nuts (26%), shellfish (22%), egg (16%), milk (13.9%) and fish (9.8%). Rosaceae fruits and crustaceans elicited 23.6% and 18.7% of the reactions, respectively. Milk and egg were the most common foods in patients < 5 years of age, whereas fruits and nuts were the most prevalent foods in patients over 5 years. The most frequent manifestations included skin reactions (65.3%), oral allergy syndrome (33.6%), digestive symptoms (24.7%) and anaphylaxis (17.9%). The clinical presentation differed among foods. Prick-tests were carried out more frequently than serum immunoglobulin E (IgE) determinations (95.9% vs. 65%). Oral challenges were performed in 13% of patients, and 72.3% of these were open. Food allergy was diagnosed on the basis of medical history and positive IgE in 60.2% of the patients, ranging from 13.7% for milk to 75% for cereals. The self perception of patients' quality of life was lower than that of the 75% of Spanish reference population . CONCLUSION: Food allergy is diagnosed in 7.4% of the patients seen in allergy clinics across Spain, and has an important impact on the quality of life of patients. The foods involved in reactions change with age. The clinical presentation changes with the food, although the skin is the most frequently affected organ.

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[232] - Hill DJ, Heine RG, Hosking CS. The diagnostic value of skin prick testing in children with food allergy. Pediatr Allergy Immunol 2004;15:435-441

The diagnostic accuracy of the skin prick test (SPT) in food allergy is controversial. We have developed diagnostic cut-off levels for SPT in children with allergy to cow milk, egg and peanut. Based on 555 open food challenges in 467 children (median age 3.0 yr) we defined food-specific SPT weal diameters that were '100% diagnostic' for allergy to cow milk (>or=8 mm), egg (>or=7 mm) and peanut (>or=8 mm). In children < 2 yr of age, the corresponding weal diameters were >or=6 mm, >or=5 mm and >or=4 mm, respectively. These SPT cut-off levels were prospectively validated in 90 consecutive children ↩

[233] - Verstege A, Mehl A, Rolinck-Werninghaus C, Staden U, Nocon M, Beyer K, et al. The predictive value of the skin prick test weal size for the outcome of oral food challenges. Clin Exp Allergy 2005;35:1220-1226

BACKGROUND: The skin prick test (SPT) is regarded as an important diagnostic measure in the diagnostic work-up of food allergy. Objective To evaluate the diagnostic capacity of the SPT in predicting the outcome of oral food challenges, and to determine decision points for the weal size and the skin index (SI) that could render double-blind, placebo-controlled food challenges unnecessary . METHODS: In 385 children (median age 22 months), 735 controlled oral challenges were performed with cow's milk (CM), hen's egg (HE), wheat and soy. Three hundred and thirty-six of 385 (87%) children suffered from atopic dermatitis. SPT was performed in all children. Diagnostic capacity, receiver-operator characteristics (ROC) curves and predictive decision points were calculated for the mean weal size and the calculated SI . RESULTS: Three hundred and twelve of 735 (43%) oral food challenges were assessed to be positive. Calculation of 95% and 99% predicted probabilities using logistic regression revealed predictive decision points of 13.0 and 17.8 mm for HE, and 12.5 and 17.3 mm for CM, respectively. However, using the SI, the corresponding cut-off levels were 2.6 and 3.7, respectively, for HE, and 2.7 and 3.7 for CM. For wheat, 95% and 99% decision points of 2.2 and 3.0 were found in children below 1 year of age . CONCLUSION: Predictive decision points for a positive outcome of food challenges can be calculated for HE and CM using weal size and SI. They may help to avoid oral food challenges.

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[234] - Martin-Blondel A, Jegu J, Lauwers-Cances V, Abbal M, Rancé F. Utilité du dosage des IgE spécifiques anti-caséine pour le diagnostic d’une allergie aux protéines du lait de vache chez l’enfant. Rev Fr Allergol 2009;49:302

Introduction.ˆ L‚allergie aux protéines de lait de vache concerne 0,1 à 7,5 des enfants en population générale. Le test de provocation par voie orale (TPO) est le test diagnostique le plus sensible et spécifique, mais il est coûteux et dangereux. Afin de réduire les indications, et les risques inhérents à sa réalisation, nous avons évalué la valeur diagnostique des tests cutanés, des dosages sériques des différentes IgE spécifiques du lait (lait total, alphalactalbumine, bétalactoglobuline, caséine) et de leur combinaison. Méthodes.ˆ Il s‚agit d‚une étude rétrospective réalisé chez les enfants vus pour TPO aux protéines de lait de vache entre septembre 2006 et août 2007. Résultats.ˆ L‚échantillon comprend 118 enfants (81 garçons, 37 filles) d‚un âge médian de 3,4 mois. Nous avons déterminé pour le test cutané à l‚extrait natif de lait une valeur seuil prédictive d‚allergie aux protéines de lait de vache de 13 mm avec une spécificité de 100 %, permettant d‚éviter la réalisation de 8 TPO. Pour les IgE spécifiques anti-caséine, la valeur seuil est de 10,6 kUI/l, avec une spécificité de 100 %, une sensibilité de 46,4 %, et ce dosage permet d‚éviter 13 TPO. Nous proposons une stratégie combinant le dosage des IgE spécifiques anti-caséine au seuil de 10,6 kUI/l, les tests cutanés à l‚extrait natif de lait au seuil de 3 mm et à l‚extrait de caséine au seuil de 4 mm. La spécificité et la valeur prédictive positive de cette stratégie sont de 100 %, évitant ainsi la réalisation de 13 TPO. Conclusion.ˆ Nos résultats démontrent l‚utilité du dosage des IgE spécifiques anti-caséine par rapport aux autres IgE spécifiques du lait. Nous proposons une stratégie combinant le dosage des IgE spécifiques anti-caséine au seuil de 10,6 kUI/l, les tests cutanés à l‚extrait natif de lait au seuil de 3 mm et à l‚extrait de caséine au seuil de 4 mm. Toutefois, leur sensibilité imparfaite rend le TPO incontournable pour de nombreuses situations cliniques.

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[235] - Vanto T, Juntunen-Backman K, Kalimo K, Klemola T, Koivikko A, Koskinen P, et al. The patch test, skin prick test, and serum milk-specific IgE as diagnostic tools in cow's milk allergy in infants. Allergy 1999;54:837-842

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[236] - Majamaa H, Moisio P, Holm K, Kautiainen H, Turjanmaa K. Cow's milk allergy: diagnostic accuracy of skin prick and patch tests and specific IgE. Allergy 1999;54:346-351

BACKGROUND: The objective of the present study was to evaluate the relevance of skin tests and the concentration of cow's milk-specific IgE antibodies in correlation with oral cow's milk challenge in infants with suspected cow's milk allergy. METHODS: The study material comprised 143 infants under the age of 2 years who had undergone a diagnostic elimination challenge because of suspected cow's milk allergy in 1996. Cow's milk-specific IgE was measured, and skin prick and patch tests were performed. RESULTS: Of the 143 oral cow's milk challenges performed, 72 (50%) were positive. Of the positive reactions, 22 involved immediate-type reactions. In 50 patients, delayed-onset reactions of eczematous or gastrointestinal type appeared. Of the infants with challenge-proven cow's milk allergy, 26% showed elevated IgE concentrations to cow's milk, 14% had a positive skin prick test, and 44% had a positive patch test for cow's milk. Interestingly, in most patch test-positive patients, the prick test for cow's milk was negative. CONCLUSIONS: Our study demonstrated that many patients with a negative prick test result had a positive patch test to cow's milk. The patch test was a more sensitive method than the prick test or RAST to detect cow's milk allergy in this study population. Our results indicate that patch testing will significantly increase the probability of early detection of cow's milk allergy. Confirmation of the diagnosis is essential in patients with negative test results but a clinical suspicion of food allergy, and in patch test-positive patients. For this purpose, the most reliable method is the elimination-challenge procedure.

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[237] - 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. Ann Allergy Asthma Immunol 2002;89(6 Suppl. 1):38-43

OBJECTIVE: To review the literature on the prevalence of beef allergy in children allergic to cow's milk and to report a series of patients with beef allergy evaluated for cow's milk allergy. DATA SOURCES: A MEDLINE search for cow's milk allergy and beef allergy was conducted. Also included in this report is a clinical evaluation of both these entities in a population of children with atopic dermatitis. STUDY SELECTION: Data from the literature were summarized. Recruited patients with beef allergy were evaluated on the basis of history, serology, skin prick tests, and double-blind, placebo-controlled food challenge (entry criterion), and presented between 1992 and 2000. RESULTS: In the literature, between 13 and 20% of children with cow's milk allergy also have beef allergy. In our personal series of patients, 28 children (18 boys and 10 girls) diagnosed with beef allergy underwent skin prick tests and double-blind, placebo-controlled food challenge, which showed that 26 (92.9%) were allergic to cow's milk. Two children nonallergic to cow's milk were the only ones who were not sensitized to bovine serum albumin. CONCLUSIONS: Most children with beef allergy are also allergic to cow's milk and should avoid the consumption of dairy products. Sensitization to bovine serum albumin is a marker of cow's milk allergy in children with beef allergy. Elimination of beef from the diet of children with cow's milk allergy should be evaluated on an individual basis after diagnostic workup.

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[238] - Sicherer SH, Morrow EH, Sampson HA. Dose-response in double-blind, placebo-controlled oral food challenges in children with atopic dermatitis. J Allergy Clin Immunol 2000;105:582-586

"BACKGROUND: Double-blind, placebo-controlled oral food challenges (DBPCFCs) are considered the ""gold standard"" for diagnosing food hypersensitivity, but the dose that elicits positive challenges, or determinants that may predict dose-response relationships, have not been reported . OBJECTIVE: Our purpose was to determine the quantity of food that elicits reactions during DBPCFCs and to evaluate parameters that may predict the provocative dose and severity of reaction . METHODS: We reviewed challenge data for all positive challenges to 6 common allergenic foods in children with atopic dermatitis evaluated for food allergy over a 13-year period. Challenge food was generally administered in 6 doses at 10- to 15-minute intervals beginning with 400 to 500 mg and completing with a total of 8 to 10 g of food. An open feeding of a larger portion followed negative challenges. At the physician's discretion, a lower starting dose was occasionally used (100 mg, 250 mg). Food-specific IgE antibody concentrations (radioallergosorbent test [RAST]) were determined on stored sera of 20% of the challenges selected randomly and 99.6% had prick skin tests (PSTs) performed to the challenged food . RESULTS: A total of 196 children (45% male; median age 5 y 9 mo; atopic dermatitis 98%, asthma 62%) had 513 positive challenges distributed as follows: egg 267, milk 117, soy 53, wheat 40, peanut 24, fish 12. The percentage of children reacting at the first dose (500 mg or less) was as follows: egg 49%, milk 55%, soy 28%, wheat 25%, peanut 26%, and fish 17%. Twenty-six milk challenges and 22 egg challenges were positive at a first dose of 250 mg; 3 milk challenges and 7 egg challenges were positive at a first dose of 100 mg. Eleven percent of the reactions that occurred on the first dose were severe. The percentage reacting after the final dose of the DBPCFC (or during open challenge) were egg 11%, milk 12%, soy 19%, wheat 12.5%, peanut 8.7%, and fish 25%. There was not a strong correlation between PST absolute wheal size or score (adjusted for histamine controls) and dose at reaction or severity of reaction (R(s) range -0.22 to 0.39 for particular foods). Serum concentration of food-specific IgE did not correlate well with the dose causing a reaction or with severity (R(s) range -0.40 to 0.55 for particular foods) . CONCLUSIONS: This food-allergic population may react to as little as 100 mg of food, possibly less, and the dose causing a reaction and the severity of reaction is not predicted by PST or RAST. Lower doses (100 mg or less) should be investigated for their appropriateness in initiating DBPCFCs."

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[239] - Lam HY, van Hoffen E, Michelsen A, Guikers K, van der Tas CHW, Bruijnzeel-Koomen CAFM, et al. Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved. Clin Exp Allergy 2008;38:995-1002

BACKGROUND: Studies on cow's milk allergy (CMA) in adults are scarce. Little is known about the clinical symptoms, eliciting doses (ED), and allergens involved . OBJECTIVE: The aim of this study was to analyse the clinical symptoms, ED and allergen recognition in adult CMA patients, compared with cow's milk (CM)-sensitized, but tolerant controls . METHODS: Adult CMA patients were evaluated by standardized questionnaires (n=30), skin prick tests (SPTs) and specific IgE for CM allergens (n=18), and a double-blind placebo-controlled food challenge (DBPCFC, n=10). A control group (n=25) of CM-sensitized, but tolerant adults was included . RESULTS: The majority of CMA patients (20/30, 67%) reported severe symptoms. In all patients participating in DBPCFC, CMA was confirmed. ED for subjective symptoms (0.3-300 mg CM protein) were significantly lower than that for objective symptoms (300-9000 mg CM protein). The severity of CMA by history and ED was not correlated with SPT or IgE. Patients had higher SPT reactivity than controls for CM, alpha-lactalbumin and beta-lactoglobulin (P=0.002, P=0.014 and P=0.004) but not for casein. Specific IgE to CM tended to be higher (P=0.068) and IgE to casein was higher in patients than that in controls (P=0.016). No difference was observed for IgE to alpha-lactalbumin and beta-lactoglobulin . CONCLUSION: Adult CMA is severe in nature. ED are low, starting from 0.3 mg CM protein. Patients with CMA recognize the same major allergens (casein and whey proteins) as controls, but display a stronger SPT and IgE reactivity.

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[240] - van der Gugten AC, den Otter M, Meijer Y, Pasmans SGAM, Knulst AC, Hoekstra MO. Usefulness of specific IgE levels in predicting cow’s milk allergy. J Allergy Clin Immunol 2008;121:531-533

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[241] - Turjanmaa K, Darsow U, Niggemann B, Rancé F, Vanto T, Werfel T. EAACI/GA2LEN Position paper: Present status of the atopy patch test. Allergy 2006;61:1377-1384

A number of scientific reports have been published on patch tests with protein allergens performed on patients with atopic eczema (AE). Evaluation of eczematous skin lesions with an atopy patch test (APT) can be used as a diagnostic tool in characterizing patients with aeroallergen- and food-triggered AE. Indications for testing with APT, choice of allergens (aeroallergens and foods), test materials and technique, including present knowledge on sensitivity and specificity, are reviewed on the basis of available literature. This position paper also points out the need for future research on the clinical use of the APT.

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[242] - de Boissieu D, Waguet JC, Dupont C. The atopy patch-test for detection of cow's milk allergy with digestive symptoms. J Pediatr 2003;142:203-205

Infants (n = 35) with digestive symptom were investigated for diagnosis of cow's milk allergy (CMA). Milk atopy patch tests (APTs) were positive in 19 of 24 CMA versus 1 of 11 in non-CMA patients (P <.001). This sensitivity (79%) and specificity (91%) suggest that the APT could improve the detection of conditions related to CMA.

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[243] - Devillers ACA, de Waard-van der Spek FB, Mulder PGH, Oranje AP. Delayed- and immediate-type reactions in the atopy patch test with food allergens in young children with atopic dermatitis. Pediatr Allergy Immunol 2008;19:53-58

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[244] - Mehl A, Rolinck-Werninghaus C, Staden U, Verstege A, Wahn U, Beyer K, et al. The atopy patch test in the diagnostic workup of suspected food-related symptoms in children. J Allergy Clin Immunol 2006;118:923-929

BACKGROUND: There is an increasing need to develop test instruments that make oral food challenges superfluous . OBJECTIVE: We sought to study the utility of atopy patch tests (APTs) in the diagnostic workup of food allergy . METHODS: We investigated 437 children (median age, 13 months; 90% with atopic dermatitis) referred for evaluation of suspected food allergy. Specific serum IgE (sIgE) measurements, skin prick tests (SPTs), APTs, and controlled oral food challenges were performed . RESULTS: We analyzed 873 oral challenges with cow's milk, hen's egg, wheat, and/or soy. One thousand seven hundred single APTs were performed. As a single parameter, the APTs showed the best specificity compared with sIgE measurements, SPTs, or both. Combining the APT with either the SPT or sIgE measurement resulted in improved sensitivity and specificity. Decision points for sIgE measurement and for the SPT showed lower values when combined with a positive APT result. Correctly bypassing an oral food challenge with combined testing, including APTs, only between 0.5% and 7% (99% predicted probability) and between 6% and 14% (using 95% predicted probability) of children would fulfill the criteria for avoiding an oral food challenge . CONCLUSION: Although the predictive capacity of the APT is improved when combined with sIgE measurement or the SPT, oral food challenges become superfluous in only 0.5% to 14% of study patients. In addition, the APT is time consuming and demands a highly experienced test evaluator. CLINICAL IMPLICATIONS: For daily clinical practice, the APT adds only a small predictive value to the standard SPT and sIgE measurement in the diagnostic workup of suspected food-related symptoms in our study population.

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[245] - de Boissieu D, Waguet JC, Dupont C. The atopy patch-test for detection of cow's milk allergy with digestive symptoms. J Pediatr 2003;142:203-205

Infants (n = 35) with digestive symptom were investigated for diagnosis of cow's milk allergy (CMA). Milk atopy patch tests (APTs) were positive in 19 of 24 CMA versus 1 of 11 in non-CMA patients (P <.001). This sensitivity (79%) and specificity (91%) suggest that the APT could improve the detection of conditions related to CMA.

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[246] - Kalach N, Soulaines P, de Boissieu D, Dupont C. A pilot study of the usefulness and safety of a ready-to-use atopy patch test (Diallertest) versus a comparator (Finn Chamber) during cow’s milk allergy in children. J Allergy Clin Immunol 2005;116:1321-1326

BACKGROUND: Patch testing is used in the diagnosis of food allergy, especially during delayed manifestations . OBJECTIVE: A ready-to-use atopy patch test (APT), the Diallertest, was compared with another APT device, the Finn Chamber, in pediatric cow's milk allergy . METHODS: This prospective study involved 49 children (34.3 +/- 17 [mean +/- SD] months of age), with cow's milk allergy manifested by atopic dermatitis (10.2%), digestive manifestations (40.8%), or both (49%). All children underwent both APT techniques, with a reading 72 hours after application, followed by a milk elimination diet for 4 to 6 weeks and open cow's milk challenge . RESULTS: A positive result was seen in 22 (44.8%) versus 13 (26.5%) patients with the ready-to-use and the comparator APTs, respectively. No side effects were recorded. Both techniques were concordant in 67.3% of patients. Of the total 41 open cow's milk challenges, 60.9% had positive results, with 8 patients lost to follow-up. The performances of the ready-to-use and comparator APTs were as follows: sensitivity, 76% (95% CI, 59.2% to 92.7%) versus 44% (95% CI, 24.5% to 63.4%; P = .02); specificity, 93.8% (95% CI, 81.9% to 100%) versus 93.8% (95% CI, 81.9% to 100%); positive predictive value, 95% (95% CI, 85.4% to 100%; 1 false-positive result) versus 91.7% (95% CI, 76% to 100%; 1 false-positive result); negative predictive value, 71.4% (95% CI, 52% to 90.7%; 6 false-negative results) versus 51.7% (95% CI, 33.5% to 69.8%; 14 false-negative results); and test accuracy, 82.9% (95% CI, 71.3% to 94.5%) versus 63.4% (95% CI, 48.6% to 78.1%; P = .05) . CONCLUSION: The ready-to-use APT exhibited a good sensitivity and specificity, with no side effects.

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[247] - Keskin O, Tuncer A, Adalioglu G, Sekerel BE, Sackesen C, Kalayci O. Evaluation of the utility of atopy patch testing, skin prick testing, and total and specific IgE assays in the diagnosis of cow's milk allergy. Ann Allergy Asthma Immunol 2005;94:553-560

BACKGROUND: Information on the utility of atopy patch testing (APT) in the diagnosis of food allergy is derived from studies of children with atopic dermatitis. OBJECTIVE: To evaluate the usefulness of APT in the diagnosis of cow's milk allergy (CMA) and to determine interleukin 4 and interferon-gamma production by peripheral blood mononuclear cells. METHODS: Thirty-seven children (median age, 11 months) with suspected CMA who had a variety of symptoms that involved many organ systems were evaluated using double-blind placebo-controlled food challenges (DBPCFCs), and the performances of milk specific IgE, skin prick testing (SPT), and APT were determined. To search for a possible relationship between the diagnostic tests and the TH1/TH2 immune response, we measured interferon-gamma and interleukin 4 levels in the supernatants of peripheral blood mononuclear cell cultures. RESULTS: Seventeen children with positive DBPCFC results and 6 with a history of anaphylaxis were diagnosed as having CMA. The combined use of APT and SPT had a sensitivity of 100% and a negative predictive value of 100% but a specificity of 50% and a positive predictive value of 76%. The addition of milk specific IgE assays to APT and SPT did not improve these values. Pattern of cytokine secretion was not associated with APT positivity or a specific response to DBPCFC. CONCLUSIONS: Atopy patch testing may be a useful adjunct to SPT in excluding CMA in children who have allergic manifestations other than atopic dermatitis. However, DBPCFCs are still necessary in the presence of positive test results.

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[248] - Roehr CC, Reibel S, Ziegert M, Sommerfeld C, Wahn U, Niggemann B. Atopy patch tests, together with determination of IgE levels, reduce the need for need for oral food challenges in children with atopic dermatitis. J Allergy Clin Immunol 2001;107:548-553

BACKGROUND: Atopic dermatitis is commonly associated with food allergy. In addition to skin prick tests (SPTs) and measurements of specific IgE levels, the atopy patch test (APT) has recently been introduced into the diagnostic procedure for food allergy . OBJECTIVE: Our aim was to evaluate whether a combination of allergologic tests could improve the prognostic value of the individual tests for positive food challenge results. We hypothesized that the combination of a positive APT result plus proof of specific IgE, a positive SPT result, or both would render double-blind, placebo-controlled, food challenges unnecessary . METHODS: One hundred seventy-three double-blind, placebo-controlled, food challenges were performed in 98 children (median age, 13 months) with atopic dermatitis. All children were subjected to SPTs, APTs, and determination of specific IgE. Sensitivity, specificity, and positive and negative predictive values were calculated . RESULTS: Ninety-five (55%) of 173 oral provocations were assessed as positive. For evaluating suspected cow's milk (CM) allergy, the APT was the best single predictive test (positive predictive value [PPV], 95%), and the combination of a positive APT result with evidence of specific IgE or an APT result together with a positive skin prick test response optimized the PPV to 100%. For hen's egg (HE) allergy, the APT was also the best single predictive test (PPV, 94%). The combination of 2 or more tests did not exceed the APT's predictive value. In both CM and HE challenges, the predictability of oral challenges depended on the level of specific IgE. For wheat allergy, the APT proved to be the most reliable test, and the PPV of 94% could not be improved by a combination with other allergologic tests . CONCLUSION: The combination of positive APT results and measurement of levels of specific IgE (CM, > or = 0.35 kU/L; HE, > or = 17.5 kU/L) makes double-blind, placebo-controlled, food challenges superfluous for suspected CM and HE allergy.

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[249] - Ruiter B, Knol EF, van Neerven RJJ, Garssen J, Bruijnzeel-Koomen CAFM, Knulst AC, et al. Maintenance of tolerance to cow's milk in atopic individuals is characterized by high levels of specific immunoglobulin G4. Clin Exp Allergy 2007;37:1103-1110

Summary Background The central role of specific IgE in cow's milk allergy (CMA) is well documented. However, less is known about the function of other immunoglobulin isotypes in allergy and tolerance to cow's milk proteins (CMPs). Objective To determine differences in the antibody responses that are associated with allergy and tolerance to cow's milk in allergic, atopic and non-atopic individuals of different age groups. Methods Nineteen infants ( < 1 year), 18 children (6-14 years) and 41 adults (21-68 years) were included. Each age group was comprised of subjects with CMA, atopic individuals without a history of CMA and non-atopic subjects. Levels of specific IgE, IgG4, IgG1 and IgA to whole cow's milk and the six most abundant individual CMPs were determined in plasma by ELISA. For comparison, specific IgE and IgG4 were measured to ovomucoid and house dust mite (HDM) in individuals allergic for the respective allergens, and in atopic and non-atopic subjects without allergy. Results In infants and children with CMA, alphas1-casein and beta-lactoglobulin induced the highest specific IgE response, whereas alphas1-casein was the most allergenic CMP in adult patients. Specific IgG4 and IgG1 responses were the highest to alphas1-casein and beta-lactoglobulin in all age groups, while kappa-casein and alpha-lactalbumin induced the highest levels of IgA. CMP-specific IgG4 was higher in atopic children and adults without CMA, as compared with non-atopic individ uals. A similar difference between tolerant atopic and non-atopic subjects was observed for IgG4 specific to ovomucoid, whereas HDM-specific IgG4 was not detectable in these subjects. Conclusion Maintenance of tolerance to cow's milk in atopic children and adults without CMA is associated with elevated levels of specific IgG4, in combination with low specific IgE. The up-regulation of specific IgG4 in tolerant atopic individuals may be related to the type of allergen and its regular dose of exposure

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[250] - Johnson J, Cronin J, Morales MB, Hogan AD. Reliability of Food-Specific Serum IgE Values in Determining Negative Outcomes in Oral Food Challenges. J Allergy Clin Immunol 2008;121:S247

RATIONALE: The purpose of this study was to determine whether low Immuno capRAST values can predict negative food challenges. METHODS: A retrospective chart review was conducted for all patients who underwent oral food challenges (OFC) between January 2003 and April 2007. Statistical analyses included descriptive statistics and frequencies and Mann-Whitney U test using SPSS 15.0. RESULTS: 202 patients underwent 268 challenges to multiple foods; 60% were male, 68% Caucasian, and the median age was 3.6 years. Patients demonstrated atopy, including atopic dermatitis (70%), allergic rhinitis (83%), and asthma (49%). 32% (n = 86) demonstrated significant allergic symptoms, resulting in a positive food challenge. Positive challenges for patients with negative CAP-RAST values, (_<.35 kilo units of allergen per liter (kUA/L)), were 32% (n=6/19) for milk, 23% (n=5/22) for egg, and 17% (n=5/29) for peanut. For values between 0.35 and 2 kUA/L, positive challenges occurred in 30% for milk (n = 8/27), 43% for egg (n = 12/28) and 47% for peanut (n = 15/32). For CAP-RAST values between 0.35 kUA/L and 5 kUA/L, there were no statistically significant differences in median IgE values for positive and negative challenges for peanut (p=0.3) or egg (p = 0.9). A statistical difference was observed for milk (p < 0.02). In failed challenges for egg, milk, and peanut, urticaria occurred in 86%, respiratory symptoms 15%, gastrointestinal symptoms 17% and cardiovascular symptoms in 1%. CONCLUSIONS: CAP-RAST levels < 0.35 kUA/L for egg, milk, and peanut do not correlate well with food challenge outcomes. Allergenspecific IgE values at low concentrations are not useful predictors of challenge outcomes.

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[251] - Lam HY, van Hoffen E, Michelsen A, Guikers K, van der Tas CHW, Bruijnzeel-Koomen CAFM, et al. Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved. Clin Exp Allergy 2008;38:995-1002

BACKGROUND: Studies on cow's milk allergy (CMA) in adults are scarce. Little is known about the clinical symptoms, eliciting doses (ED), and allergens involved . OBJECTIVE: The aim of this study was to analyse the clinical symptoms, ED and allergen recognition in adult CMA patients, compared with cow's milk (CM)-sensitized, but tolerant controls . METHODS: Adult CMA patients were evaluated by standardized questionnaires (n=30), skin prick tests (SPTs) and specific IgE for CM allergens (n=18), and a double-blind placebo-controlled food challenge (DBPCFC, n=10). A control group (n=25) of CM-sensitized, but tolerant adults was included . RESULTS: The majority of CMA patients (20/30, 67%) reported severe symptoms. In all patients participating in DBPCFC, CMA was confirmed. ED for subjective symptoms (0.3-300 mg CM protein) were significantly lower than that for objective symptoms (300-9000 mg CM protein). The severity of CMA by history and ED was not correlated with SPT or IgE. Patients had higher SPT reactivity than controls for CM, alpha-lactalbumin and beta-lactoglobulin (P=0.002, P=0.014 and P=0.004) but not for casein. Specific IgE to CM tended to be higher (P=0.068) and IgE to casein was higher in patients than that in controls (P=0.016). No difference was observed for IgE to alpha-lactalbumin and beta-lactoglobulin . CONCLUSION: Adult CMA is severe in nature. ED are low, starting from 0.3 mg CM protein. Patients with CMA recognize the same major allergens (casein and whey proteins) as controls, but display a stronger SPT and IgE reactivity.

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[252] - Moneret-Vautrin DA, Morisset M, Lemerdy P, Hatahet R, Frentz P, Cuny JM. Are low levels of specific IgE useful in diagnosing clinically relevant food sensitization ? Eur Ann Allergy Clin Immunol 2006;38:307-309

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[253] - Sampson HA, Ho DG. Relationship between food-specific IgE concentrations and the risk of positive food challenges in children and adolescents. J Allergy Clin Immunol 1997;100:444-451

"BACKGROUND: The double-blind, placebo-controlled food challenge (DBPCFC) is the ""gold standard"" for diagnosis of food hypersensitivity. Skin prick tests and RASTs are sensitive indicators of food-specific IgE antibodies but poor predictors of clinical reactivity. Previous studies suggested that high concentrations of food-specific IgE antibody were predictive of food-induced clinical symptoms. Because the CAP System FEIA (Pharmacia Diagnostics, Uppsala, Sweden) provides a quantitative assessment of allergen-specific IgE antibody, this study was undertaken to determine the potential utility of the CAP System FEIA in diagnosis of IgE-mediated food hypersensitivity . METHODS: Sera from 196 patients with food allergy were analyzed for specific IgE antibodies to egg, milk, peanut, soy, wheat, and fish by CAP System FEIA. Sera were randomly selected from 300 stored samples of children and adolescents who had been evaluated by history, skin prick tests, and DBPCFCs. The study population was highly atopic; all patients had atopic dermatitis, and approximately 50% had asthma and allergic rhinitis at the time of initial evaluation. The performance characteristics of the CAP System FEIA were compared with those of skin prick tests and the outcome of DBPCFCs or ""convincing"" histories of anaphylactic reactions . RESULTS: The prevalence of specific food allergies in the study population varied from 22% for wheat to 73% for egg. Allergy to egg, milk, peanut, and soy accounted for 87% of confirmed reactions. The performance characteristics of skin prick tests and CAP System FEIA (egg, milk, peanut, fish) were comparable, with excellent sensitivity and negative predictive accuracy but poor specificity and positive predictive accuracy. The performance characteristics of the CAP System FEIA for soy and wheat were poor. For egg, milk, peanut, and fish allergy, diagnostic levels of IgE, which could predict clinical reactivity in this population with greater than 95% certainty, were identified: egg, 6 kilounits of allergen-specific IgE per liter (kU[A]/L); milk, 32 kU(A)/L; peanut, 15 kU(A)/L; and fish, 20 kU(A)/L . CONCLUSIONS: When compared with the outcome of DBPCFCs, results of CAP System FEIA are generally comparable to those of skin prick tests in predicting symptomatic food hypersensitivity. Furthermore, by measuring the concentrations of food-specific IgE antibodies with the CAP System FEIA, it is possible to identify a subset of patients who are highly likely (>95%) to experience clinical reactions to egg, milk, peanut, or fish. This could eliminate the need to perform DBPCFCs in a significant number of patients suspected of having IgE-mediated food allergy."

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[254] - Sampson HA, Ho DG. Relationship between food-specific IgE concentrations and the risk of positive food challenges in children and adolescents. J Allergy Clin Immunol 1997;100:444-451

"BACKGROUND: The double-blind, placebo-controlled food challenge (DBPCFC) is the ""gold standard"" for diagnosis of food hypersensitivity. Skin prick tests and RASTs are sensitive indicators of food-specific IgE antibodies but poor predictors of clinical reactivity. Previous studies suggested that high concentrations of food-specific IgE antibody were predictive of food-induced clinical symptoms. Because the CAP System FEIA (Pharmacia Diagnostics, Uppsala, Sweden) provides a quantitative assessment of allergen-specific IgE antibody, this study was undertaken to determine the potential utility of the CAP System FEIA in diagnosis of IgE-mediated food hypersensitivity . METHODS: Sera from 196 patients with food allergy were analyzed for specific IgE antibodies to egg, milk, peanut, soy, wheat, and fish by CAP System FEIA. Sera were randomly selected from 300 stored samples of children and adolescents who had been evaluated by history, skin prick tests, and DBPCFCs. The study population was highly atopic; all patients had atopic dermatitis, and approximately 50% had asthma and allergic rhinitis at the time of initial evaluation. The performance characteristics of the CAP System FEIA were compared with those of skin prick tests and the outcome of DBPCFCs or ""convincing"" histories of anaphylactic reactions . RESULTS: The prevalence of specific food allergies in the study population varied from 22% for wheat to 73% for egg. Allergy to egg, milk, peanut, and soy accounted for 87% of confirmed reactions. The performance characteristics of skin prick tests and CAP System FEIA (egg, milk, peanut, fish) were comparable, with excellent sensitivity and negative predictive accuracy but poor specificity and positive predictive accuracy. The performance characteristics of the CAP System FEIA for soy and wheat were poor. For egg, milk, peanut, and fish allergy, diagnostic levels of IgE, which could predict clinical reactivity in this population with greater than 95% certainty, were identified: egg, 6 kilounits of allergen-specific IgE per liter (kU[A]/L); milk, 32 kU(A)/L; peanut, 15 kU(A)/L; and fish, 20 kU(A)/L . CONCLUSIONS: When compared with the outcome of DBPCFCs, results of CAP System FEIA are generally comparable to those of skin prick tests in predicting symptomatic food hypersensitivity. Furthermore, by measuring the concentrations of food-specific IgE antibodies with the CAP System FEIA, it is possible to identify a subset of patients who are highly likely (>95%) to experience clinical reactions to egg, milk, peanut, or fish. This could eliminate the need to perform DBPCFCs in a significant number of patients suspected of having IgE-mediated food allergy."

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[255] - Majamaa H, Moisio P, Holm K, Kautiainen H, Turjanmaa K. Cow's milk allergy: diagnostic accuracy of skin prick and patch tests and specific IgE. Allergy 1999;54:346-351

BACKGROUND: The objective of the present study was to evaluate the relevance of skin tests and the concentration of cow's milk-specific IgE antibodies in correlation with oral cow's milk challenge in infants with suspected cow's milk allergy. METHODS: The study material comprised 143 infants under the age of 2 years who had undergone a diagnostic elimination challenge because of suspected cow's milk allergy in 1996. Cow's milk-specific IgE was measured, and skin prick and patch tests were performed. RESULTS: Of the 143 oral cow's milk challenges performed, 72 (50%) were positive. Of the positive reactions, 22 involved immediate-type reactions. In 50 patients, delayed-onset reactions of eczematous or gastrointestinal type appeared. Of the infants with challenge-proven cow's milk allergy, 26% showed elevated IgE concentrations to cow's milk, 14% had a positive skin prick test, and 44% had a positive patch test for cow's milk. Interestingly, in most patch test-positive patients, the prick test for cow's milk was negative. CONCLUSIONS: Our study demonstrated that many patients with a negative prick test result had a positive patch test to cow's milk. The patch test was a more sensitive method than the prick test or RAST to detect cow's milk allergy in this study population. Our results indicate that patch testing will significantly increase the probability of early detection of cow's milk allergy. Confirmation of the diagnosis is essential in patients with negative test results but a clinical suspicion of food allergy, and in patch test-positive patients. For this purpose, the most reliable method is the elimination-challenge procedure.

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[256] - Vanto T, Juntunen-Backman K, Kalimo K, Klemola T, Koivikko A, Koskinen P, et al. The patch test, skin prick test, and serum milk-specific IgE as diagnostic tools in cow's milk allergy in infants. Allergy 1999;54:837-842

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[257] - Sampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol 2001;107:891-896

BACKGROUND: The double-blind, placebo-controlled food challenge is considered the gold standard for diagnosing food allergy. However, in a retrospective analysis of children and adolescents with atopic dermatitis and food allergy, discrete food-specific IgE concentrations were established that could predict clinical reactivity to egg, milk, peanut, and fish with greater than 95% certainty. OBJECTIVE: The purpose of this investigation was to determine the utility of these 95% predictive decision points in a prospective evaluation of food allergy. METHODS: Sera from 100 consecutive children and adolescents referred for evaluation of food allergy were analyzed for specific IgE antibodies to egg, milk, peanut, soy, wheat, and fish by using the Pharmacia CAP System FEIA. Food-specific IgE values were compared with history and the results of skin prick tests and food challenges to determine the efficacy of previously established 95% predictive decision points in identifying patients with increased probability of reacting during a specific food challenge. RESULTS: One hundred children (62% male; median age, 3.8 years; range, 0.4-14.3 years) were evaluated for food allergy. The diagnosis of food allergy was established by means of history or oral food challenge. On the basis of the previously established 95% predictive decision points for egg, milk, peanut, and fish allergy, greater than 95% of food allergies diagnosed in this prospective study were correctly identified by quantifying serum food-specific IgE concentrations. CONCLUSION: In a prospective study of children and adolescents referred for evaluation of food allergy, previously established 95% predictive decision points of food-specific IgE antibody concentrations for 4 major food allergens were effective in predicting clinical reactivity. Quantification of food-specific IgE is a useful test for diagnosing symptomatic allergy to egg, milk, peanut, and fish in the pediatric population and could eliminate the need to perform double-blind, placebo-controlled food challenges in a significant number of children.

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[258] - Garcia-Ara C, Boyano-Martinez T, Diaz-Pena JM, Martin-Munoz F, Reche-Frutos M, Martin-Esteban M. Specific IgE levels in the diagnosis of immediate hypersensitivity to cows' milk protein in the infant. J Allergy Clin Immunol 2001;107:185-190

A milk-free diet with substitute formula should be established when immediate symptomatic hypersensitivity to cows' milk protein (CMP) is diagnosed, and therefore an accurate diagnosis is very important. OBJECTIVE: This study aims to find the optimal cutoff values for specific IgE antibody levels that discriminate between allergic and tolerant infants by using cows' milk and its principal proteins as allergens. METHODS: A prospective study was carried out on 170 patients under 1 year old (mean, 4.8 month.5These patients were seen consecutively over a 4-year period in our outpatient clinic and for the first time because of a reaction suggesting immediate hypersensitivity after ingestion of cows' milk formula. A clinical history, prick test with cows' milk and its proteins (alpha-lact-albumin, beta- lactoglobulin, and casein), determination of specific IgE antibodies with the CAP system FEIA for the same allergens as for the prick test, and a challenge test according to the diagnostic protocol were performed in all of the children. A study of validity of the prick test (cutoff point, 3 mm) and CAP system by using different cutoff points in the specific IgE values for cows' milk and its proteins were also analyzed. RESULTS: Prevalence of immediate symptomatic hypersensitivity to CMP in this study was 44%. When both the whole milk and its principal milk proteins were used in the prick test, the negative predictive value was very high, and a negative value excluded allergy in 97% of the patients. When the different cutoff points of the specific IgE for milk were analyzed, 2.5 KU(A)/L had a positive predictive value of 90% and 5 KU(A)/L had a positive predictive value of 95%. CONCLUSIONS: When diagnosing immediate hypersensitivity to CMP in infants, negative skin test responses exclude allergy in most of the patients. If the prick test response is positive, specific IgE levels for cows' milk may be helpful. If these values are 2.5 KU(A)/L or greater, the challenge test should not be performed because of its high positive predictive value (90%).

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[259] - Roehr CC, Reibel S, Ziegert M, Sommerfeld C, Wahn U, Niggemann B. Atopy patch tests, together with determination of IgE levels, reduce the need for need for oral food challenges in children with atopic dermatitis. J Allergy Clin Immunol 2001;107:548-553

BACKGROUND: Atopic dermatitis is commonly associated with food allergy. In addition to skin prick tests (SPTs) and measurements of specific IgE levels, the atopy patch test (APT) has recently been introduced into the diagnostic procedure for food allergy . OBJECTIVE: Our aim was to evaluate whether a combination of allergologic tests could improve the prognostic value of the individual tests for positive food challenge results. We hypothesized that the combination of a positive APT result plus proof of specific IgE, a positive SPT result, or both would render double-blind, placebo-controlled, food challenges unnecessary . METHODS: One hundred seventy-three double-blind, placebo-controlled, food challenges were performed in 98 children (median age, 13 months) with atopic dermatitis. All children were subjected to SPTs, APTs, and determination of specific IgE. Sensitivity, specificity, and positive and negative predictive values were calculated . RESULTS: Ninety-five (55%) of 173 oral provocations were assessed as positive. For evaluating suspected cow's milk (CM) allergy, the APT was the best single predictive test (positive predictive value [PPV], 95%), and the combination of a positive APT result with evidence of specific IgE or an APT result together with a positive skin prick test response optimized the PPV to 100%. For hen's egg (HE) allergy, the APT was also the best single predictive test (PPV, 94%). The combination of 2 or more tests did not exceed the APT's predictive value. In both CM and HE challenges, the predictability of oral challenges depended on the level of specific IgE. For wheat allergy, the APT proved to be the most reliable test, and the PPV of 94% could not be improved by a combination with other allergologic tests . CONCLUSION: The combination of positive APT results and measurement of levels of specific IgE (CM, > or = 0.35 kU/L; HE, > or = 17.5 kU/L) makes double-blind, placebo-controlled, food challenges superfluous for suspected CM and HE allergy.

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[260] - Saarinen KM, Suomalainen H, Savilhati E. Diagnostic value of skin-prick and patch tests and serum eosinophil cationic protein and cow's milk-specific IgE in infants with cow's milk allergy. Clin Exp Allergy 2001;31:423-429

The diagnosis of cow's milk allergy is based on a clinical response to an elimination-challenge test with cow's milk. We studied the usefulness of the skin-prick and patch tests and measurement of cow's milk-specific IgE and eosinophil cationic protein in serum as diagnostic tools for cow's milk allergy in a cohort of 6209 unselected infants followed from birth for the development of cow's milk allergy. Of the 239 infants challenged with cow's milk, 118 showed a positive and 121 a negative response at a mean age of 6.9 months. A positive reaction to a skin-prick test with cow's milk (> or = 3 mm) was seen in 72 (61%) and 29 (24%) infants with positive and negative challenges, elevated serum cow's milk-specific IgE (> or = 0.7 kU/L) in 52 (45%) and 15 (13%) infants, a positive reaction to patch test with cow's milk protein fractions in 26 (26%) and eight (8%) infants, and elevated serum eosinophil cationic protein (> or = 20 microg/L) in 22 (21%) and seven (13%) infants, respectively. Parallel use of the four tests with the above-mentioned cut-off values correctly classified 73% of the infants with a sensitivity of 0.76 and a specificity of 0.67. An immediate reaction to cow's milk challenge correlated with skin prick test positivity and elevated serum milk-specific IgE, and tended to correlate with patch test positivity. No single test or parallel use of the four tests could predict the challenge outcome acceptably in this prospectively followed, unselected cohort of 6209 infants. A positive reaction to one or more tests needs to be confirmed by a challenge test and a negative response to all four tests does not rule out the possibility of cow's milk allergy.

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[261] - Ricci G, Capelli M, Miniero R, Menna G, Zannarini L, Dillon P, et al. A comparison of different allergometric tests, skin prick test, Pharmacia UniCAP and ADVIA Centaur, for diagnosis of allergic diseases in children. Allergy 2003;58:38-45

BACKGROUND: The diagnosis of allergic disease is performed by skin prick tests (SPT) or through the demonstration of specific IgE in a blood sample via an in vitro test. The measurement of IgE concentration against allergens provides critical information in clinical allergy. Standardized and reproducible methods contribute to the quality of diagnosis and treatment of allergic disease . METHODS: In this study we evaluated the performance of a new specific IgE method, developed by ALK-Abellò for Bayer Diagnostics to run on their ADVIA Centaur immunoassay system. One hundred and fifty-one children with allergic diseases (both food and inhalant allergies) were tested for specific IgE (sIgE) via SPT and in vitro tests (UniCAP system, Pharmacia, and ADVIA Centaur immunoassay system, Bayer Diagnostics) and the test results were correlated with the clinical data . RESULTS: Statistical analysis revealed no significant difference between the two in vitro tests compared with clinical history. The sensitivities and specificities are similar, but the UniCAP system method has higher sensitivity. In the children with cow's milk allergy, the UniCAP system has sensitivity of 91% and specificity of 70%; the ADVIA Centaur immunoassay has sensitivity of 82% and specificity of 74%. In hen's egg allergy, UniCAP system has 94% sensitivity and 64% specificity, and the ADVIA Centaur system has 88% sensitivity and 52% specificity. In inhalant allergies, the two methods show statistically similar performances for both grass pollen allergies (UniCAP sensitivity 100%, specificity 73%; ADVIA Centaur sensitivity 95%, specificity 79%) and in the dust mites allergies (UniCAP sensitivity 91%, specificity 62%; ADVIA Centaur sensitivity 86%, specificity 64%). In cat allergies, the systems showed equivalent results (UniCAP sensitivity 100%, specificity 71%; ADVIA Centaur sensitivity 100%, specificity 70%). Using the UniCAP system, the geometric mean of sIgE values in children with clinical allergy is significantly higher than in sensitized ones. The ADVIA Centaur system shows a similar trend with the exclusion of cow's milk and Dermatophagoides farinae allergens. With this last method the mean value of sIgE is higher in sensitized than in symptomatic children . CONCLUSION: The new ADVIA Centaur method compares favorably with the results obtained on the UniCAP system. If other studies continue to confirm this data, then the advantages are numerous: the use of only a small quantity of serum (25 micro l per allergen), rapid turnaround time, minimal hands-on time, and no interference from IgG.

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[262] - Garcia-Ara MC, Boyano-Martinez MT, Diaz-Pena JM, Martin-Muñoz MF, Martin-Esteban M. Cow’s milk-specific immunoglobulin E levels as predictors of clinical reactivity in the follow-up of the cow’s milk allergy infants. Clin Exp Allergy 2004;34:866-870

BACKGROUND: IgE-mediated cow's milk proteins (CMPs) allergy shows a tendency to disappear with age. The sooner tolerance is detected, the earlier the substitute diets can be suspended and the quicker family emotional hardship is alleviated . OBJECTIVE: To analyse the specific IgE levels to cow's milk and its proteins, which help to separate tolerant from no tolerant children in the follow-up of infants with allergy to cow's milk. PATIENTS AND METHODS: Sixty-six infants diagnosed with IgE-mediated allergy to CMPs were included in this prospective follow-up study. Periodic reassessments were carried out every 6 months until they were 2-years old and then, annually, until tolerance arose or until the last reassessment in which tolerance had not been achieved. Non-tolerant infants were followed, at least, for a period of 3 years. In each visit, the same skin tests and determination of specific IgE (CAP System FEIA) for milk and its proteins were carried out. The open challenge test was repeated unless a clear transgression to milk, which came to be positive, had taken place within the previous 3 months in each of the follow-up visits. Specific IgE levels to milk and its proteins, in different moments of the follow-up were analysed by means of the receiver-operating characteristic curve to predict clinical reactivity . RESULTS: Throughout the follow-up 45 (68%) infants became tolerant. The follow-up mean for tolerant infants was 21.2 months whereas for non-tolerant infants it was 58 months. The specific IgE levels which were predictors of the clinical reactivity (positive predictive value (PPV)> or =90%), grew as the age of the infants increased: 1.5, 6 and 14 kU(A)/L for milk in the age range 13-18 and 19-24 months and in the third year, respectively. Specific IgE levels to casein: 0.6, 3 and 5 kU(A)/L, respectively, predicted clinical reactivity (PPV> or =90%) in the different analysed moments of the follow-up. The cut-off points: 2.7, 9 and 24 kU(A)/L for milk and 2, 4.2 and 9 kU(A)/L for casein, respectively, predicted clinical reactivity with an accuracy > or =95% corresponding to a specificity of 90% . CONCLUSIONS: Monitorization of specific IgE concentration for milk and casein by means of the CAP system in allergic children to CMPs allows us to predict, to a high degree of probability, clinical reactivity. Age factor must be taken into account to evaluate the specific IgE levels which are predictors of tolerance or clinical reactivity.

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[263] - Celik-Bilgili S, Mehl A, Verstege A, Staden U, Nocon M, Beyer K, et al. The predictive value of specific immunoglobulin E levels in serum for the outcome of oral food challenges. Clin Exp Allergy 2005;35:268-273

Summary Background Specific serum IgE is considered as one of the important diagnostic measures in the diagnostic work-up of food allergy. Objective To evaluate the role of specific serum IgE in predicting the outcome of oral food challenges, and to determine threshold concentrations of specific serum IgE that could render double-blind, placebo-controlled food challenges unnecessary. Methods In 501 children (median age 13 months), 992 controlled oral challenges were performed with cow's milk (CM), hen's egg (HE), wheat and soy. 440/501 (88%) children suffered from atopic dermatitis. For all children, specific IgE concentrations in serum were determined. Sensitivity, specificity, positive and negative predictive values, receiver operator characteristics-curves as well as predictive decision points were calculated. Results Four hundred and forty-five out of 992 oral food challenges with allergens were assessed as positive. Sensitivity of specific serum IgE was 97% for HE, 83% for CM, 69% for soy, and 79% for wheat. Specificity was 51% for HE, 53% for CM, 50% for soy, and 38% for wheat. Calculating 90%, 95% and 99% predicted probabilities using logistic regression revealed predictive decision points of 6.3, 12.6, and 59.2 kU/L for HE, respectively. Subdividing our children in those of below or above 1 year of age resulted in a markedly different predicted probability for HE. For CM, only the 90% predicted probability (88.8 kU/L) could be calculated. No decision points could be determined for CM, wheat and soy. Conclusion In general, specific serum IgE levels showed a correlation with the outcome of positive oral food challenges for CM and HE. Meaningful predictive decision points can be calculated for HE, which may help to avoid oral food challenges in some cases. However, data need to be ascertained for each allergen separately. Furthermore, the age of the patient population under investigation must also be taken into account.

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[264] - Komata T, Söderström L, Borres MP, Tachimoto H, Ebisawa M. The predictive relationship of food-specific serum IgE concentrations to challenge outcomes for egg and milk varies by patient age. J Allergy Clin Immunol 2007;119:1272-1274

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[265] - van der Gugten AC, den Otter M, Meijer Y, Pasmans SGAM, Knulst AC, Hoekstra MO. Usefulness of specific IgE levels in predicting cow’s milk allergy. J Allergy Clin Immunol 2008;121:531-533

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[266] - Ott H, Baron JM, Heise R, Ocklenburg C, Stanzel S, Merk HF, et al. Clinical usefulness of microarray-based IgE detection in children with suspected food allergy. Allergy 2008;63:1521-1528

BACKGROUND: Component-resolved diagnostics using microarray technology has recently been introduced into clinical allergology, but its applicability in children with food allergy has hardly been investigated so far. The aim of this study was to evaluate the utility of microarray-based IgE detection in the diagnostic workup of food allergy and to compare this new diagnostic tool with established methods of allergen-specific IgE detection . METHODS: We investigated 130 infants and children with suspected allergy to cow's milk (CM) or hen's egg (HE). Serum IgE measurements, skin prick tests, allergen microarray assays and controlled oral food challenges with HE and CM were performed . RESULTS: We analyzed 145 oral challenges that served as reference parameters for assay performance assessment. On this basis, the panel of microarrayed allergen components was shown to represent a comprehensive repertoire of clinically relevant CM and HE proteins. Additionally, the implemented CM and HE components respectively sufficed for equivalent test performance as compared to the corresponding fluorescence enzyme immunoassay extract and skin testing. However, component-resolved diagnostics for HE and CM allergy did not make oral food challenges superfluous. Clinical IgE decision points predicting positive oral food challenges could be calculated for both in vitro test methods . CONCLUSIONS: Allergen microarrays provide a new tool to diagnose symptomatic CM and HE allergy. They show performance characteristics comparable to the current diagnostic tests and may be indicated in small children in whom only small blood volumes are obtainable. However, they are not capable of replacing double-blind, placebo-controlled food challenges in most cases.

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[267] - Hill DJ, Heine RG, Hosking CS. The diagnostic value of skin prick testing in children with food allergy. Pediatr Allergy Immunol 2004;15:435-441

The diagnostic accuracy of the skin prick test (SPT) in food allergy is controversial. We have developed diagnostic cut-off levels for SPT in children with allergy to cow milk, egg and peanut. Based on 555 open food challenges in 467 children (median age 3.0 yr) we defined food-specific SPT weal diameters that were '100% diagnostic' for allergy to cow milk (>or=8 mm), egg (>or=7 mm) and peanut (>or=8 mm). In children < 2 yr of age, the corresponding weal diameters were >or=6 mm, >or=5 mm and >or=4 mm, respectively. These SPT cut-off levels were prospectively validated in 90 consecutive children ↩

[268] - Garcia-Ara MC, Boyano-Martinez MT, Diaz-Pena JM, Martin-Muñoz MF, Martin-Esteban M. Cow’s milk-specific immunoglobulin E levels as predictors of clinical reactivity in the follow-up of the cow’s milk allergy infants. Clin Exp Allergy 2004;34:866-870

BACKGROUND: IgE-mediated cow's milk proteins (CMPs) allergy shows a tendency to disappear with age. The sooner tolerance is detected, the earlier the substitute diets can be suspended and the quicker family emotional hardship is alleviated . OBJECTIVE: To analyse the specific IgE levels to cow's milk and its proteins, which help to separate tolerant from no tolerant children in the follow-up of infants with allergy to cow's milk. PATIENTS AND METHODS: Sixty-six infants diagnosed with IgE-mediated allergy to CMPs were included in this prospective follow-up study. Periodic reassessments were carried out every 6 months until they were 2-years old and then, annually, until tolerance arose or until the last reassessment in which tolerance had not been achieved. Non-tolerant infants were followed, at least, for a period of 3 years. In each visit, the same skin tests and determination of specific IgE (CAP System FEIA) for milk and its proteins were carried out. The open challenge test was repeated unless a clear transgression to milk, which came to be positive, had taken place within the previous 3 months in each of the follow-up visits. Specific IgE levels to milk and its proteins, in different moments of the follow-up were analysed by means of the receiver-operating characteristic curve to predict clinical reactivity . RESULTS: Throughout the follow-up 45 (68%) infants became tolerant. The follow-up mean for tolerant infants was 21.2 months whereas for non-tolerant infants it was 58 months. The specific IgE levels which were predictors of the clinical reactivity (positive predictive value (PPV)> or =90%), grew as the age of the infants increased: 1.5, 6 and 14 kU(A)/L for milk in the age range 13-18 and 19-24 months and in the third year, respectively. Specific IgE levels to casein: 0.6, 3 and 5 kU(A)/L, respectively, predicted clinical reactivity (PPV> or =90%) in the different analysed moments of the follow-up. The cut-off points: 2.7, 9 and 24 kU(A)/L for milk and 2, 4.2 and 9 kU(A)/L for casein, respectively, predicted clinical reactivity with an accuracy > or =95% corresponding to a specificity of 90% . CONCLUSIONS: Monitorization of specific IgE concentration for milk and casein by means of the CAP system in allergic children to CMPs allows us to predict, to a high degree of probability, clinical reactivity. Age factor must be taken into account to evaluate the specific IgE levels which are predictors of tolerance or clinical reactivity.

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[269] - Nowak-Wegrzyn A, Bloom KA, Sicherer SH, Shreffler WG, Noone S, Wanich N, et al. Tolerance to extensively heated milk in children with cow’s milk allergy. J Allergy Clin Immunol 2008;122:342-347

BACKGROUND: Cow's milk allergy is the most common childhood food allergy. Previously we noted that children who outgrew their milk allergy had milk-specific IgE antibodies primarily directed against conformational epitopes; those with persistent milk allergy also had IgE antibodies directed against specific sequential epitopes . OBJECTIVE: Because high temperature largely destroys conformational epitopes, we hypothesized that some children with milk allergy would tolerate extensively heated (baked) milk products . METHODS: Children with milk allergy were challenged with heated milk products; heated milk-tolerant subjects were subsequently challenged with unheated milk. Heated milk-tolerant, unheated milk-reactive subjects ingested heated milk products for 3 months and were then re-evaluated. Immune responses were assessed in all subjects; growth and intestinal permeability were followed in heated milk-tolerant subjects . RESULTS: One hundred children (mean age, 7.5 years; range, 2.1-17.3 years) underwent heated milk challenges. Sixty-eight subjects tolerated extensively heated milk only, 23 reacted to heated milk, and 9 tolerated both heated and unheated milk. Heated milk-reactive subjects had significantly larger skin prick test wheals and higher milk-specific and casein-specific IgE levels than other groups. At 3 months, subjects ingesting heated milk products had significantly smaller skin prick test wheals and higher casein-IgG(4) compared with baseline; other immunologic parameters, growth, and intestinal permeability were not significantly different. Heated milk-reactive subjects had more severe symptoms during heated milk challenge than heated milk-tolerant subjects experienced during their unheated milk challenge . CONCLUSION: The majority (75%) of children with milk allergy tolerate heated milk.

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[270] - Niggemann B, Beyer K. Diagnostic pitfalls in food allergy in children. Allergy 2005;60:104-107

Currently, the diagnostic work-up of suspected food allergy includes skin prick tests, the measurement of food specific immunoglobulin E (IgE), and the atopy patch test, and double-blind, placebo-controlled food challenges. However, all of these methods, even double-blind, placebo-controlled food challenges (DBPCFC), may sometimes be misleading. This overview describes several pitfalls for standard diagnostic methods such as problems with irritative skin reactions mimicking IgE-mediated symptoms, the problem of non-IgE-mediated reactions, pitfalls arising from the way foods are prepared or processed, effects of the route of exposure, the role of augmentation factors lowering the threshold value for clinical reactions, the noncomparability of specific IgE decision points, the influence of the timing of diagnostic measures. In addition, the problem of alternative diagnostic measures is discussed. In conclusion, there are several pitfalls in the diagnostic work-up of food allergy, which may be misleading for the physician. Properly performed controlled oral food challenges still represent the gold standard for implementing specific diets in food allergic individuals in order to avoid both unjustified diets, which may lead to severe impairments in growth and development, and to avoid unnecessary symptoms if an underlying food allergy is not correctly identified as a cause for the symptoms of the patient.

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[271] - Saarinen KM, Suomalainen H, Savilhati E. Diagnostic value of skin-prick and patch tests and serum eosinophil cationic protein and cow's milk-specific IgE in infants with cow's milk allergy. Clin Exp Allergy 2001;31:423-429

The diagnosis of cow's milk allergy is based on a clinical response to an elimination-challenge test with cow's milk. We studied the usefulness of the skin-prick and patch tests and measurement of cow's milk-specific IgE and eosinophil cationic protein in serum as diagnostic tools for cow's milk allergy in a cohort of 6209 unselected infants followed from birth for the development of cow's milk allergy. Of the 239 infants challenged with cow's milk, 118 showed a positive and 121 a negative response at a mean age of 6.9 months. A positive reaction to a skin-prick test with cow's milk (> or = 3 mm) was seen in 72 (61%) and 29 (24%) infants with positive and negative challenges, elevated serum cow's milk-specific IgE (> or = 0.7 kU/L) in 52 (45%) and 15 (13%) infants, a positive reaction to patch test with cow's milk protein fractions in 26 (26%) and eight (8%) infants, and elevated serum eosinophil cationic protein (> or = 20 microg/L) in 22 (21%) and seven (13%) infants, respectively. Parallel use of the four tests with the above-mentioned cut-off values correctly classified 73% of the infants with a sensitivity of 0.76 and a specificity of 0.67. An immediate reaction to cow's milk challenge correlated with skin prick test positivity and elevated serum milk-specific IgE, and tended to correlate with patch test positivity. No single test or parallel use of the four tests could predict the challenge outcome acceptably in this prospectively followed, unselected cohort of 6209 infants. A positive reaction to one or more tests needs to be confirmed by a challenge test and a negative response to all four tests does not rule out the possibility of cow's milk allergy.

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[272] - van Thuijl AOJ, Loeffen Y, van Aalderen WMC, Sprikkelman AB. Cow’s milk allergy: A Diagnostic Challenge. Pediatr Allergy Immunol 2008;19:275

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[273] - Villard-Truc F, Gomez SA, Deschildre A, Rancé F. Test de provocation par voie orale aux aliments chez l'enfant. Quand, pour qui et comment? 2- Sélection des patients. Rev Fr Allergol Immunol Clin 2006;46:610-624

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[274] - Miceli Sopo S, Radzik D, Calvani M. The predictive value of specific immunoglobulin E levels for the first diagnosis of cow’s milk allergy. A critical analysis of pediatric literature. Pediatr Allergy Immunol 2007;18:575-582

Investigators have tried to identify a level of seric specific immunoglobulin E (IgE) that had a sufficient predictive value to diagnose a food allergy without having will resort to the food challenge. To search in literature, all the studies that have estimated the possibility to identify a level of seric specific cow milk IgE with a positive predictive value (PPV) of 95% for the first diagnosis of cow's milk allergy (CMA) in pediatric age. We have identified six studies, nearly all studies suffer from relevant methodological bias. Proposed cut-off are all different. The studied pediatric populations were highly selected. Also neglecting the methodological bias of the studies and the great difference of value between the proposed cut-off, it always remains to consider that the pre-test probability of having a CMA between the children enrolled in the six studies included in this review is particularly high. The likelihood ratio helps to transfer the results of a study on a diagnostic test just to our population, and it is more realistic rather than to entrust itself to the PPV or the negative predictive value, that are much influenced from the prevalence of the disease in the studied population.

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[275] - Besler M, Eigenmann P, Schwartz RH. Cow's milk (Bos domesticus). Internet Symp Food Allergens 2002;4(1):19-106

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[276] - Wal JM. Cow's milk proteins/allergens. Ann Allergy Asthma Immunol 2002;89(6 Suppl. 1):3-10

OBJECTIVE: The primary objective of this review is to provide updated data on the structure and function of the main cow's milk proteins (CMPs) identified as allergens and on the characterization of their epitopes. DATA SOURCES: The review represents a synthesis of basic literature and most relevant original recent publications on both topics of clinical and epidemiologic aspects of milk allergy and of milk protein's bio- and immunochemistry. STUDY SELECTION: The expert opinion of the author was used to select the relevant data for the review. RESULTS: Most CMPs are potential allergens, even the proteins present at very low concentration. There are both conformational and linear epitopes, widely spread all along the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule which comprise highly conserved sequences responsible for immunoglobulin E cross-reactivity with corresponding milk proteins of other mammals, including human beings. Those sequential epitopes have also been proposed as good markers of persistent allergy to CMPs. CONCLUSIONS: No specific structure nor function is associated with allergenicity of CMPs. Variability and heterogeneity of the human immunoglobulin E response preclude anticipating the allergenic potential of any CMP or fragment thereof, as well as justify the need for being careful before using peptides for desensitization or proposing any milk protein hydrolysate in a diet for highly allergenic children.

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[277] - Shek LPC, Bardina L, Castro R, Sampson HA, Beyer K. Humoral and cellular responses to cow milk proteins in patients with milk-induced IgE-mediated and non-IgE-mediated disorders. Allergy 2005;60:912-919

BACKGROUND: Cow milk allergy (CMA) is one of the most common food allergies in childhood. Patients with CMA present with a wide range of immunoglobulin (Ig)E- and non-IgE-mediated clinical syndromes. Limited information is known about the specific humoral and cellular responses to cow milk proteins in these various forms of CMA . OBJECTIVE: The aim of the study was to determine IgE, IgA, IgG1 and IgG4 antibody levels and lymphocyte proliferative responses to the major cow milk allergens in patients with IgE- and non-IgE-mediated CMA . METHODS: One hundred and forty cow milk allergic patients, 6 months to 22 years of age, were included in the study. One hundred and thirteen patients had IgE-mediated CMA, 11 had milk protein-induced enterocolitis syndrome and 16 had allergic eosinophilic gastroenteritis. Twenty-one patients without food allergy, 8 months to 18 years of age, served as controls. Serum IgE, IgA, IgG1 and IgG4 antibodies to alpha-, beta-, and kappa-casein, alpha-lactalbumin and beta-lactoglobulin were measured using enzyme-linked immunosorbent assays. For a subset of these patients, we performed lymphocyte proliferation assays to the various milk allergens . RESULTS: Patients with IgE-mediated CMA had higher specific IgE concentrations to casein compared with whey proteins (P < 0.001). In this group of patients, there was a positive correlation between IgE levels and levels of the other isotypes for all four milk proteins (P < 0.001). In general, the caseins were the more allergenic and antigenic proteins in all groups of patients. Patients with enterocolitis syndrome produced less milk protein-specific IgG4 (P < 0.05) and had a trend for higher IgA antibody levels when compared to the control group. Lymphocyte proliferative responses in all groups with CMA were significantly higher than controls (P < 0.05), although this response was similar in patients with IgE- and non-IgE-mediated CMA . CONCLUSION: There is a distinct pattern of humoral antibody response in the different forms of CMA. Patients with IgE-mediated CMA have an elevated polyisotypic response to cow milk protein. The relative lack of specific IgG4 production in patients with enterocolitis syndrome may be involved in the pathogenesis of the disease. In general, caseins appear to be the predominant allergen in patients with CMA.

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[278] - Noormahomed MT, Bourrier T, Albertini M, Boutte P. La persistance de l'allergie au lait de vache: à propos de 20 cas. Rev Fr Allergol Immunol Clin 2003;43:314-321

Objectif. - L'allergie au lait de vache est connue pour disparaître dans la majorité des cas (85 à 90 %) mais peut parfois persister. Le but de cette étude est d'analyser l'histoire naturelle de cette allergie. Patients et méthode. - Cette étude rétrospective a colligé parmi 68 enfants présentant une allergie au lait et ayant subi un test de réintroduction, ceux qui ont présenté une persistance de leur allergie après l'âge de 2 ans. Nous avons inclus 20 enfants âgés de 2 à 14 ans : 16 garçons et 4 filles. Le diagnostic de persistance d'allergie était prouvé par le test de réintroduction par voie orale ou par ingestion accidentelle de lait. Résultats. - Sur les 20 cas (30 %) qui présentaient une allergie persistante à l'âge de 2 ans, 7 ont acquis une tolérance à un âge variant de 3 ans 6 mois à 9 ans 7 mois. On observe une nette prédominance de garçons (sex-ratio 4/1), une atopie familiale dans 75 % des cas et une autre sensibilisation alimentaire dans 60 % cas. Les manifestations initiales amenant au diagnostic étaient digestives dans seulement 20 % des cas. Nous avons constaté des manifestations respiratoires secondaires chez 75 % des enfants mais rarement liées au lait. Des réactions sévères ont pu être observées chez 2 patients. Un enfant a déclenché une réaction sévère après ingestion de lait de chèvre. Un taux d'IgE spécifique pour le lait de vache et la caséine > 2,5 kU/l était toujours corrélé à une positivité du test de réintroduction. Cependant un taux faible des RASTS ne permettait pas de prédire une tolérance. Conclusion. - L'allergie au lait de vache n'est pas toujours une allergie transitoire. Dans les cas d'allergie persistante, on peut suivre le taux d'IgE spécifique pour le lait de vache et ne proposer un test de réintroduction que lorsque ce taux devient inférieur à 2,5 kU/l.

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[279] - Fiocchi A, Bouygue GR, Isoardi P, Ranzini C, Terracciano L, Martelli A. How many specific IgE determinations are needed to diagnose children with cow's mlk allergy ? ACAAI Annual Meeting, New Orleans, 7-12 Nov. 2003, Poster n° P45

In diagnosing cow?s milk allergy (CMA), predictive decision points, combinations of methods and threshold challenge doses have been used to improve accuracy and reduce the need for double-blinded challenges. The RAST performance characteristics of the main allergenic fractions of cow?s milk, however, have not been assessed to discriminate between diagnostic and screening purposes in CMA. The aim of this study was to determine the differential utilities of RAST with these allergens in CMA workup. Eighty-three infants and children (aged 0.25 to 9 y; median 1.9 y) referred for suspected CMA were prospectively evaluated. As part of the diagnostic work-up, sera were submitted to specific IgE determination (CAP System, Pharmacia, S) for whole milk, alpha-lactoalbumin, beta-lactoglobulin and casein. The performance characteristics of IgE determinations using a cutpoint of 0.35 kUA/L to predict cow?s milk allergy were assessed by sensitivity analysis. RAST with whole milk (sen=1, spe=0.5; +vePV=0.7, -vePV=1) and casein (sen=0.95, spe=0.68; +vePV=0.7, -vePV=0.92) have better rule-out utility than diagnostic value. Both alpha-lactoalbumin (sen=0.5, spe=0.84; +vePV=0.8, -vePV=0.6) and beta-lactoglobulin (sen=0.7, spe= 0.8; +vePV=0.8, -vePV=0.7) have good diagnostic utility and poorer screening potential. We conclude that the tradeoff between the various discriminating powers of whole cow?s milk and cow?s milk fractions confirmed the use of four RAST in the workup of CMA in children but that both alpha-lactoalbumin and beta-lactoglobulin, being more specific and having higher false-negative rates, may be more suitable for screening settings.

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[280] - Natale M, Bisson C, Monti G, Peltran A, Garoffo LP, Valentini S, et al. Cow's milk allergens identification by two-dimensional immunoblotting and mass spectrometry. Mol Nutr Food Res 2004;48:363-369

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.

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[281] - Wal JM. Cow's milk proteins/allergens. Ann Allergy Asthma Immunol 2002;89(6 Suppl. 1):3-10

OBJECTIVE: The primary objective of this review is to provide updated data on the structure and function of the main cow's milk proteins (CMPs) identified as allergens and on the characterization of their epitopes. DATA SOURCES: The review represents a synthesis of basic literature and most relevant original recent publications on both topics of clinical and epidemiologic aspects of milk allergy and of milk protein's bio- and immunochemistry. STUDY SELECTION: The expert opinion of the author was used to select the relevant data for the review. RESULTS: Most CMPs are potential allergens, even the proteins present at very low concentration. There are both conformational and linear epitopes, widely spread all along the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule which comprise highly conserved sequences responsible for immunoglobulin E cross-reactivity with corresponding milk proteins of other mammals, including human beings. Those sequential epitopes have also been proposed as good markers of persistent allergy to CMPs. CONCLUSIONS: No specific structure nor function is associated with allergenicity of CMPs. Variability and heterogeneity of the human immunoglobulin E response preclude anticipating the allergenic potential of any CMP or fragment thereof, as well as justify the need for being careful before using peptides for desensitization or proposing any milk protein hydrolysate in a diet for highly allergenic children.

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[282] - Martorell A, Plaza AM, Bone J, Nevot S, Ara CG, Echeverria L, et al. Cow's milk protein allergy. A multi-centre study: clinical and epidemiological aspects. Allergol Immunopathol (Madr) 2006;34:46-53

Background: Due to the age when it becomes apparent and the treatment needed, cow's milk proteins (CMP) allergy requires an accurate diagnosis to avoid labelling infants falsely as allergic and subjecting them to unnecessary diets. The objective of this multi-centre study carried out at the Allergy Units of 14 Children's Hospitals was to discover the epidemiological, clinical and evolutionary characteristics of cow's milk protein allergy (CMPA). Methods and results: Infants suspected of CMPA who attended allergy clinics at the hospitals taking part during the study period were studied and a detailed clinical history was collected on all of them. Prick tests were done with cow's milk and its proteins and specific IgE anti-bodies were determined by means of CAP with the same allergens as the Prick test. The challenge test with cow's milk was carried out unless contraindicated by the diagnostic protocol. Two different challenge regimens were used: one of them carried out in 3 days and the other in one day. 409 infants with suspected CMPA were included and the diagnostic challenge test was performed on 286 patients (70 %) and not carried out on 123, as it was not indicated according to the protocol. IgE-mediated allergy was confirmed in 234 infants (58 %) and in 15 (4 %) non-IgE-mediated hypersensitivity was diagnosed. The two challenge regimens were equally secure. The average age when the reaction to cow's milk formula took place was 3.5 months (10 days-10 months). The symptoms appeared in the first week of introduction in 95 % of cases and appeared in 60 % with the first feeding with the formula. The most frequent clinical signs were cutaneous in 94 % of cases and the majority of cases appeared within 30 minutes of the feed. 99 % had been breast fed and 44 % had received some cow's milk supplement during the lactation period. Sensitization to egg not given in the feed was noted in 30 % and to beef in 29 %, being well tolerated in all of these.

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[283] - Cavagni G, D'Urbano L, Donnanno S, Artesani M, Pellegrino K, Luciano R et al. Allergy to cow's milk and egg: the performance of a component-based allergen microarray in clinical practice. Allergy 2009;64(Suppl. 90):567

Background and aim. Currently, the diagnosis of food allergy is based on allergen-specific history, on the measurement of allergenspecific IgE (sIgE) using natural allergen extracts for in vivo and in vitro tests and on the food challenge test (FCT). The diagnostic specificity of sIgE is still discussed. Although cut-off values of sIgE levels measured with the ImmunoCap System˙ have been proposed1, the food challenge test (FCT) still represents the diagnostic gold standard. The aim of this study was to compare the performance of the measurement of sIgE with a component-based allergen microarray (ISAC˙ version CRD89) and that of the standard ImmunoCAP System˙ in children with allergy to cow's milk (CM) or hen's egg (HE) proteins that underwent FCT. Patients and methods. We enrolled 104 children, with clinically suspected diagnosis of allergy to CM (58) and HE (46). In these patients we performed skin prick test (SPT, Lofarma), and sIgE measurement with the CAP˙ and ISAC˙ systems and open FCT. The performance of IgE reactivity to each component of microarray, of the ImmunoCap system and of the SPT was evaluated by ROC analysis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of SPT and of the two methods for sIgE measurement were evaluated as predictors of a positive diagnostic FCT performed in all patients. Results. FCT was positive in 32/58 (55%) patients with suspected CM allergy and in 22/46 (44%) with suspected HE allergy. IgE reactivities to Bos d 8 (27/58, 46.5%) and Bos d 4 (16/58, 27.6%) were more frequent, while very few patients showed sIgE to Bos d 7 (6/58, 10.3%) or lactoferrin (6/58, 10.3%). IgE reactivities to Gal d 1 (18/46, 39.1%), Gal d 2 (24/46, 52.1%) and Gal d 4 (17/46, 36.9%) were more frequent, while very few patients showed sIgE to Gal d 3 (6/46, 13.0%) or Gal d 5 (2/46, 4.3%). Comparisons among the results of the various tests did not show statistically significant differences. For CM, the highest AUC were observed when casein was used as the target allergen in the various tests. For HE, the highest AUC were observed with sIgE to Gal d 1 and the combination of Gal d 1 + Gal d 3. Based on ROC analysis and on the selection of a cut-off value with the highest possible PPV, sIgE to Bos d 8 >0.61 had PPV=96% and NPV=78%; similarly, sIgE to Gal d 1 > 0.86 had PPV=88% and NPV=83% and sIgE to the combination of Gal d 1 + Gal d 3 > 0.64 had PPV=87% and NPV=80%. Conclusions. In patients with severe allergy to CM or to HE, the microarray allergen test appears to have a good clinical performance in predicting the outcome of the FCT. 1. Sampson HA. Food allergy ˆ accurately identifying clinical reactivity. Allergy 2005;60 (s79):19-24

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[284] - Ruiter B, Knol EF, van Neerven RJJ, Garssen J, Bruijnzeel-Koomen CAFM, Knulst AC, et al. Maintenance of tolerance to cow's milk in atopic individuals is characterized by high levels of specific immunoglobulin G4. Clin Exp Allergy 2007;37:1103-1110

Summary Background The central role of specific IgE in cow's milk allergy (CMA) is well documented. However, less is known about the function of other immunoglobulin isotypes in allergy and tolerance to cow's milk proteins (CMPs). Objective To determine differences in the antibody responses that are associated with allergy and tolerance to cow's milk in allergic, atopic and non-atopic individuals of different age groups. Methods Nineteen infants ( < 1 year), 18 children (6-14 years) and 41 adults (21-68 years) were included. Each age group was comprised of subjects with CMA, atopic individuals without a history of CMA and non-atopic subjects. Levels of specific IgE, IgG4, IgG1 and IgA to whole cow's milk and the six most abundant individual CMPs were determined in plasma by ELISA. For comparison, specific IgE and IgG4 were measured to ovomucoid and house dust mite (HDM) in individuals allergic for the respective allergens, and in atopic and non-atopic subjects without allergy. Results In infants and children with CMA, alphas1-casein and beta-lactoglobulin induced the highest specific IgE response, whereas alphas1-casein was the most allergenic CMP in adult patients. Specific IgG4 and IgG1 responses were the highest to alphas1-casein and beta-lactoglobulin in all age groups, while kappa-casein and alpha-lactalbumin induced the highest levels of IgA. CMP-specific IgG4 was higher in atopic children and adults without CMA, as compared with non-atopic individ uals. A similar difference between tolerant atopic and non-atopic subjects was observed for IgG4 specific to ovomucoid, whereas HDM-specific IgG4 was not detectable in these subjects. Conclusion Maintenance of tolerance to cow's milk in atopic children and adults without CMA is associated with elevated levels of specific IgG4, in combination with low specific IgE. The up-regulation of specific IgG4 in tolerant atopic individuals may be related to the type of allergen and its regular dose of exposure

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[285] - Lam HY, van Hoffen E, Michelsen A, Guikers K, van der Tas CHW, Bruijnzeel-Koomen CAFM, et al. Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved. Clin Exp Allergy 2008;38:995-1002

BACKGROUND: Studies on cow's milk allergy (CMA) in adults are scarce. Little is known about the clinical symptoms, eliciting doses (ED), and allergens involved . OBJECTIVE: The aim of this study was to analyse the clinical symptoms, ED and allergen recognition in adult CMA patients, compared with cow's milk (CM)-sensitized, but tolerant controls . METHODS: Adult CMA patients were evaluated by standardized questionnaires (n=30), skin prick tests (SPTs) and specific IgE for CM allergens (n=18), and a double-blind placebo-controlled food challenge (DBPCFC, n=10). A control group (n=25) of CM-sensitized, but tolerant adults was included . RESULTS: The majority of CMA patients (20/30, 67%) reported severe symptoms. In all patients participating in DBPCFC, CMA was confirmed. ED for subjective symptoms (0.3-300 mg CM protein) were significantly lower than that for objective symptoms (300-9000 mg CM protein). The severity of CMA by history and ED was not correlated with SPT or IgE. Patients had higher SPT reactivity than controls for CM, alpha-lactalbumin and beta-lactoglobulin (P=0.002, P=0.014 and P=0.004) but not for casein. Specific IgE to CM tended to be higher (P=0.068) and IgE to casein was higher in patients than that in controls (P=0.016). No difference was observed for IgE to alpha-lactalbumin and beta-lactoglobulin . CONCLUSION: Adult CMA is severe in nature. ED are low, starting from 0.3 mg CM protein. Patients with CMA recognize the same major allergens (casein and whey proteins) as controls, but display a stronger SPT and IgE reactivity.

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[286] - Bogdanovic J, Skripak JM, Matsui EC, Burks AW, Wood RA, Hamilton RG. Agreement of Total vs. Component Milk-Specific IgE ImmunoCAP Measurements. J Allergy Clin Immunol 2009;123:S33

RATIONALE: To determine the agreement between ImmunoCAP cow‚s milk-specific IgE measurements and IgE antibody responses to individual allergic milk protein components. METHODS: Sera from 30 children (6-16 years) with cow‚s milk allergy, 20 of which received milk oral immunotherapy (MOIT), were analyzed for IgE anti-cow‚s milk-(F2), alpha-lactalbumin-(ALA), beta-lactoglobulin- (BLG) and casein-(CAS) by Phadia ImmunoCAP. IgE anti-cow‚s milk levels alone were compared to the sum of individual IgE anti-alpha-lactalbumin; beta-lactoglobulin;casein in kUa/L. Bias/inaccuracy was assessed by deviation from an ideal ratio of 1.0. All IgE antibody levels >50 kUa/L were re-analyzed at dilutions to insure accuracy. RESULTS: Assuming the sum of IgE anti-ALA-BLG-CAS should accurately reflect, or possibly underestimate the total milk specific IgE, IgE anti-milk/summed IgE anti-ALA-BLG-CAS ratios for pre MOIT sera ranged from 2.17-1.74 (n = 2; over-estimate possibly due to missing IgE anti-BSA/lactoferrin), 1.04-0.90 (n = 3; equivalence) and 0.77-0.42 (n = 25, underestimate). In MOIT-treated subjects, IgE anti-milk/IgE anti-ALA-BLG-CAS ratios remained generally constant within each subject over a year of MOIT despite fluctuations in absolute IgE anti-milk levels and marked increases in IgG anti-milk. CONCLUSIONS: Patient-dependent bias can occur in which IgE antimilk measurements alone may underestimate the actual level of IgE antibody reactive with milk components by up to 2.4 fold. While the concordance between the levels of milk-specific IgE antibody and the summed individual IgE anti-milk components is good for a minority of milk allergic patients, there is a majority where underestimation, possibly due to limited molar excess of components on the milk ImmunoCAP, may be substantial and clinically relevant.

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[287] - Plaza AM, Piquer M, Giner MT, Martin Mateos MA, Sierra JI. Diagnosis of Cow's Milk Allergy: Are Low Levels of Specific IgE Significant ? J Allergy Clin Immunol 2007;119(1 suppl):S194

AIM: To minimize the patients selected to undergo oral food challenge (OFC), two in vitro immunoassays were analyzed for their diagnostic efficiency in identifying allergic patients. Specific IgE detection down to 0.1 kU/L was also assessed to determine if low level detection improves identification of allergic patients. METHODS: OFC was performed on 60 consecutive patients who visited our department for a possible cow's milk allergy. Two different methods for measurements of allergen specific IgE were used: the CAP system FEIA, and the IMMULITE2000 3gAllergy. Data management and statistical analysis were performed in MedCalc version 8.2.1.0. RESULTS: 20 patients demonstrated a negative response to the OFC and 35 experienced a positive reaction. 5 parent's patients did not give their authorizations for the challenge. Using a 0,35 kU/L decision point, the sensitivity, specificity, positive predictive value, and negative predictive value, were 74,3%, 75%, 83,9% and 62,5%, respectively, for the UniCAP system and the values were 77,1%, 75%, 84,4% and 65,2%, respectively, for the IMMULITE2000. The ROC analysis showed the following results for milk, alpha-lactolbumin, beta-lactoglobulin and casein, 0.87, 0.4, 0.47 and 0.35 kU/L for the UniCAP100, and 0.24, 0.42, 0.46 and 0.13 kU/L, for the IMMULITE2000. In all cases sensitivities in the IMMULITE2000 are higher, maintaining specificities above 85%. The area under the curve is closer to the optimal point of 1 for the IMMULITE2000 3 g Allergy test. CONCLUSIONS: The use of in vitro diagnostic tools alone can not entirely prevent unnecessary OFC. A suggestive clinical history and levels of IgE below 0,35 kU/L for milk and casein allergens should be considered of clinical importance.

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[288] - Cavagni G, D'Urbano L, Donnanno S, Artesani M, Pellegrino K, Luciano R et al. Allergy to cow's milk and egg: the performance of a component-based allergen microarray in clinical practice. Allergy 2009;64(Suppl. 90):567

Background and aim. Currently, the diagnosis of food allergy is based on allergen-specific history, on the measurement of allergenspecific IgE (sIgE) using natural allergen extracts for in vivo and in vitro tests and on the food challenge test (FCT). The diagnostic specificity of sIgE is still discussed. Although cut-off values of sIgE levels measured with the ImmunoCap System˙ have been proposed1, the food challenge test (FCT) still represents the diagnostic gold standard. The aim of this study was to compare the performance of the measurement of sIgE with a component-based allergen microarray (ISAC˙ version CRD89) and that of the standard ImmunoCAP System˙ in children with allergy to cow's milk (CM) or hen's egg (HE) proteins that underwent FCT. Patients and methods. We enrolled 104 children, with clinically suspected diagnosis of allergy to CM (58) and HE (46). In these patients we performed skin prick test (SPT, Lofarma), and sIgE measurement with the CAP˙ and ISAC˙ systems and open FCT. The performance of IgE reactivity to each component of microarray, of the ImmunoCap system and of the SPT was evaluated by ROC analysis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of SPT and of the two methods for sIgE measurement were evaluated as predictors of a positive diagnostic FCT performed in all patients. Results. FCT was positive in 32/58 (55%) patients with suspected CM allergy and in 22/46 (44%) with suspected HE allergy. IgE reactivities to Bos d 8 (27/58, 46.5%) and Bos d 4 (16/58, 27.6%) were more frequent, while very few patients showed sIgE to Bos d 7 (6/58, 10.3%) or lactoferrin (6/58, 10.3%). IgE reactivities to Gal d 1 (18/46, 39.1%), Gal d 2 (24/46, 52.1%) and Gal d 4 (17/46, 36.9%) were more frequent, while very few patients showed sIgE to Gal d 3 (6/46, 13.0%) or Gal d 5 (2/46, 4.3%). Comparisons among the results of the various tests did not show statistically significant differences. For CM, the highest AUC were observed when casein was used as the target allergen in the various tests. For HE, the highest AUC were observed with sIgE to Gal d 1 and the combination of Gal d 1 + Gal d 3. Based on ROC analysis and on the selection of a cut-off value with the highest possible PPV, sIgE to Bos d 8 >0.61 had PPV=96% and NPV=78%; similarly, sIgE to Gal d 1 > 0.86 had PPV=88% and NPV=83% and sIgE to the combination of Gal d 1 + Gal d 3 > 0.64 had PPV=87% and NPV=80%. Conclusions. In patients with severe allergy to CM or to HE, the microarray allergen test appears to have a good clinical performance in predicting the outcome of the FCT. 1. Sampson HA. Food allergy ˆ accurately identifying clinical reactivity. Allergy 2005;60 (s79):19-24

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[289] - Fiocchi A, Bouygue GR, Isoardi P, Ranzini C, Terracciano L, Martelli A. How many specific IgE determinations are needed to diagnose children with cow's mlk allergy ? ACAAI Annual Meeting, New Orleans, 7-12 Nov. 2003, Poster n° P45

In diagnosing cow?s milk allergy (CMA), predictive decision points, combinations of methods and threshold challenge doses have been used to improve accuracy and reduce the need for double-blinded challenges. The RAST performance characteristics of the main allergenic fractions of cow?s milk, however, have not been assessed to discriminate between diagnostic and screening purposes in CMA. The aim of this study was to determine the differential utilities of RAST with these allergens in CMA workup. Eighty-three infants and children (aged 0.25 to 9 y; median 1.9 y) referred for suspected CMA were prospectively evaluated. As part of the diagnostic work-up, sera were submitted to specific IgE determination (CAP System, Pharmacia, S) for whole milk, alpha-lactoalbumin, beta-lactoglobulin and casein. The performance characteristics of IgE determinations using a cutpoint of 0.35 kUA/L to predict cow?s milk allergy were assessed by sensitivity analysis. RAST with whole milk (sen=1, spe=0.5; +vePV=0.7, -vePV=1) and casein (sen=0.95, spe=0.68; +vePV=0.7, -vePV=0.92) have better rule-out utility than diagnostic value. Both alpha-lactoalbumin (sen=0.5, spe=0.84; +vePV=0.8, -vePV=0.6) and beta-lactoglobulin (sen=0.7, spe= 0.8; +vePV=0.8, -vePV=0.7) have good diagnostic utility and poorer screening potential. We conclude that the tradeoff between the various discriminating powers of whole cow?s milk and cow?s milk fractions confirmed the use of four RAST in the workup of CMA in children but that both alpha-lactoalbumin and beta-lactoglobulin, being more specific and having higher false-negative rates, may be more suitable for screening settings.

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[290] - Castro APM, Gushken AKF, Pastorino AC, Ciccone E, Brandao AC, Jacob CMA. What Specific IgE Levels to Milk Protein Fractions May Contribute to Cow's Milk (CM) Allergy Diagnosis? AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°161

RATIONALE: Specific IgE levels to milk protein fractions have been evaluated in association with specific IgE to.whole milk protein, but their importance is not well established for CM allergy diagnosis METHODS: We evaluated 23 patients (14 male, mean 4yrs 2mo, range 11mo - 14y) with CM IgE mediated allergy confirmed by anaphylaxis or positive DBPCFC. All of them performed Pharmacia ImmunoCAP(r) system to whole milk (WM), casein, alpha-lactoalbumin and beta-lactoglobulin . A group of 23 equivalent healthy children was considered as control. Sensitivity (Se), specificity (Sp), PPV, NPV for all levels from 0,35 to 15 kU/L were determined. As the objective of this analysis was to consider the diagnosis and not only a screening test, PPV and NPV were the main valorized measures RESULTS: For alpha-lactoalbumin, the best result considering PPV = 100%, was ImmunoCap level of 3 kU/L (Se =65,2%, Sp= 100%, PPV = 100%, NPV= 74,1%). For beta-lactoglobulin the best level was 2 kU/L (Se =56,5%, Sp= 100%, PPV = 100%, NPV= 69,7%) and for casein the best level was 4 kU/L (Se =78,2%, Sp= 100%, PPV = 100%, NPV= 82,1%). Considering specific IgE to WM =11kU/L as a decision point for CM allergy diagnosis, five patients with IgE < 11kU/L presented positive protein fraction IgE CONCLUSIONS: This study showed that the decisions points for protein fractions were lower than to WM protein and they can improve the diagnosis in patients whose specific IgE levels to whole milk are lower than the established decision point. More studies are necessary to confirm this hypothesis

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[291] - Garcia-Ara MC, Boyano-Martinez MT, Diaz-Pena JM, Martin-Muñoz MF, Martin-Esteban M. Cow’s milk-specific immunoglobulin E levels as predictors of clinical reactivity in the follow-up of the cow’s milk allergy infants. Clin Exp Allergy 2004;34:866-870

BACKGROUND: IgE-mediated cow's milk proteins (CMPs) allergy shows a tendency to disappear with age. The sooner tolerance is detected, the earlier the substitute diets can be suspended and the quicker family emotional hardship is alleviated . OBJECTIVE: To analyse the specific IgE levels to cow's milk and its proteins, which help to separate tolerant from no tolerant children in the follow-up of infants with allergy to cow's milk. PATIENTS AND METHODS: Sixty-six infants diagnosed with IgE-mediated allergy to CMPs were included in this prospective follow-up study. Periodic reassessments were carried out every 6 months until they were 2-years old and then, annually, until tolerance arose or until the last reassessment in which tolerance had not been achieved. Non-tolerant infants were followed, at least, for a period of 3 years. In each visit, the same skin tests and determination of specific IgE (CAP System FEIA) for milk and its proteins were carried out. The open challenge test was repeated unless a clear transgression to milk, which came to be positive, had taken place within the previous 3 months in each of the follow-up visits. Specific IgE levels to milk and its proteins, in different moments of the follow-up were analysed by means of the receiver-operating characteristic curve to predict clinical reactivity . RESULTS: Throughout the follow-up 45 (68%) infants became tolerant. The follow-up mean for tolerant infants was 21.2 months whereas for non-tolerant infants it was 58 months. The specific IgE levels which were predictors of the clinical reactivity (positive predictive value (PPV)> or =90%), grew as the age of the infants increased: 1.5, 6 and 14 kU(A)/L for milk in the age range 13-18 and 19-24 months and in the third year, respectively. Specific IgE levels to casein: 0.6, 3 and 5 kU(A)/L, respectively, predicted clinical reactivity (PPV> or =90%) in the different analysed moments of the follow-up. The cut-off points: 2.7, 9 and 24 kU(A)/L for milk and 2, 4.2 and 9 kU(A)/L for casein, respectively, predicted clinical reactivity with an accuracy > or =95% corresponding to a specificity of 90% . CONCLUSIONS: Monitorization of specific IgE concentration for milk and casein by means of the CAP system in allergic children to CMPs allows us to predict, to a high degree of probability, clinical reactivity. Age factor must be taken into account to evaluate the specific IgE levels which are predictors of tolerance or clinical reactivity.

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[292] - Gaudin JC, Rabesona H, Choiset Y, Yeretssian G, Chobert JM, Sakanyan V, et al. Assessment of the immunoglobulin E-mediated immune response to milk-specific proteins in allergic patients using microarrays. Clin Exp Allergy 2008;38:686-693

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available . METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins . RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients . CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.

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[293] - Cavagni G, D'Urbano L, Donnanno S, Artesani M, Pellegrino K, Luciano R et al. Allergy to cow's milk and egg: the performance of a component-based allergen microarray in clinical practice. Allergy 2009;64(Suppl. 90):567

Background and aim. Currently, the diagnosis of food allergy is based on allergen-specific history, on the measurement of allergenspecific IgE (sIgE) using natural allergen extracts for in vivo and in vitro tests and on the food challenge test (FCT). The diagnostic specificity of sIgE is still discussed. Although cut-off values of sIgE levels measured with the ImmunoCap System˙ have been proposed1, the food challenge test (FCT) still represents the diagnostic gold standard. The aim of this study was to compare the performance of the measurement of sIgE with a component-based allergen microarray (ISAC˙ version CRD89) and that of the standard ImmunoCAP System˙ in children with allergy to cow's milk (CM) or hen's egg (HE) proteins that underwent FCT. Patients and methods. We enrolled 104 children, with clinically suspected diagnosis of allergy to CM (58) and HE (46). In these patients we performed skin prick test (SPT, Lofarma), and sIgE measurement with the CAP˙ and ISAC˙ systems and open FCT. The performance of IgE reactivity to each component of microarray, of the ImmunoCap system and of the SPT was evaluated by ROC analysis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of SPT and of the two methods for sIgE measurement were evaluated as predictors of a positive diagnostic FCT performed in all patients. Results. FCT was positive in 32/58 (55%) patients with suspected CM allergy and in 22/46 (44%) with suspected HE allergy. IgE reactivities to Bos d 8 (27/58, 46.5%) and Bos d 4 (16/58, 27.6%) were more frequent, while very few patients showed sIgE to Bos d 7 (6/58, 10.3%) or lactoferrin (6/58, 10.3%). IgE reactivities to Gal d 1 (18/46, 39.1%), Gal d 2 (24/46, 52.1%) and Gal d 4 (17/46, 36.9%) were more frequent, while very few patients showed sIgE to Gal d 3 (6/46, 13.0%) or Gal d 5 (2/46, 4.3%). Comparisons among the results of the various tests did not show statistically significant differences. For CM, the highest AUC were observed when casein was used as the target allergen in the various tests. For HE, the highest AUC were observed with sIgE to Gal d 1 and the combination of Gal d 1 + Gal d 3. Based on ROC analysis and on the selection of a cut-off value with the highest possible PPV, sIgE to Bos d 8 >0.61 had PPV=96% and NPV=78%; similarly, sIgE to Gal d 1 > 0.86 had PPV=88% and NPV=83% and sIgE to the combination of Gal d 1 + Gal d 3 > 0.64 had PPV=87% and NPV=80%. Conclusions. In patients with severe allergy to CM or to HE, the microarray allergen test appears to have a good clinical performance in predicting the outcome of the FCT. 1. Sampson HA. Food allergy ˆ accurately identifying clinical reactivity. Allergy 2005;60 (s79):19-24

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[294] - Cerecedo I, Dieguez M, Muriel A, Garcia balda A, Terrados S, Sanchez Cano M, et al. Usefulness of Diagnostic Tests in Cows' Milk Allergy. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°185

RATIONALE: Different cutoff values for skin prik test (SPT) and specific IgE levels have been previously given to predict a positive food challenge test in milk (CM) allergy METHODS: Atransversal study including CM allergic children. After six months of milk-excluding-diet a complete anamnesis, SPT and specific IgE with CM and fractions were determinated. Simpled-blind controlled food challenge (SBCFC) were performed in children with specific IgE lower than 2.5 KU/l. The others continued with milk-excluding-diet and after 6 months were asked for any transgression in diet. We considered this transgresions as a food oral chalenge test at home RESULTS: 162 children were included. Only 85 children had specific IgE lower than 2.5KU/l and a SBCFC was performed. 54 tolerated (63.5%). 77 continued without CM.13 had a symptomatic transgresion (possitive chalenge). Best cutoff points for SPT to milk and fractions were 3mm, except for alfalactoalbumine (6mm). The cutoff value 2,5 Ku/l of CM specific-IgE in our study had a specificty of 79,2% and a negative predictive value of 60% Differences in accuracy were obteined to SPT and milk specific IgE between older and younger than one year. CM SPT ROC area was 0.54 in <1 year and 0.80 in >1year, and milk specific IgE ROC area was 0.40 in <1 year and 0.75 in >1 year CONCLUSIONS: The accuracy of previously given 2,5 Ku/l cutoff point for specific IgE in the patient management in clinical practice was similar to published studies in clinical research conditionts. Test accuracy was higher in children older than 1 year.

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[295] - James JM, Sampson HA. Immunologic changes associated with the development of tolerance in children with cow milk allergy. J Pediatr 1992;121:371-377

The purpose of this study was to determine whether cow milk-specific antibody responses correlated with the development of clinical tolerance in cow milk-allergic children. Double-blind, placebo-controlled food challenges were performed annually in 29 patients with cow milk allergy. Clinical reactivity was lost in 11 (38%) of 29 patients. The median age for all patients at the time of diagnosis by these food challenges was 3 years; more than 80% of patients in each group had atopic dermatitis as part of their presenting symptoms. Casein-specific and beta-lactoglobulin-specific IgE, IgG, IgG1, and IgG4 antibody concentrations were analyzed in all patients at regular intervals. In the patients becoming clinically tolerant to cow milk, the IgE-specific antibody concentrations and IgE/IgG-specific ratios for both milk proteins were lower initially and decreased significantly with time, in comparison with those in the group who retained clinical sensitivity. The concentrations of IgG1- and IgG4-specific antibody to casein and the IgE/IgG1 and IgE/IgG4 ratios for both casein and beta-lactoglobulin were significantly less in the patients losing clinical reactivity. No differences in the IgG-specific concentrations were observed in either group at any of the evaluation times noted above. Monitoring similar casein-specific and beta-lactoglobulin-specific IgE concentrations and IgE/IgG ratios may help predict which patients will ultimately lose their clinical reactivity to cow milk.

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[296] - Sicherer SH, Sampson HA. Cow's milk protein-specific IgE concentrations in two age groups of milk-allergic children and in children achieving clinical tolerance. Clin Exp Allergy 1999;29:507-512

BACKGROUND: IgE-mediated cow's milk protein allergy (CMPA) is usually outgrown in children by the age of 3 years. The immunological responses to cow's milk proteins in children who achieve tolerance, in comparison with those who remain allergic, however, are not well described. OBJECTIVE: To compare the level of cow's milk protein-specific IgE among children with documented CMPA under the age of 3 years, another group over the age of 9 years (persistent allergy), and in another group of children in whom clinical tolerance developed. METHODS: Stored sera from children with CMPA were analysed for IgE antibodies specific for whole cow's milk, casein, whey, alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), and bovine serum albumin (BSA) using the Pharmacia CAP System FEIA. RESULTS: Within each group of CMPA children, the concentration of specific IgE antibody to casein proteins was not significantly different from that to whey proteins. However, children in the group with CMPA over 9 years of age had significantly greater concentrations of whole milk (P = 0.02) and casein-specific (P = 0. 04) IgE antibodies compared with the group of children with CMPA under age 3 years. Children under the age of 3 years had a higher median concentration of casein-specific IgE (20.2 vs. 5.5, P = 0.04) than another group of 11 children (mean age 3.5 years), who later lost their milk allergy. Out of 16 children who lost sensitivity to milk, 75% had milk-specific IgE levels below 14.3 kUA/L (median, 1.3 kUA/L). CONCLUSIONS: Although a dominant allergenic milk protein fraction was not identified within either of the two age groups, those with persistent CMPA over age 9 years had significantly elevated levels of milk and casein-specific IgE compared with younger children with CMPA

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[297] - Cerecedo I, Zamora J, Shreffler WG, Lin J, Bardina L, Dieguez MC, et al. Mapping of the IgE and IgG4 sequential epitopes of milk allergens with a peptide microarray–based immunoassay. J Allergy Clin Immunol 2008;122:589-594

BACKGROUND: Peptide microarray analysis is a novel method that can provide useful information on the nature of specific allergies . OBJECTIVE: We sought to determine the specificity and diversity of IgE and IgG4 antibodies binding to sequential epitopes of alpha(s1)-, alpha(s2)-, beta-, and kappa-caseins and beta-lactoglobulin by using a peptide microarray-based immunoassay . METHODS: A microarray immunoassay was performed with sera from 31 children with IgE-mediated milk allergy (16 with positive oral milk challenge results [ie, the reactive group] and 15 with negative oral milk challenge results [ie, the tolerant group]). A library of peptides, consisting of 20 amino acids (AAs) overlapping by 17 (3-offset), corresponding to the primary sequences of alpha(s1)-, alpha(s2)-, beta-, and kappa-caseins and beta-lactoglobulin was printed on epoxy-coated slides. A region was defined as an epitope if it was statistically associated with reactive groups and recognized by at least 75% of reactive patients . RESULTS: By using this method, a total of 10 epitopes were identified: alpha(s1), AAs 28 to 50, 75% reactive and 26.7% tolerant; alpha(s2), AAs 1 to 20, 75% reactive and 13.3% tolerant; AAs 13 to 32, 75% reactive and 26.7% tolerant; AAs 67 to 86, 75% reactive and 33.3% tolerant; and AAs 181 to 207, 75% reactive and 20% tolerant; beta-casein, AAs 25 to 50, 75% reactive and 33.3% tolerant, AAs 52 to 74, 81.3% reactive and 26.7% tolerant; and AAs 154 to 173, 75% reactive and 33.3% tolerant; beta-lactoglobulin, AAs 58 to 77, 81.3% reactive and 40% tolerant; and kappa-casein, AAs 34 to 53, 87.5% reactive and 40% tolerant . CONCLUSION: Several regions have been defined as epitopes, which showed differential recognition patterns between reactive and tolerant patients. Further studies are needed to validate the utility of this assay in clinical practice.

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[298] - Ott H, Baron JM, Heise R, Ocklenburg C, Stanzel S, Merk HF, et al. Clinical usefulness of microarray-based IgE detection in children with suspected food allergy. Allergy 2008;63:1521-1528

BACKGROUND: Component-resolved diagnostics using microarray technology has recently been introduced into clinical allergology, but its applicability in children with food allergy has hardly been investigated so far. The aim of this study was to evaluate the utility of microarray-based IgE detection in the diagnostic workup of food allergy and to compare this new diagnostic tool with established methods of allergen-specific IgE detection . METHODS: We investigated 130 infants and children with suspected allergy to cow's milk (CM) or hen's egg (HE). Serum IgE measurements, skin prick tests, allergen microarray assays and controlled oral food challenges with HE and CM were performed . RESULTS: We analyzed 145 oral challenges that served as reference parameters for assay performance assessment. On this basis, the panel of microarrayed allergen components was shown to represent a comprehensive repertoire of clinically relevant CM and HE proteins. Additionally, the implemented CM and HE components respectively sufficed for equivalent test performance as compared to the corresponding fluorescence enzyme immunoassay extract and skin testing. However, component-resolved diagnostics for HE and CM allergy did not make oral food challenges superfluous. Clinical IgE decision points predicting positive oral food challenges could be calculated for both in vitro test methods . CONCLUSIONS: Allergen microarrays provide a new tool to diagnose symptomatic CM and HE allergy. They show performance characteristics comparable to the current diagnostic tests and may be indicated in small children in whom only small blood volumes are obtainable. However, they are not capable of replacing double-blind, placebo-controlled food challenges in most cases.

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[299] - Cavagni G, D'Urbano L, Donnanno S, Artesani M, Pellegrino K, Luciano R et al. Allergy to cow's milk and egg: the performance of a component-based allergen microarray in clinical practice. Allergy 2009;64(Suppl. 90):567

Background and aim. Currently, the diagnosis of food allergy is based on allergen-specific history, on the measurement of allergenspecific IgE (sIgE) using natural allergen extracts for in vivo and in vitro tests and on the food challenge test (FCT). The diagnostic specificity of sIgE is still discussed. Although cut-off values of sIgE levels measured with the ImmunoCap System˙ have been proposed1, the food challenge test (FCT) still represents the diagnostic gold standard. The aim of this study was to compare the performance of the measurement of sIgE with a component-based allergen microarray (ISAC˙ version CRD89) and that of the standard ImmunoCAP System˙ in children with allergy to cow's milk (CM) or hen's egg (HE) proteins that underwent FCT. Patients and methods. We enrolled 104 children, with clinically suspected diagnosis of allergy to CM (58) and HE (46). In these patients we performed skin prick test (SPT, Lofarma), and sIgE measurement with the CAP˙ and ISAC˙ systems and open FCT. The performance of IgE reactivity to each component of microarray, of the ImmunoCap system and of the SPT was evaluated by ROC analysis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of SPT and of the two methods for sIgE measurement were evaluated as predictors of a positive diagnostic FCT performed in all patients. Results. FCT was positive in 32/58 (55%) patients with suspected CM allergy and in 22/46 (44%) with suspected HE allergy. IgE reactivities to Bos d 8 (27/58, 46.5%) and Bos d 4 (16/58, 27.6%) were more frequent, while very few patients showed sIgE to Bos d 7 (6/58, 10.3%) or lactoferrin (6/58, 10.3%). IgE reactivities to Gal d 1 (18/46, 39.1%), Gal d 2 (24/46, 52.1%) and Gal d 4 (17/46, 36.9%) were more frequent, while very few patients showed sIgE to Gal d 3 (6/46, 13.0%) or Gal d 5 (2/46, 4.3%). Comparisons among the results of the various tests did not show statistically significant differences. For CM, the highest AUC were observed when casein was used as the target allergen in the various tests. For HE, the highest AUC were observed with sIgE to Gal d 1 and the combination of Gal d 1 + Gal d 3. Based on ROC analysis and on the selection of a cut-off value with the highest possible PPV, sIgE to Bos d 8 >0.61 had PPV=96% and NPV=78%; similarly, sIgE to Gal d 1 > 0.86 had PPV=88% and NPV=83% and sIgE to the combination of Gal d 1 + Gal d 3 > 0.64 had PPV=87% and NPV=80%. Conclusions. In patients with severe allergy to CM or to HE, the microarray allergen test appears to have a good clinical performance in predicting the outcome of the FCT. 1. Sampson HA. Food allergy ˆ accurately identifying clinical reactivity. Allergy 2005;60 (s79):19-24

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[300] - Higa M, Lui C, Melo R, Watanabe L, Gushken A, Castro A et al. Could Cow's Milk Fractions Contribute to Improve Diagnosis of IgE Mediated Cow's Milk Allergy ?. J Allergy Clin Immunol 2008;121:S240

RATIONALE: The contribution of IgE specific cow‚s milk fractions measurement are not well established in cow‚s milk allergy diagnosis. The aim is to evaluate its contribution especially in patients with negative whole milk specific IgE. METHODS: It were included patients with IgE mediated cow‚s milk allergy (clinical history, laboratorial data associated to anaphylaxis and/or positive DBPCFC). Skin prick-test was considered positive with wheal >= 3 mm and ImmunoCAP values >= 3,5 kUA/ml. The patients who presented negative skin prick test and/or ImmunoCap to whole milk were submitted to SPT or ImmunoCAP to CM fractions. RESULTS: Specific IgE by ImmunoCAP were done in 70 patients, 43 (61,4%) were positive and 27 (38,6%) were negative. Among negatives patients, 7 (25,9%) presented at least one positive fraction (5 alphalactoalbumin, 3 betalactoglobulin and 1 casein). Regarding SPT from 101 tests, 78 (77,2%) were positive to whole milk and 23 (22,8%) negatives. From negative patients, 18 (78,2%) presented at least one positive fraction (12 alphalactoalbumin, 13 betalactoglobulin and 9 casein). Considering association between ImmunoCAP and SPT it was found 14 patients that presented positive SPT to whole milk when ImmunoCAP was negative. CONCLUSION: Diagnosis of cow‚s milk allergy can be improved associating research of IgE cow‚s milk fractions. SPT to whole milk may also contibute for diagnosis specially in patients with negative ImmunoCAP.

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[301] - Calvani M, Alessandri C, Frediani T, Lucarelli S, Miceli Sopo S, Panetta V, et al. Correlation between skin prick test using commercial extract of cow’s milk protein and fresh milk and food challenges. Pediatr Allergy Immunol 2007;18:583-588

The skin prick test (SPT) is regarded as an important diagnostic measure in the diagnostic work-up of cow's milk protein allergy. It is not known whether commercial extracts have any advantage over fresh milk. The aims of the study were to (i) compare the diagnostic capacity of SPTs for the three main cow's milk proteins (alpha-lactalbumin, casein and beta-lactoglobulin) with fresh milk and (ii) determine a cut-off that discriminates between allergic and tolerant children in a controlled food challenge. A study was carried out on 104 children consecutively attending two paediatric allergy clinics for suspected cow's milk allergy. A clinical history, SPTs with fresh cow's milk and commercial extracts of its three main proteins and a challenge test were performed on all the children. A study of the validity of the prick test was also performed by taking different cut-off points for fresh milk and its proteins. Twenty-eight of 104 challenge tests (26.9%) were positive. At a cut-off point of 3 mm, fresh milk showed the greatest negative predictive value (98%), whereas casein showed the greatest positive predictive value (PPV, 85%). Calculation of 95% predicted probabilities using logistic regression revealed predictive decision points of 12 mm for lactalbumin, 9 mm for casein, 10 mm for beta-lactoglobulin and 15 mm for fresh cow's milk. We found that the greater the number of positive SPTs for milk proteins, the more likely the positive response to challenge. Having a positive SPT for all three milk proteins had PPV of 92.3% and would seem more clinically useful than any cut-off. Both fresh milk and cow's milk extract of the three main proteins could be useful in the diagnostic work-up of cow's milk allergy. Finding positivity to all three cow's milk proteins seems to be a simpler and more useful way of avoiding oral food challenges.

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[302] - Martin-Blondel A, Jegu J, Lauwers-Cances V, Abbal M, Rancé F. Utilité du dosage des IgE spécifiques anti-caséine pour le diagnostic d’une allergie aux protéines du lait de vache chez l’enfant. Rev Fr Allergol 2009;49:302

Introduction.ˆ L‚allergie aux protéines de lait de vache concerne 0,1 à 7,5 des enfants en population générale. Le test de provocation par voie orale (TPO) est le test diagnostique le plus sensible et spécifique, mais il est coûteux et dangereux. Afin de réduire les indications, et les risques inhérents à sa réalisation, nous avons évalué la valeur diagnostique des tests cutanés, des dosages sériques des différentes IgE spécifiques du lait (lait total, alphalactalbumine, bétalactoglobuline, caséine) et de leur combinaison. Méthodes.ˆ Il s‚agit d‚une étude rétrospective réalisé chez les enfants vus pour TPO aux protéines de lait de vache entre septembre 2006 et août 2007. Résultats.ˆ L‚échantillon comprend 118 enfants (81 garçons, 37 filles) d‚un âge médian de 3,4 mois. Nous avons déterminé pour le test cutané à l‚extrait natif de lait une valeur seuil prédictive d‚allergie aux protéines de lait de vache de 13 mm avec une spécificité de 100 %, permettant d‚éviter la réalisation de 8 TPO. Pour les IgE spécifiques anti-caséine, la valeur seuil est de 10,6 kUI/l, avec une spécificité de 100 %, une sensibilité de 46,4 %, et ce dosage permet d‚éviter 13 TPO. Nous proposons une stratégie combinant le dosage des IgE spécifiques anti-caséine au seuil de 10,6 kUI/l, les tests cutanés à l‚extrait natif de lait au seuil de 3 mm et à l‚extrait de caséine au seuil de 4 mm. La spécificité et la valeur prédictive positive de cette stratégie sont de 100 %, évitant ainsi la réalisation de 13 TPO. Conclusion.ˆ Nos résultats démontrent l‚utilité du dosage des IgE spécifiques anti-caséine par rapport aux autres IgE spécifiques du lait. Nous proposons une stratégie combinant le dosage des IgE spécifiques anti-caséine au seuil de 10,6 kUI/l, les tests cutanés à l‚extrait natif de lait au seuil de 3 mm et à l‚extrait de caséine au seuil de 4 mm. Toutefois, leur sensibilité imparfaite rend le TPO incontournable pour de nombreuses situations cliniques.

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[303] - Schrander JJ, Oudsen S, Forget PP, Kuijten RH. Follow up study of cow's milk protein intolerant infants. Eur J Pediatr 1992;151:783-785

Over a period of 4 years, 88 infants with cow's milk protein intolerance (CMPI) were followed prospectively in order to evaluate the persistence of CMPI and its relationship between either serum IgE levels or RAST results for cow's milk. After exclusion of lactose intolerance, two positive cow's milk elimination challenge tests were considered diagnostic for CMPI. At the age of 1, 2, 3 and 4 years respectively, 85%, 78%, 49% and 33% of the children still were cow's milk intolerant. Initial serum values of IgE greater than or equal to 10 kU/l indicated a late development of tolerance to cow's milk proteins. At the age of 4 years, 90% of infants with initial IgE levels less than 10 kU/l had become tolerant to cow's milk while this was the case for only 47% of infants with initial IgE levels greater than or equal to 10 kU/l. Initial RAST results for cow's milk bore no obvious relationship to outcome

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[304] - Skripak JM, Matsui EC, Mudd K, Wood RA. The natural history of IgE-mediated cow's milk allergy. J Allergy Clin Immunol 2007;120:1172-1177

BACKGROUND: Cow's milk allergy (CMA) is the most common food allergy in infants and young children, affecting 2% to 3% of the general population. Most studies have shown the prognosis of developing tolerance to cow's milk to be good, with most outgrowing their allergy by age 3 years . OBJECTIVE: To define the natural course of CMA and identify the factors that best predict outcome in a large referral population of children with CMA . METHODS: Clinical history, test results, and final outcome were collected on 807 patients with IgE-mediated CMA. Patients were considered tolerant after they passed a challenge or experienced no reactions in the past 12 months and had a cow's milk IgE (cm-IgE) level <3 kU/L . RESULTS: Rates of resolution were 19% by age 4 years, 42% by age 8 years, 64% by age 12 years, and 79% by 16 years. Patients with persistent allergy had higher cm-IgE levels at all ages to age 16 years. The highest cm-IgE for each patient, defined as peak cm-IgE, was found to be highly predictive of outcome (P < .001). Coexisting asthma (P < .001) and allergic rhinitis (P < .001) were also significant predictors of outcome . CONCLUSION: The prognosis for CMA in this population is worse than previously reported. However, some patients developed tolerance during adolescence, indicating that follow-up and re-evaluation of CMA patients is important in their care. cm-IgE level is highly predictive of outcome. CLINICAL IMPLICATIONS: The increasing potential for persistence of CMA, along with cm-IgE level's effect on prognosis, should be considered when counseling families regarding expected clinical course.

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[305] - Rottem M, Shostak D, Foldi S. The Predictive Value of Specific Immunoglobulin E on the Outcome of Milk Allergy. IMAJ 2008;10:862-864

BACKGROUND: Cow's milk allergy is the most prevalent food hypersensitivity, affecting 2-3% of infants, but it tends to resolve with age. Cow's milk-specific immunoglobulin E in the serum is an important measure in the diagnosis and follow-up of infants and children with cow's milk allergy . OBJECTIVES: To examine the relation between CmsIgE and the probability of resolution of milk allergy . METHODS: CMsIgE was determined in the serum of 1800 infants and children referred for the evaluation of possible milk allergy. All children with CmslgE of 1 kU/L or above were followed at the allergy clinic and, according to their condition, underwent milk challenge. The diagnosis of cow's milk allergy was made on the basis of a significant and specific history or a positive oral food challenge. Subsequently, oral tolerance was defined as an uneventful oral challenge . RESULTS: A total of 135 infants and children had milk-specific IgE greater than 1 kU/L. Forty-one percent of children still had clinical milk allergy after the age of 3 years. Sixty-eight percent of children older than 3 years with persistence of cow's milk allergy had milk-specific IgE > 3 IU/ml before the age of 1 year. Furthermore, 70% of children who at 3 years old had resolved their cow's milk allergy had milk-specific IgE that was lower than 3 IU/ml before the age of 1 year. The positive predictive value of CmsIgE > 3 IU/ml to persistent cow's milk allergy at age 3 years was 82.6% (P = 0.001), with a sensitivity of 67.9% and specificity of 70.4% . CONCLUSIONS: Milk-specific IgE concentration in the first year of life can serve as a predictor of the persistence of milk allergy.

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[306] - Sicherer SH, Sampson HA. Cow's milk protein-specific IgE concentrations in two age groups of milk-allergic children and in children achieving clinical tolerance. Clin Exp Allergy 1999;29:507-512

BACKGROUND: IgE-mediated cow's milk protein allergy (CMPA) is usually outgrown in children by the age of 3 years. The immunological responses to cow's milk proteins in children who achieve tolerance, in comparison with those who remain allergic, however, are not well described. OBJECTIVE: To compare the level of cow's milk protein-specific IgE among children with documented CMPA under the age of 3 years, another group over the age of 9 years (persistent allergy), and in another group of children in whom clinical tolerance developed. METHODS: Stored sera from children with CMPA were analysed for IgE antibodies specific for whole cow's milk, casein, whey, alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), and bovine serum albumin (BSA) using the Pharmacia CAP System FEIA. RESULTS: Within each group of CMPA children, the concentration of specific IgE antibody to casein proteins was not significantly different from that to whey proteins. However, children in the group with CMPA over 9 years of age had significantly greater concentrations of whole milk (P = 0.02) and casein-specific (P = 0. 04) IgE antibodies compared with the group of children with CMPA under age 3 years. Children under the age of 3 years had a higher median concentration of casein-specific IgE (20.2 vs. 5.5, P = 0.04) than another group of 11 children (mean age 3.5 years), who later lost their milk allergy. Out of 16 children who lost sensitivity to milk, 75% had milk-specific IgE levels below 14.3 kUA/L (median, 1.3 kUA/L). CONCLUSIONS: Although a dominant allergenic milk protein fraction was not identified within either of the two age groups, those with persistent CMPA over age 9 years had significantly elevated levels of milk and casein-specific IgE compared with younger children with CMPA

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[307] - Vanto T, Heppilä S, Juntunen-Backman K, Kalimo K, Klemola T, Korpela R, et al. Prediction of the development of tolerance to milk in children with cow's milk hypersensitivity. J Pediatr 2004;144:218-222

OBJECTIVES: To investigate whether the development of tolerance to cow's milk (CM) by aged 4 years can be predicted with a skin prick test (SPT) and measurements of total or specific immunoglobulin E (IgE) in the serum, taken at the time of diagnosis of cow's milk hypersensitivity (CMH). STUDY DESIGN: Infants with immediate (n=95) or delayed (n=67) challenge reactions to CM were prospectively followed to aged 4 years. CMH status was assessed annually by CM challenges . RESULTS: By aged 2, 3, and 4 years, children with delayed reactions developed tolerance to CM faster than those with immediate reactions: 64%, 92%, and 96% versus 31%, 53%, and 63%, respectively. A wheal size of <5 mm in SPT correctly identified 83% of 124 infants who developed tolerance to CM by aged 4 years, and a wheal size of >or=5 mm in SPT correctly identified 71% of 39 infants with persistent CMH. Milk-specific IgE <2 kU/L correctly identified 82% of infants who developed tolerance to CM, and milk-specific IgE >or=2 kU/L correctly identified 71% of infants with persistent CMH . CONCLUSION: SPT and milk-specific IgE in the serum are useful prognostic indicators of the development of tolerance to CM in infants with CMH.

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[308] - Chatchatee P, Järvinen KM, Bardina L, Beyer K, Sampson HA. Identification of IgE- and IgG-binding epitopes on alpha S1-casein: Differences in patients with persistent and transient cow's milk allergy. J Allergy Clin Immunol 2001;107:379-383

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of children less than 2 years of age, but about 80% become clinically tolerant within the first 3 years of life. Casein is one of the major allergens responsible for CMA and seems to play an important role in persistent allergy. Previous studies on egg allergy suggested that linear epitopes are associated with long-lasting food allergy. OBJECTIVE: The aim of the study was to identify IgE- and IgG-binding epitopes on alpha(s1)-casein and to determine whether the patterns of epitope recognition are associated with the natural history of CMA. METHODS: According to the known amino acid (AA) sequence, 96 overlapping decapeptides representing the entire length of alpha(s1)-casein were synthesized on a cellulose-derived membrane. Sera from 24 children with milk allergy were used to identify IgE- and IgG-binding epitopes. RESULTS: Six major and 3 minor IgE-binding, as well as 5 major and 1 minor IgG-binding, regions on alpha(s1)-casein were identified. Two IgE-binding regions (AA 69-78 and AA 173-194) were recognized by the majority of patients over 9 years of age with persistent allergy (67% and 100%, respectively) but by none of the children less than 3 years of age who are likely to outgrow CMA. No differences in IgG binding between the groups were observed. CONCLUSION: There appears to be a difference in epitope recognition between patients with different natural histories of CMA. Screening for IgE antibodies to these epitopes may be useful in identifying children who will have persistent milk hypersensitivity.

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[309] - Vila L, Beyer K, Järvinen KM, Chatchatee P, Bardina L, Sampson HA. Role of conformational and linear epitopes in the achievement of tolerance in cow's milk allergy. Clin Exp Allergy 2001;31:1599-1606

Background Cow's milk (CM) is one of the leading causes of food allergy in children. However, approximately 85% of milk-allergic children become clinically tolerant to CM within the first 3 years of life. The mechanisms involved in the achievement of tolerance remain unknown. Objective To study whether IgE antibodies from children with persistent cow's milk allergy (CMA) differ from children who become clinically tolerant in their ability to recognize linear and conformational epitopes of as1- and b-casein. Methods Thirty-six milk-allergic children were included in the study: 11 of the children became clinically tolerant, and 25 had persistent CMA. Blood was obtained from all patients during the time they showed clinical reactions to milk challenge. Six non-milk-allergic children served as controls. Specific IgE antibodies against linear (denatured) as well as conformational (native) milk proteins were determined by probing dot-blots with patients' sera. In addition, selected decapeptides from as1- and b-casein, previously found to be suggestive of persistent CMA, were synthesized on a cellulose-derivatized membrane and probed with individual sera from 10 patients who outgrew CMA and from 10 patients with persistent CMA. Results Analysis of immunodot-blots showed that, in comparison to tolerant patients, milk-allergic children with persistent symptoms had a significantly higher ratio of specific IgE antibodies to linearized than to native a- and b-casein (P < 0.005 and P < 0.02, respectively). Comparing the selected decapeptides, six of the 10 patients with persistent allergy recognized the peptide corresponding to amino acids 69_78 from as1-casein while none of the patients who outgrew CMA had IgE binding to this epitope. Conclusion Patients with persistent milk allergy possess higher detectable levels of IgE antibodies to linear epitopes from as1- and b-casein than children who have achieved tolerance. Specific IgE binding to particular linear epitopes in as1-casein may be a predictive factor for persistence of CMA.

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[310] - Järvinen KM, Busse PJ, Vila L, Beyer K, Sampson HA. Recognition of IgE Binding Regions of Bovine S2-Casein Differs in Patients With Persistent and Transient Cow's Milk Allergy. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°878

Cow's milk allergy (CMA) is the most common cause of food allergy in the first years of life. While the majority of children with CMA outgrow their allergy by the age of 5 years, a small proportion will retain CMA into adulthood. We have previously mapped the major IgE binding regions of three milk caseins (s1-,-, and -casein) and two whey proteins (-lactalbumin and -lactoglobulin). We identified certain epitopes within these proteins that are recognized by IgE antibodies from children with persistent CMA but not those of children with transient CMA. The present study was performed to identify the IgE-binding epitopes of s2-casein, and to compare the pattern of epitope recognition between children with persistent CMA and those with transient milk allergy. According to the known amino acid (AA) sequence of s2-casein, 99 overlapping decapeptides, offset by two, were synthesized on a cellulose-derivatized membrane (SPOTs membrane, Genosys Biotechnologies, Inc, Woodlands). Sera from 13 cow's milk allergic children (IgE to cow's milk >100 kUA/L) were used to detect the IgE binding regions. Thereafter, a core of decapeptides representing each of the major IgE binding regions of s2-casein, were synthesized on SPOTs membrane. Sera from 10 patients, who retained CMA beyond the age of 7 years (IgE to cow's milk >100 kUA/L), and sera from 10 patients, who subsequently gained tolerance to cow's milk (IgE to cow's milk <0.35 to 39.2 kUA/L), were used to compare the pattern of recognition of these IgE binding regions. The sera utilized were drawn at the time of clinical reactivity. A healthy individual with negative prick skin tests to milk and/or milk specific IgE antibodies < 0.35 kUA /L, and documented clinical tolerance to cow's milk served as a control. Five major IgE binding regions, corresponding to amino acids (AA) 31-44, 83-100, 143-156, 157-170, and 165-184 were detected on s2-casein. The five decapeptides (AA 33-42, 87-96, 145-154, 159-168, and 171-180), each representing the core epitope of one of the major IgE binding regions, were recognized by 40%, 80%, 80%, 90%, and 80% of the patients with persistent CMA, and by 0%, 30%, 0%, 0%, and 10% of the patients with transient CMA. The sera from a healthy control showed no binding to the epitopes. Patients with persistent CMA recognize several specific sequential IgE binding epitopes on s2-casein, whereas patients who will lose their reactivity recognize only 2 „common‰ allergenic epitopes. The presence of serum IgE antibodies to regions corresponding to AA 133, 145-154, and 159-168 of s2-casein may be used as a screening instrument to identify patients who will not outgrow CMA.

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[311] - Järvinen KM, Beyer K, Vila L, Chatchatee P, Busse PJ, Sampson HA. B-cell epitopes as a screening instrument for persistent cow's milk allergy. J Allergy Clin Immunol 2002;110:293-297

Background: Cow's milk is one of the most common causes of food allergy in the first years of life. We recently defined IgE-binding epitopes of all 6 major cow's milk proteins (s1-, s2-, -, and -casein; -lactalbumin; and -lactoglobulin) and had some evidence suggesting that IgE antibodies from patients with persistent cow's milk allergy (CMA) recognize different epitopes on cow's milk proteins than do those from patients who were likely to outgrow their allergy. Objective: In this study we sought to assess whether recognition of IgE antibodies of certain epitopes of cow's milk proteins would clearly separate the patients with life-long CMA from those who will become clinically tolerant to cow's milk. Methods: According to the known IgE-binding regions of cow's milk proteins, 25 decapeptides of s1-casein, s2-casein, -casein, -lactalbumin, and -lactoglobulin, comprising the core epitopes, were synthesized on a cellulose-derivatized membrane. Sera from 10 patients with persistent CMA and 10 patients who subsequently outgrew their milk allergy were used to investigate the differences in epitope recognition. Results: Five IgE-binding epitopes (2 on s1-casein, 1 on s2-casein, and 2 on -casein) were not recognized by any of the patients with transient CMA but showed binding by the majority of the patients with persistent allergy. The presence of IgE antibodies against at least 1 of 3 epitopes (amino acid [AA] 123-132 on s1-casein, AA 171-180 on s2-casein, and AA 155-164 on -casein) identified all patients with persistent CMA. Conclusions: The presence of IgE antibodies to distinct allergenic epitopes of cow's milk proteins can be used as a marker of persistent CMA. Prospective studies are needed to investigate the usefulness of these informative epitopes in predicting life-long CMA in young children.

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[312] - Busse PJ, Järvinen KM, Vila K, Sampson HA. Identification of Sequential IgE-Binding Epitopes on Bovine alpha-s2-Casein in Cow's Milk Allergic Patients. Int Arch Allergy Immunol 2002;129:93-96

Background: Caseins are the major allergens responsible for cow's milk allergy (CMA). We have previously identified the IgE-binding epitopes of the major cow's milk (CM) proteins except for s2-casein. Methods: Overlapping decapeptides representing the entire length of alpha-s2-casein were synthesized on a cellulose-derivatized membrane. Sera from 13 CM-allergic children, 4-15 years of age, with a median level of CM-specific IgE >100 kU/l (range 33.7 to > 100 kU/l) were used to identify IgE-binding epitopes. Results: Four major and six minor sequential IgE-binding regions were identified on s2-casein. The first major region is located in the middle of the protein at amino acids (AA) 83-100, and the other three major regions are located in the carboxy terminal portion of the protein at AA 143-158, 157-172 and 165-188. The minor IgE-binding regions were identified at AA 31-44, 43-56, 93-106, 105-114, 117-128, and 191-200. Conclusion: We identified 10 sequential IgE-binding regions on alpha-s2-casein and performed the first crucial step in the development of immunotherapeutic interventions for CMA.

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[313] - Beyer K, Jarvinen KM, Bardina L, Mishoe M, Turjanmaa K, Niggemann B, et al. IgE-binding peptides coupled to a commercial matrix as a diagnostic instrument for persistent cow’s milk allergy. J Allergy Clin Immunol 2005;116:704-705

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[314] - Chatchatee P, Jarvinen KM, Bardina L, Vila L, Beyer K, Sampson HA. Identification of IgE and IgG binding epitopes on beta- and kappa-casein in cow's milk allergic patients. Clin Exp Allergy 2001;31:1256-1262

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of children aged less than 2 years of age. Although beta- and kappa-casein are considered among the major allergens responsible for CMA, no data are available on their allergenic epitopes in humans. OBJECTIVE: The aim of the study was to identify IgE- and IgG-binding epitopes on beta- and kappa-casein and to determine whether the pattern of epitope recognition is associated with the natural history of CMA. METHODS: Overlapping decapeptides representing the entire length of beta- and kappa-casein, respectively, were synthesized on a cellulose-derivatized membrane. Sera from 15 milk-allergic children, 4-18 years of age, with high levels of specific IgE antibodies to cow's milk were used to identify IgE- and IgG-binding epitopes. In addition, IgE epitopes were screened with pooled or individual sera from younger patients aged less than 3 years and who had low levels of specific serum IgE, who are likely to outgrow CMA. RESULTS: Six major and three minor IgE-bindi ng epitopes, as well as eight major and one minor IgG binding regions, were identified on beta-casein. Eight major IgE-binding epitopes, as well as two major and two minor IgG-binding epitopes, were detected on kappa- casein. Three of the IgE binding regions on beta-casein and six on kappa-casein were recognized by the majority of patients in the older age group, but not by the younger patients. CONCLUSION: Information regarding the immunodominant epitopes in beta- and kappa-casein may be important for understanding the pathophysiology and natural history of CMA. Differences in epitope recognition may be useful in identifying children who will have persistent milk hypersensitivity

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[315] - Han N, Järvinen KM, Cocco RR, Busse PJ, Sampson HA, Beyer K. Identification of amino acids critical for IgE-binding to sequential epitopes of bovine kappa-casein and the similarity of these epitopes to the corresponding human kappa-casein sequence. Allergy 2008;63:198-204

BACKGROUND: The delineation of allergenic (i.e. IgE-binding) epitopes in cow's milk proteins and the amino acids (AAs) critical for IgE-binding is necessary to understand better the structural properties of an allergen and to develop more efficacious immunotherapeutic reagents. Furthermore, this information may enable us to understand better cross-sensitivity between different allergens . METHODS: Eleven peptides, 10-14 AAs in length, representing the IgE-binding epitopes of kappa-casein were synthesized on a derivatized cellulose membrane with single AA substitutions at each position. Membranes were incubated with pooled sera from 15 milk-allergic patients and individual sera from 10 of the patients included in the pool . RESULTS: For 10/11 allergenic peptides, one to five different single AA substitutions resulted in elimination of IgE-binding of pooled patient sera. Overall at least one mutated peptide could be found for these 10 IgE-binding sites that resulted in a reduction of IgE-binding in at least 80% of the patients who recognized the native protein. Furthermore, the IgE-binding region at AA104-112 on bovine kappa-casein showed a high degree of similarity with the human kappa-casein, respectively, including the AAs critical for IgE-binding . CONCLUSION: This finding suggests that critical AAs should be assessed with both pooled and individual patient sera to account for the B-cell epitope heterogeneity between patients, with cow's milk allergy. In addition, we identified two potentially cross-reactive peptides between bovine and human caseins of unknown clinical relevance.

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[316] - Cocco RR, Jarvinen KM, Beyer K, Sampson HA. Mutational analysis of IgE epitopes of bovine alpha-s1-casein, a major cow’s milk protein. EAACI 21th Congress, Naples, 1-5 June, 2002, Communication n°57

Background: Cow‚s milk is one of the most common food allergens in childhood, and as1- casein is a major cow‚s milk allergenic protein. We have previously identified eight major and two minor IgE-binding epitopes on as1-casein. The goal of our study was to identify the critical amino acids for IgE binding within these epitopes. Determination of amino acids critical for IgE binding provides information necessary to alter the as1-casein cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE-binding capacity compared to native protein. Methods: The amino acids (AA) essential to IgE binding were determined by synthesizing peptides of 10ˆ14AA in length on a derivatized cellulose membrane with single AA substitutions (alanine or glutamine) at each position. The peptides generated were as follows: (1) AA 17ˆ30, (2) AA 23ˆ36, (3) AA 68ˆ78, (4) AA 93ˆ102, (5) AA 109ˆ120, (6) AA 123ˆ132, (7) AA 139ˆ152, (8) AA 159ˆ172, (9) AA 173ˆ 186, and (10) AA 179ˆ192. After blocking with human serum albumin, the membranes were incubated with a serum pool derived from 15 cow‚s milk allergic patients. IgE antibody binding was detected utilizing an immunoenzymatic method with biotinylated goat anti-human IgE and peroxidaseˆstreptavidin conjugate. Membranes were developed with a chemiluminescent detection method. The optical density of each individual peptide spot on X-ray film was quantitated. Healthy normal serum was used as a control. Results: Single amino acid changes within epitopes 1, 2, 3, 5, 6 and 10 led to abrogated IgE binding. For the second and eighth epitope, only one critical amino acid position was found, whereas for the other epitopes up to four positions, each resulted in a similar abrogation of IgE binding. Generally, the critical AA positions tended to be located in the centre of the epitope and involved aromatic AA predominantly. There was no increase in IgE binding in any epitope following AA substitutions. Conclusion: These results imply that single AA substitutions in critical positions within IgE-binding epitopes result in abrogated binding of milk-specific IgE antibodies by milkallergic patient sera. These mutated peptides may be used in future immunotherapeutic interventions.

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[317] - Cocco RR, Jarvinen KM, Han N, Chatchatee P, Sampson HA, Beyer K. Mutational Analysis of IgE-Binding Epitopes of the Major Cow's Milk Allergen alpha-Casein Showed a Heterogenous Pattern of Critical Amino Acids between Individual Patients and Pooled Sera. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°1134

RATIONALE: In order to alter a cDNA to encode -casein with reduced IgE-binding capacity for vaccine development, critical amino acids (AA) necessary for IgE-binding B-cell epitopes were identified. METHODS: Eleven peptides, 10 to 14 AA in length, were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with pooled sera from 15 cow's milk allergic patients and individual sera from six of the patients. IgE antibody binding was detected utilizing an immunoenzymatic method. The ODs of the peptide spots were quantitated on x-ray film. In case no decrease in binding was gained with single substitutions, peptides with double AA substitutions were generated and labeled. RESULTS: Using pooled patient sera, single AA substitution led to a total abrogation of IgE binding in seven of eleven peptides. One to six critical AA were identified per epitope. For the remaining peptides double substitution led to a clear reduction in IgE binding. However, in four of the eleven peptides, modification of the critical AA identified with pooled sera did not result in abrogation of binding with at least one individual patient serum. For these patients AAs other than those implicated by the patient pool were identified, indicating a more heterogeneous pattern in IgE recognition than expected. CONCLUSIONS: For future immunotherapeutic interventions with mutated peptides, critical AA should be determined with individual patient sera to account for the B-cell epitope heterogeneity between patients.

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[318] - Cocco RR, Järvinen KM, Sampson HA, Beyer K. Mutational analysis of major, sequential IgE-binding epitopes in alpha-s1-casein, a major cow’s milk allergen. J Allergy Clin Immunol 2003;112:433-437

BACKGROUND: Allergy to cow's milk is common in early childhood, and no therapy other than avoidance exists. In murine models of peanut allergy, immunotherapy with mutated, engineered, proteins appears promising . OBJECTIVE: We sought to identify the critical amino acids (AAs) for immunoglobulin E (IgE) binding within the major B-cell epitopes of alpha(s1)-casein, a major cow's milk allergen. This will provide the necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE-binding capacity . METHODS: For mutational analysis of the IgE-binding epitopes, peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single or multiple AA substitutions. Membranes were immunolabeled with pooled sera from 15 cow's-milk-allergic patients and with 8 individual sera . RESULTS: With the pooled sera, substitution of a single AA led to complete abrogation of IgE binding to 2 of 8 peptides and diminished binding in the remainder. Substitution of multiple AAs led to an abrogation of binding in the remaining peptides. In 4 of the 8 peptides, the critical AA identified with pooled sera did not result in significant reduction of IgE binding with 1 or more individual patients. For these patients, other critical AAs were identified, indicating a more heterogeneous pattern in IgE recognition . CONCLUSION: This study indicates that single or multiple AA substitutions within IgE-binding epitopes result in reduced binding of milk-specific IgE antibodies by patients' sera. However, for future immunotherapeutic interventions with mutated peptides, critical AAs should be evaluated with individual patient sera to determine B-cell-epitope heterogeneity.

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[319] - Cocco RR, Järvinen KM, Han N, Beyer K, Sampson HA. Mutational analysis of immunoglobulin E-binding epitopes of beta-casein and beta-lactoglobulin showed a heterogeneous pattern of critical amino acids between individual patients and pooled sera. Clin Exp Allergy 2007;37:831-838

Background For immunotherapeutic approaches, 'critical' amino acids (AAs) within allergenic epitopes are replaced with alternate AAs to eliminate IgE antibody binding. Objective To determine the critical AAs for IgE binding in beta-casein and beta-lactoglobulin (BLG). Methods Peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with a pool of sera from 15 cow's milk-allergic patients and individual sera from six of the 15 patients. In cases where no decrease in binding occurred with a single AA substitution, peptides with two AA substitutions were generated and labelled. Results Using pooled patient sera, single AA substitutions led to complete elimination of binding to six of 11 peptides for beta-casein and to all six peptides for BLG. Substituting two AAs led to an elimination of binding to four of the remaining five beta-casein epitopes. However, in three of the 11 modified beta-casein peptides and five of the six BLG peptides, no decrease in IgE binding occurred in at least one individual patient. For these patients, critical AAs other than those defined by the patient serum pool were identified, indicating a heterogeneous pattern of IgE recognition. Conclusion These results indicate that AAs critical for IgE binding are more heterogeneous than initially defined by pooled milk-allergic patient sera. For future immunotherapeutic interventions with mutated peptides, critical AAs should also be identified with individual patient sera to account for heterogeneity of IgE binding between patients.

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[320] - Ruiter B, Tregoat V, M'Rabet L, Garssen J, Knol E, van Hoffen E. Identification of immunodominant T cell epitopes in alpha s1 casein using T cells from cow's milk allergic, atopic and non-atopic children. Allergy Clin Immunol Int 2005;17(Suppl. 1):31

Background: In western countries, approximately 1.5 % of infants suffer from IgE-mediated allergic reactions against cow‚s milk proteins. Most children develop clinical and immunological tolerance, but about 15 % is still allergic by the age of 10 years. Little is known about the T cell responses to individual cow‚s milk proteins. In this study, T cell responses were determined to as1-casein, the most abundant milk protein. Furthermore, T cell epitopes were identified in this protein. Methods: Casein-specific polyclonal T cell lines (TCL, n = 33) and as1-casein-specific T cell clones (TCC, n = 20) from PBMCs of 29 children were tested. The subjects were characterized as persistent or transient cow's milk allergic (CMA), atopic (but not CMA) or non-atopic. T cell responses were measured to as1-casein and to overlapping synthetic peptides (18-mers), spanning the as1 casein molecule. Proliferation was measured by incorporation of 3Hthymidine, and cytokine production (IL-10, IL-13 and IFNg) was determined by ELISA. Results: as1-Casein induced high proliferation in T cells from all three subject groups. TCL from atopic children produced higher levels of IL-10 in response to as1-casein than TCL from CMA or non-atopic children. Using overlapping peptides, the region in as1-casein encoded by AA 133-156 was found to be most immunogenic. No differences were found in peptide recognition between the subject groups. At cytokine level, differences between the peptides were mainly observed for IFNg. The ratio of IL-10/IFNg in response to the immunodominant peptides was highest in the atopic group. Conclusion: The immunodominant part of as1-casein is encoded by AA 133-156. TCL from the atopic group produce a more regulatory cytokine pattern in response to whole as1-casein and to the immunodominant region than TCL from the CMA and non-atopic group. Some T cell epitopes in as1-casein appear to be different from previously reported IgE-epitopes, of which some are relevant in persistent CMA. The identified T cell epitopes may be a useful target in developing immunotherapy.

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[321] - Schulmeister U, Hochwallner H, Swoboda I, Focke-Tejkl M, Geller B, Nystrand M et al. Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen. J Immunol 2009;182:7019-7029

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alphaS1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alphaS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alphaS1-casein were determined with recombinant fragments and synthetic peptides spanning the alphaS1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of ralphaS1-casein and the alphaS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcepsilonRI, which had been loaded with the patients' serum IgE. Our results demonstrate that ralphaS1-casein as well as alphaS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact ralphaS1-casein induced strong basophil degranulation. These results suggest that primarily intact alphaS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alphaS1-casein as well as alphaS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

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[322] - Nowak-Wegrzyn A, Bloom KA, Sicherer SH, Shreffler WG, Noone S, Wanich N, et al. Tolerance to extensively heated milk in children with cow’s milk allergy. J Allergy Clin Immunol 2008;122:342-347

BACKGROUND: Cow's milk allergy is the most common childhood food allergy. Previously we noted that children who outgrew their milk allergy had milk-specific IgE antibodies primarily directed against conformational epitopes; those with persistent milk allergy also had IgE antibodies directed against specific sequential epitopes . OBJECTIVE: Because high temperature largely destroys conformational epitopes, we hypothesized that some children with milk allergy would tolerate extensively heated (baked) milk products . METHODS: Children with milk allergy were challenged with heated milk products; heated milk-tolerant subjects were subsequently challenged with unheated milk. Heated milk-tolerant, unheated milk-reactive subjects ingested heated milk products for 3 months and were then re-evaluated. Immune responses were assessed in all subjects; growth and intestinal permeability were followed in heated milk-tolerant subjects . RESULTS: One hundred children (mean age, 7.5 years; range, 2.1-17.3 years) underwent heated milk challenges. Sixty-eight subjects tolerated extensively heated milk only, 23 reacted to heated milk, and 9 tolerated both heated and unheated milk. Heated milk-reactive subjects had significantly larger skin prick test wheals and higher milk-specific and casein-specific IgE levels than other groups. At 3 months, subjects ingesting heated milk products had significantly smaller skin prick test wheals and higher casein-IgG(4) compared with baseline; other immunologic parameters, growth, and intestinal permeability were not significantly different. Heated milk-reactive subjects had more severe symptoms during heated milk challenge than heated milk-tolerant subjects experienced during their unheated milk challenge . CONCLUSION: The majority (75%) of children with milk allergy tolerate heated milk.

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[323] - Wanich N, Nowak-Wegrzyn A, Sampson HA, Shreffler WG. Allergen-specific basophil suppression associated with clinical tolerance in patients with milk allergy. J Allergy Clin Immunol 2009;123:789-794

BACKGROUND: Children with milk allergy who tolerate heat-denatured milk (HM) have less severe reactions and outgrow the condition earlier than those who react to HM, which might be related to differences in IgE-dependent effector cell function . OBJECTIVE: We sought to apply a novel assay to test the hypothesis that HM-tolerant children have suppressed IgE-mediated basophil responses . METHODS: Allergic, HM-tolerant, outgrown, or control subjects were defined based on oral food challenges. Whole blood cells were stimulated in vitro with a range of milk allergen doses in the presence or absence of autologous serum or with dilutions of autologous serum. Activated basophils were identified by means of flow cytometry as CD63(bright)CD123+CD203c+HLA-DR(-)CD41a(-) . RESULTS: HM-tolerant subjects' basophils were significantly less responsive to milk allergen stimulation at all doses than were basophils from HM-reactive (allergic) individuals. In the absence of autologous serum, HM-tolerant subjects' basophils were significantly more reactive at low allergen concentrations. To a lesser extent, autologous serum also inhibited IL-3- and anti-IgE-induced, but not N-formyl-methionyl-leucyl-phenylalanine-induced, responses. The allergen-specific responsiveness of HM-tolerant subjects' basophils increased with dilution of autologous serum with normal pooled serum . CONCLUSION: Children with milk allergy with a favorable prognosis have evidence of extrinsically suppressed allergen-specific effector cell reactivity.

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[324] - Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzyn A, Sampson HA. Association of allergen-specific regulatory T cells with the onset of clinical tolerance to milk protein. J Allergy Clin Immunol 2009;123:43-52

BACKGROUND: About 70% of children with milk allergy tolerate extensively heated milk (HM) products and outgrow their allergy earlier than those who react to HM . OBJECTIVE: To test the hypothesis that HM-tolerant children have a higher precursor frequency of adaptive allergen-specific regulatory T (Treg) cells . METHODS: Allergic, HM-tolerant, outgrown, or control subjects were defined by oral food challenge. PBMCs were cultured with purified caseins and controls for 7 days, and proliferating CD25(+)CD27(+) Treg cells were identified by flow cytometry. Proliferating cells were also characterized for their expression of FoxP3, CTLA 4, CD45RO, and CD127. Allergen-specific Treg cell origin and function were assessed by depletion of CD25(hi) cells before culture . RESULTS: There was a higher percentage (median [25th% to 75th%], 16.85% [7.1-31.7]) of proliferating allergen-specific CD25(+)CD27(+) T cells from cultures of HM-tolerant subjects (n = 18) than subjects with allergy (n = 8; 4.91% [2.6-7.5]; P < .01). Control subjects with no history of milk allergy (n = 7) also had low percentages of these cells (2.9% [2.4-6.0]), whereas outgrown subjects (n = 7) had intermediate percentages (9.0% [2.7-16.4]). There were no significant differences between the patient groups in the frequency of polyclonal Treg cells or allergen-specific effector T cells. Allergen-specific Treg cells were found to be FoxP3(+)CD25(hi)CD27(+), cytotoxic T lymphocyte-associated antigen 4(+), CD45RO(+)CD127(-) and were derived from circulating CD25(hi) T cells. Depletion of the CD25(hi) cells before in vitro culture significantly enhanced allergen-specific effector T-cell expansion . CONCLUSION: A higher frequency of milk allergen-specific Treg cells correlates with a phenotype of mild clinical disease and favorable prognosis.

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[325] - Nowak-Wegrzyn A, Bloom KA, Sicherer SH, Shreffler WG, Noone S, Wanich N, et al. Tolerance to extensively heated milk in children with cow’s milk allergy. J Allergy Clin Immunol 2008;122:342-347

BACKGROUND: Cow's milk allergy is the most common childhood food allergy. Previously we noted that children who outgrew their milk allergy had milk-specific IgE antibodies primarily directed against conformational epitopes; those with persistent milk allergy also had IgE antibodies directed against specific sequential epitopes . OBJECTIVE: Because high temperature largely destroys conformational epitopes, we hypothesized that some children with milk allergy would tolerate extensively heated (baked) milk products . METHODS: Children with milk allergy were challenged with heated milk products; heated milk-tolerant subjects were subsequently challenged with unheated milk. Heated milk-tolerant, unheated milk-reactive subjects ingested heated milk products for 3 months and were then re-evaluated. Immune responses were assessed in all subjects; growth and intestinal permeability were followed in heated milk-tolerant subjects . RESULTS: One hundred children (mean age, 7.5 years; range, 2.1-17.3 years) underwent heated milk challenges. Sixty-eight subjects tolerated extensively heated milk only, 23 reacted to heated milk, and 9 tolerated both heated and unheated milk. Heated milk-reactive subjects had significantly larger skin prick test wheals and higher milk-specific and casein-specific IgE levels than other groups. At 3 months, subjects ingesting heated milk products had significantly smaller skin prick test wheals and higher casein-IgG(4) compared with baseline; other immunologic parameters, growth, and intestinal permeability were not significantly different. Heated milk-reactive subjects had more severe symptoms during heated milk challenge than heated milk-tolerant subjects experienced during their unheated milk challenge . CONCLUSION: The majority (75%) of children with milk allergy tolerate heated milk.

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[326] - Lidman PGL, Watson WTA, Simons FER, Becker AB. Reactions to Food in Children Recurs After Negative Oral Challenge. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°492

Rationale To determine if patients sensitized to food remain tolerant after negative food challenge. Methods We assessed children who had allergic reactions, positive epicutaneous skin test, and/or positive capFEIA to peanut, egg or milk and subsequently had a negative food challenge between 1996 to 2003. Families were contacted by phone and questioned as to the frequency of ingestion of the food(s) in question and if any further reactions had occurred. Results There were 302 children <16 years of age with negative food challenges during this period. We randomly reviewed 107 patients (35%) with 121 challenges. 14 (12%) <6 months from challenge were excluded. 28 (23%) were lost to follow-up. Complete data were available for 59 (47%). 40/59 (68%) regularly ate the food with no problem. 4/59 (7%) subsequently reacted, all with egg (4/18; 22%), and all to raw or less well cooked egg; 1/4 completely avoided egg and 3/4 tolerated egg in baked goods. In spite of no reaction, 2/26 previously peanut allergic continued to completely avoid peanut and 10/26 had peanut < weekly. All 15 patients with negative milk challenge tolerated and continued to regularly consume milk. Conclusions Subjects with negative egg challenge were more likely to react again than subjects with negative peanut or milk challenge. Peanut allergy remains a concern for many patients, even with a negative challenge. Allergists should consider a second challenge for egg and/or peanut allergic patients.

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[327] - Lam HY, van Hoffen E, Michelsen A, Guikers K, van der Tas CHW, Bruijnzeel-Koomen CAFM, et al. Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved. Clin Exp Allergy 2008;38:995-1002

BACKGROUND: Studies on cow's milk allergy (CMA) in adults are scarce. Little is known about the clinical symptoms, eliciting doses (ED), and allergens involved . OBJECTIVE: The aim of this study was to analyse the clinical symptoms, ED and allergen recognition in adult CMA patients, compared with cow's milk (CM)-sensitized, but tolerant controls . METHODS: Adult CMA patients were evaluated by standardized questionnaires (n=30), skin prick tests (SPTs) and specific IgE for CM allergens (n=18), and a double-blind placebo-controlled food challenge (DBPCFC, n=10). A control group (n=25) of CM-sensitized, but tolerant adults was included . RESULTS: The majority of CMA patients (20/30, 67%) reported severe symptoms. In all patients participating in DBPCFC, CMA was confirmed. ED for subjective symptoms (0.3-300 mg CM protein) were significantly lower than that for objective symptoms (300-9000 mg CM protein). The severity of CMA by history and ED was not correlated with SPT or IgE. Patients had higher SPT reactivity than controls for CM, alpha-lactalbumin and beta-lactoglobulin (P=0.002, P=0.014 and P=0.004) but not for casein. Specific IgE to CM tended to be higher (P=0.068) and IgE to casein was higher in patients than that in controls (P=0.016). No difference was observed for IgE to alpha-lactalbumin and beta-lactoglobulin . CONCLUSION: Adult CMA is severe in nature. ED are low, starting from 0.3 mg CM protein. Patients with CMA recognize the same major allergens (casein and whey proteins) as controls, but display a stronger SPT and IgE reactivity.

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[328] - Tikkanen S, Kokkonen J, Juntti H, Niinimaki A. Status of children with cow's milk allergy in infancy by 10 years of age. Acta Paediatr 2000;89:1174-1180

To assess the development of milk protein tolerance and atopic diseases in children diagnosed for cow's milk allergy (CMA) in infancy, we conducted re-examinations of 56 CMA subjects at the age of 10 y using 204 age-matched controls. The children underwent clinical examinations and skin prick tests (SPT), and their IgE-specific antibodies to milk and five other food allergens were determined. By the age of 10 y, all but four subjects had become tolerant to at least small amounts of milk protein. However, gastrointestinal symptoms relating to more abundant milk consumption were reported by 45% of the study subjects and 15% of the controls (p < 0.001). The incidence figures for asthma, allergic rhinitis and dermatitis, as well as the occurrence of recurrent otitis, were three to four times higher than in the controls. Positive SPTs were seen in two-thirds of the subjects, the figure being highest (83%) in those with dermatitis onset CMA. Seven subjects showed positive titres of IgE-class milk-specific antibodies, and five showed a clinical response. CONCLUSION: This re-examination study showed that CMA in infancy, even when properly treated, has significant clinical consequences by posing special risks for respiratory atopy and persistence of atopic dermatitis as well as positive SPT and recurrent ear infections. However, each of these clinical manifestations seems to have an independent curriculum unrelated to the persistence of CMA itself.

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[329] - Hidvegi E, Cserhati E, Kereki E, Savilahti E, Arato A. Serum immunoglobulin E, IgA, and IgG antibodies to different cow's milk proteins in children with cow's milk allergy: Association with prognosis and clinical manifestations. Pediatr Allergy Immunol 2002;13:255-261

Diverse pathogenic mechanisms elicit different clinical manifestations in cow's milk allergy (CMA). Our aim was to determine the concentration of serum immunoglobulin levels to different cow's milk proteins in patients with CMA and to determine how these values were related to clinical symptoms and prognosis. Fifty children (mean age 10.9 months, range: 1-34 months) with previously confirmed CMA were enrolled in this study. All had various clinical manifestations of CMA, including gastrointestinal, skin, and respiratory symptoms. At the diagnosis of CMA the serum total and the milk-specific immunoglobulin (Ig)E values were measured by enzyme immunoassay and fluoroimmunoassay, respectively, while the relative levels of serum IgA and IgG antibodies against different cow's milk proteins were determined by a sensitive enzyme-linked immunosorbent assay (ELISA). The results were compared to those of 30 non-atopic age-matched control children. On average, after 9.2 months (range 2-31 months) on a milk-free diet, a repeated challenge was performed in 38 children. At the re-challenge, 12 patients had clinical symptoms while the remaining 26 children were symptom-free. The IgG antibody level to bovine serum albumin (BSA) was significantly lower in the patients than in the controls (median: 0.36 vs. 2.94, p < 0.01). There was a close correlation among all individual IgA and IgG antibodies to different cow's milk proteins. The anti--casein IgG level (of 2.10) in children with a positive reaction at the re-challenge was significantly higher than in those with a negative reaction (0.89) (p < 0.05). The total IgE serum concentration was also significantly higher in those who had symptoms at the re-challenge compared to those who did not have any reaction at this time (22.9 vs. 6.8 kU/l, geometric mean, p < 0.02). There was no association between the clinical manifestations and the IgG and IgA antibody levels to the cow's milk proteins studied, except for the anti-BSA IgA level, which was higher in patients with gastrointestinal symptoms. The serum total IgE and anti--casein IgG levels could have prognostic values; their increase at the beginning of the disease may indicate the development of tolerance to cow's milk only at a later age and after a longer duration of CMA. However, as there is considerable overlap among the values observed in different groups of patients, there is a limitation of these tests for predicting the prognosis.

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[330] - James JM, Sampson HA. Immunologic changes associated with the development of tolerance in children with cow milk allergy. J Pediatr 1992;121:371-377

The purpose of this study was to determine whether cow milk-specific antibody responses correlated with the development of clinical tolerance in cow milk-allergic children. Double-blind, placebo-controlled food challenges were performed annually in 29 patients with cow milk allergy. Clinical reactivity was lost in 11 (38%) of 29 patients. The median age for all patients at the time of diagnosis by these food challenges was 3 years; more than 80% of patients in each group had atopic dermatitis as part of their presenting symptoms. Casein-specific and beta-lactoglobulin-specific IgE, IgG, IgG1, and IgG4 antibody concentrations were analyzed in all patients at regular intervals. In the patients becoming clinically tolerant to cow milk, the IgE-specific antibody concentrations and IgE/IgG-specific ratios for both milk proteins were lower initially and decreased significantly with time, in comparison with those in the group who retained clinical sensitivity. The concentrations of IgG1- and IgG4-specific antibody to casein and the IgE/IgG1 and IgE/IgG4 ratios for both casein and beta-lactoglobulin were significantly less in the patients losing clinical reactivity. No differences in the IgG-specific concentrations were observed in either group at any of the evaluation times noted above. Monitoring similar casein-specific and beta-lactoglobulin-specific IgE concentrations and IgE/IgG ratios may help predict which patients will ultimately lose their clinical reactivity to cow milk.

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[331] - Cerecedo I, Zamora J, Shreffler WG, Lin J, Bardina L, Dieguez MC, et al. Mapping of the IgE and IgG4 sequential epitopes of milk allergens with a peptide microarray–based immunoassay. J Allergy Clin Immunol 2008;122:589-594

BACKGROUND: Peptide microarray analysis is a novel method that can provide useful information on the nature of specific allergies . OBJECTIVE: We sought to determine the specificity and diversity of IgE and IgG4 antibodies binding to sequential epitopes of alpha(s1)-, alpha(s2)-, beta-, and kappa-caseins and beta-lactoglobulin by using a peptide microarray-based immunoassay . METHODS: A microarray immunoassay was performed with sera from 31 children with IgE-mediated milk allergy (16 with positive oral milk challenge results [ie, the reactive group] and 15 with negative oral milk challenge results [ie, the tolerant group]). A library of peptides, consisting of 20 amino acids (AAs) overlapping by 17 (3-offset), corresponding to the primary sequences of alpha(s1)-, alpha(s2)-, beta-, and kappa-caseins and beta-lactoglobulin was printed on epoxy-coated slides. A region was defined as an epitope if it was statistically associated with reactive groups and recognized by at least 75% of reactive patients . RESULTS: By using this method, a total of 10 epitopes were identified: alpha(s1), AAs 28 to 50, 75% reactive and 26.7% tolerant; alpha(s2), AAs 1 to 20, 75% reactive and 13.3% tolerant; AAs 13 to 32, 75% reactive and 26.7% tolerant; AAs 67 to 86, 75% reactive and 33.3% tolerant; and AAs 181 to 207, 75% reactive and 20% tolerant; beta-casein, AAs 25 to 50, 75% reactive and 33.3% tolerant, AAs 52 to 74, 81.3% reactive and 26.7% tolerant; and AAs 154 to 173, 75% reactive and 33.3% tolerant; beta-lactoglobulin, AAs 58 to 77, 81.3% reactive and 40% tolerant; and kappa-casein, AAs 34 to 53, 87.5% reactive and 40% tolerant . CONCLUSION: Several regions have been defined as epitopes, which showed differential recognition patterns between reactive and tolerant patients. Further studies are needed to validate the utility of this assay in clinical practice.

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[332] - Hidvegi E, Cserhati E, Kereki E, Savilahti E, Arato A. Serum immunoglobulin E, IgA, and IgG antibodies to different cow's milk proteins in children with cow's milk allergy: Association with prognosis and clinical manifestations. Pediatr Allergy Immunol 2002;13:255-261

Diverse pathogenic mechanisms elicit different clinical manifestations in cow's milk allergy (CMA). Our aim was to determine the concentration of serum immunoglobulin levels to different cow's milk proteins in patients with CMA and to determine how these values were related to clinical symptoms and prognosis. Fifty children (mean age 10.9 months, range: 1-34 months) with previously confirmed CMA were enrolled in this study. All had various clinical manifestations of CMA, including gastrointestinal, skin, and respiratory symptoms. At the diagnosis of CMA the serum total and the milk-specific immunoglobulin (Ig)E values were measured by enzyme immunoassay and fluoroimmunoassay, respectively, while the relative levels of serum IgA and IgG antibodies against different cow's milk proteins were determined by a sensitive enzyme-linked immunosorbent assay (ELISA). The results were compared to those of 30 non-atopic age-matched control children. On average, after 9.2 months (range 2-31 months) on a milk-free diet, a repeated challenge was performed in 38 children. At the re-challenge, 12 patients had clinical symptoms while the remaining 26 children were symptom-free. The IgG antibody level to bovine serum albumin (BSA) was significantly lower in the patients than in the controls (median: 0.36 vs. 2.94, p < 0.01). There was a close correlation among all individual IgA and IgG antibodies to different cow's milk proteins. The anti--casein IgG level (of 2.10) in children with a positive reaction at the re-challenge was significantly higher than in those with a negative reaction (0.89) (p < 0.05). The total IgE serum concentration was also significantly higher in those who had symptoms at the re-challenge compared to those who did not have any reaction at this time (22.9 vs. 6.8 kU/l, geometric mean, p < 0.02). There was no association between the clinical manifestations and the IgG and IgA antibody levels to the cow's milk proteins studied, except for the anti-BSA IgA level, which was higher in patients with gastrointestinal symptoms. The serum total IgE and anti--casein IgG levels could have prognostic values; their increase at the beginning of the disease may indicate the development of tolerance to cow's milk only at a later age and after a longer duration of CMA. However, as there is considerable overlap among the values observed in different groups of patients, there is a limitation of these tests for predicting the prognosis.

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[333] - Villard-Truc F, Gomez SA, Deschildre A, Rancé F. Test de provocation par voie orale aux aliments chez l'enfant. Quand, pour qui et comment? 2- Sélection des patients. Rev Fr Allergol Immunol Clin 2006;46:610-624

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[334] - James JM, Sampson HA. Immunologic changes associated with the development of tolerance in children with cow milk allergy. J Pediatr 1992;121:371-377

The purpose of this study was to determine whether cow milk-specific antibody responses correlated with the development of clinical tolerance in cow milk-allergic children. Double-blind, placebo-controlled food challenges were performed annually in 29 patients with cow milk allergy. Clinical reactivity was lost in 11 (38%) of 29 patients. The median age for all patients at the time of diagnosis by these food challenges was 3 years; more than 80% of patients in each group had atopic dermatitis as part of their presenting symptoms. Casein-specific and beta-lactoglobulin-specific IgE, IgG, IgG1, and IgG4 antibody concentrations were analyzed in all patients at regular intervals. In the patients becoming clinically tolerant to cow milk, the IgE-specific antibody concentrations and IgE/IgG-specific ratios for both milk proteins were lower initially and decreased significantly with time, in comparison with those in the group who retained clinical sensitivity. The concentrations of IgG1- and IgG4-specific antibody to casein and the IgE/IgG1 and IgE/IgG4 ratios for both casein and beta-lactoglobulin were significantly less in the patients losing clinical reactivity. No differences in the IgG-specific concentrations were observed in either group at any of the evaluation times noted above. Monitoring similar casein-specific and beta-lactoglobulin-specific IgE concentrations and IgE/IgG ratios may help predict which patients will ultimately lose their clinical reactivity to cow milk.

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[335] - Perry TT, Matsui EC, Conover-Walker MK, Wood RA. The relationship of allergen-specific IgE levels and oral food challenge outcome. J Allergy Clin Immunol 2004;114:144-149

Background Oral food challenges remain the gold standard for the diagnosis of food allergy. However, clear clinical and laboratory guidelines have not been firmly established to determine when oral challenges should be performed. Objective : We sought to determine the value of food-specific IgE levels in predicting challenge outcome. Method s : A retrospective chart review of 604 food challenges in 391 children was performed. All children had food-specific IgE levels measured by means of CAP-RAST before challenge. Data were analyzed to determine the relationship between food-specific IgE levels and challenge outcome, as well as the relationship between other clinical parameters and challenge outcome. Result s : Forty-five percent of milk challenges were passed compared with 57% for egg, 59% for peanut, 67% for wheat, and 72% for soy. Specific IgE levels were higher among patients who failed challenges than among those who passed (P .03 for each food). When seeking a specific IgE level at which a 50% pass rate could be expected, a cutoff level of 2 kUA/L was determined for milk, egg, and peanut. Data were less clear for wheat and soy. Coexistent eczema or asthma was associated with failed egg challenges, but other atopic disease was otherwise not associated with challenge outcome. Conclusions : Allergen-specific IgE concentrations to milk, egg, and peanut and, to a lesser extent, wheat and soy serve as useful predictors of challenge outcome and should be considered when selecting patients for oral challenge to these foods.

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[336] - Garcia-Ara MC, Boyano-Martinez MT, Diaz-Pena JM, Martin-Muñoz MF, Martin-Esteban M. Cow’s milk-specific immunoglobulin E levels as predictors of clinical reactivity in the follow-up of the cow’s milk allergy infants. Clin Exp Allergy 2004;34:866-870

BACKGROUND: IgE-mediated cow's milk proteins (CMPs) allergy shows a tendency to disappear with age. The sooner tolerance is detected, the earlier the substitute diets can be suspended and the quicker family emotional hardship is alleviated . OBJECTIVE: To analyse the specific IgE levels to cow's milk and its proteins, which help to separate tolerant from no tolerant children in the follow-up of infants with allergy to cow's milk. PATIENTS AND METHODS: Sixty-six infants diagnosed with IgE-mediated allergy to CMPs were included in this prospective follow-up study. Periodic reassessments were carried out every 6 months until they were 2-years old and then, annually, until tolerance arose or until the last reassessment in which tolerance had not been achieved. Non-tolerant infants were followed, at least, for a period of 3 years. In each visit, the same skin tests and determination of specific IgE (CAP System FEIA) for milk and its proteins were carried out. The open challenge test was repeated unless a clear transgression to milk, which came to be positive, had taken place within the previous 3 months in each of the follow-up visits. Specific IgE levels to milk and its proteins, in different moments of the follow-up were analysed by means of the receiver-operating characteristic curve to predict clinical reactivity . RESULTS: Throughout the follow-up 45 (68%) infants became tolerant. The follow-up mean for tolerant infants was 21.2 months whereas for non-tolerant infants it was 58 months. The specific IgE levels which were predictors of the clinical reactivity (positive predictive value (PPV)> or =90%), grew as the age of the infants increased: 1.5, 6 and 14 kU(A)/L for milk in the age range 13-18 and 19-24 months and in the third year, respectively. Specific IgE levels to casein: 0.6, 3 and 5 kU(A)/L, respectively, predicted clinical reactivity (PPV> or =90%) in the different analysed moments of the follow-up. The cut-off points: 2.7, 9 and 24 kU(A)/L for milk and 2, 4.2 and 9 kU(A)/L for casein, respectively, predicted clinical reactivity with an accuracy > or =95% corresponding to a specificity of 90% . CONCLUSIONS: Monitorization of specific IgE concentration for milk and casein by means of the CAP system in allergic children to CMPs allows us to predict, to a high degree of probability, clinical reactivity. Age factor must be taken into account to evaluate the specific IgE levels which are predictors of tolerance or clinical reactivity.

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[337] - Perry TT, Matsui EC, Conover-Walker MK, Wood RA. The relationship of allergen-specific IgE levels and oral food challenge outcome. J Allergy Clin Immunol 2004;114:144-149

Background Oral food challenges remain the gold standard for the diagnosis of food allergy. However, clear clinical and laboratory guidelines have not been firmly established to determine when oral challenges should be performed. Objective : We sought to determine the value of food-specific IgE levels in predicting challenge outcome. Method s : A retrospective chart review of 604 food challenges in 391 children was performed. All children had food-specific IgE levels measured by means of CAP-RAST before challenge. Data were analyzed to determine the relationship between food-specific IgE levels and challenge outcome, as well as the relationship between other clinical parameters and challenge outcome. Result s : Forty-five percent of milk challenges were passed compared with 57% for egg, 59% for peanut, 67% for wheat, and 72% for soy. Specific IgE levels were higher among patients who failed challenges than among those who passed (P .03 for each food). When seeking a specific IgE level at which a 50% pass rate could be expected, a cutoff level of 2 kUA/L was determined for milk, egg, and peanut. Data were less clear for wheat and soy. Coexistent eczema or asthma was associated with failed egg challenges, but other atopic disease was otherwise not associated with challenge outcome. Conclusions : Allergen-specific IgE concentrations to milk, egg, and peanut and, to a lesser extent, wheat and soy serve as useful predictors of challenge outcome and should be considered when selecting patients for oral challenge to these foods.

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[338] - Vanto T, Heppilä S, Juntunen-Backman K, Kalimo K, Klemola T, Korpela R, et al. Prediction of the development of tolerance to milk in children with cow's milk hypersensitivity. J Pediatr 2004;144:218-222

OBJECTIVES: To investigate whether the development of tolerance to cow's milk (CM) by aged 4 years can be predicted with a skin prick test (SPT) and measurements of total or specific immunoglobulin E (IgE) in the serum, taken at the time of diagnosis of cow's milk hypersensitivity (CMH). STUDY DESIGN: Infants with immediate (n=95) or delayed (n=67) challenge reactions to CM were prospectively followed to aged 4 years. CMH status was assessed annually by CM challenges . RESULTS: By aged 2, 3, and 4 years, children with delayed reactions developed tolerance to CM faster than those with immediate reactions: 64%, 92%, and 96% versus 31%, 53%, and 63%, respectively. A wheal size of <5 mm in SPT correctly identified 83% of 124 infants who developed tolerance to CM by aged 4 years, and a wheal size of >or=5 mm in SPT correctly identified 71% of 39 infants with persistent CMH. Milk-specific IgE <2 kU/L correctly identified 82% of infants who developed tolerance to CM, and milk-specific IgE >or=2 kU/L correctly identified 71% of infants with persistent CMH . CONCLUSION: SPT and milk-specific IgE in the serum are useful prognostic indicators of the development of tolerance to CM in infants with CMH.

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[339] - Shek LPC, Soderstrom L, Ahlstedt S, Beyer K, Sampson HA. Determination of food specific IgE levels over time can predict the development of tolerance in cow's milk and hen's egg allergy. J Allergy Clin Immunol 2004;114:387-391

Background The majority of children with cow's milk and hen's egg allergy develop clinical tolerance with time. However, there are no good indices to predict when and in whom this occurs. Objective The aim of this study was to determine if monitoring food specific IgE levels over time could be used as a predictor for determining when patients develop clinical tolerance. Methods Eighty-eight patients with hen's egg and 49 patients with cow's milk allergy who underwent repeated double-blind, placebo-controlled food challenges were included in the study. Using the Pharmacia CAP-System FEIA, specific IgE (sIgE) levels to cow's milk and hen's egg were retrospectively determined from stored serum samples obtained at the time of the food challenges. Logistic regression was used to evaluate the relationship between tolerance development and the decrease in sIgE levels over a specific time period between the two challenges. Results Twenty-eight of the 66 egg-allergic and 16 of the 33 milk-allergic patients lost their allergy over time. For egg, the decrease in sIgE levels (P=.0014) was significantly related to the probability of developing clinical tolerance, with the duration between challenges having an influence (P=.06). For milk there also was a significant relationship between the decrease in sIgE levels (P=.0175) and the probability of developing tolerance to milk but no significant contribution with regard to time. Stratification into 2 age groups, those below 4 years of age and those above 4 years of age at time of first challenge, had an effect, with the younger age group being more likely to develop clinical tolerance in relation to the rate of decrease in sIgE. The median food sIgE level at diagnosis was significantly less for the group developing „tolerance‰ to egg (P < .001), and a similar trend was seen for milk allergy (P=.06). Using these results, we developed a model for predicting the likelihood of developing tolerance in milk and egg allergy based on the decrease in food sIgE over time. Conclusion We found that the rate of decrease in food sIgE levels over time was predictive for the likelihood of developing tolerance in milk and egg allergy. Using the likelihood estimates from this study could aid clinicians in providing prognostic information and in timing subsequent food challenges, thereby decreasing the number of premature and unnecessary double-blind, placebo-controlled food challenges

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[340] - Sampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol 2001;107:891-896

BACKGROUND: The double-blind, placebo-controlled food challenge is considered the gold standard for diagnosing food allergy. However, in a retrospective analysis of children and adolescents with atopic dermatitis and food allergy, discrete food-specific IgE concentrations were established that could predict clinical reactivity to egg, milk, peanut, and fish with greater than 95% certainty. OBJECTIVE: The purpose of this investigation was to determine the utility of these 95% predictive decision points in a prospective evaluation of food allergy. METHODS: Sera from 100 consecutive children and adolescents referred for evaluation of food allergy were analyzed for specific IgE antibodies to egg, milk, peanut, soy, wheat, and fish by using the Pharmacia CAP System FEIA. Food-specific IgE values were compared with history and the results of skin prick tests and food challenges to determine the efficacy of previously established 95% predictive decision points in identifying patients with increased probability of reacting during a specific food challenge. RESULTS: One hundred children (62% male; median age, 3.8 years; range, 0.4-14.3 years) were evaluated for food allergy. The diagnosis of food allergy was established by means of history or oral food challenge. On the basis of the previously established 95% predictive decision points for egg, milk, peanut, and fish allergy, greater than 95% of food allergies diagnosed in this prospective study were correctly identified by quantifying serum food-specific IgE concentrations. CONCLUSION: In a prospective study of children and adolescents referred for evaluation of food allergy, previously established 95% predictive decision points of food-specific IgE antibody concentrations for 4 major food allergens were effective in predicting clinical reactivity. Quantification of food-specific IgE is a useful test for diagnosing symptomatic allergy to egg, milk, peanut, and fish in the pediatric population and could eliminate the need to perform double-blind, placebo-controlled food challenges in a significant number of children.

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[341] - Perry TT, Matsui EC, Conover-Walker MK, Wood RA. The relationship of allergen-specific IgE levels and oral food challenge outcome. J Allergy Clin Immunol 2004;114:144-149

Background Oral food challenges remain the gold standard for the diagnosis of food allergy. However, clear clinical and laboratory guidelines have not been firmly established to determine when oral challenges should be performed. Objective : We sought to determine the value of food-specific IgE levels in predicting challenge outcome. Method s : A retrospective chart review of 604 food challenges in 391 children was performed. All children had food-specific IgE levels measured by means of CAP-RAST before challenge. Data were analyzed to determine the relationship between food-specific IgE levels and challenge outcome, as well as the relationship between other clinical parameters and challenge outcome. Result s : Forty-five percent of milk challenges were passed compared with 57% for egg, 59% for peanut, 67% for wheat, and 72% for soy. Specific IgE levels were higher among patients who failed challenges than among those who passed (P .03 for each food). When seeking a specific IgE level at which a 50% pass rate could be expected, a cutoff level of 2 kUA/L was determined for milk, egg, and peanut. Data were less clear for wheat and soy. Coexistent eczema or asthma was associated with failed egg challenges, but other atopic disease was otherwise not associated with challenge outcome. Conclusions : Allergen-specific IgE concentrations to milk, egg, and peanut and, to a lesser extent, wheat and soy serve as useful predictors of challenge outcome and should be considered when selecting patients for oral challenge to these foods.

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[342] - Thong BYH, Hourihane JOB. Monitoring of IgE-mediated food allergy in chidhood. Acta Paediatr 2004;93:759-764

"BACKGROUND: The prevalence of IgE-mediated food allergy (FA) in childhood varies from 6% to 8% in the first year of life compared to 1% to 2% in adults. In contrast to adults, FA in childhood, often part of the ""allergic march"", resolves in more than 85% of children, especially those with hypersensitivity to cow's milk and egg. AIM: This paper explains the rationale for continuing care for childhood FA and describes how children should be monitored for resolution/persistence of FA . METHODS: A clinical, multidisciplinary approach and management algorithm based on relevant, peer-reviewed original research articles and reviews using the keywords anaphylaxis, atopic eczema, children, milk allergy, double-blind placebo-controlled food challenge, egg allergy, epinephrine, failure to thrive, food allergy, food challenge, food hypersensitivity, immunoglobulin E, nutrition, natural history, paediatrics, peanut allergy, prevalence, psychosocial factors, quality of life, radioallergosorbent test, and tolerance from years 1966 to 2003 in MEDLINE. Additional studies were identified from article reference lists . RESULTS: A combination of outcome measures, a multidisciplinary approach involving a dietitian and allergy nurse specialist, and a management algorithm are useful tools in clinical management . CONCLUSIONS: Prospective studies of non-selected children, optimally from birth cohorts, are needed to evaluate the effects of such management programmes regarding FA in childhood."

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[343] - Martorell A, García Ara MC, Plaza AM, Boné J, Nevot S, Echeverria L, et al. The predictive value of specific immunoglobulin E levels in serum for the outcome of the development of tolerance in cow's milk allergy. Allergol Immunopathol (Madr) 2008;36:325-330

Background: Immunoglobulin E-mediated allergy to cow's milk protein (CMP) tends to subside over years of follow-up. The gold standard for detecting such allergy has been the oral challenge test. The development of some other test for determining the correct timing of the oral challenge test would avoid unnecessary patient discomfort. The aim of this study was to determine whether monitoring cow's milk (CM) specific IgE levels over time can be used as a predictor for determining when patients develop clinical tolerance. Methods: A prospective 4-year follow-up study was made of 170 patients with IgE-mediated allergy to CMP, involving periodic evaluations (12, 18, 24, 36 and 48 months) with the determination of casein and CM specific IgE on each visit, along with CM challenge testing. ROC curves were used to analyse the sensitivity, specificity and predictive values of the casein and CM specific IgE levels versus the challenge test outcomes at the different moments of follow-up. Results: In the course of follow-up, 140 infants (82 %) became tolerant. Specific IgE levels to CM: 2.58, 2.5, 2.7, 2.26, 5 kUA/l and to casein: 0.97, 1.22, 3, 2.39, 2.73 kUA/l, respectively, predicted clinical reactivity (greatest diagnostic efficiency values) at the different analysed moments of follow-up (12, 18, 24, 36 and 48 months). Conclusions: Quantification of CMP specific IgE is a useful test for diagnosing symptomatic allergy to CM in the paediatric population, and could eliminate the need to perform oral challenges tests in a significant number of children.

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[344] - Vassilopoulou E, Konstantinou G, Kassimos D, Douladiris N, Xepapadaki P, Manoussakis E et al. Reintroduction of cow's milk in milk-allergic children: safety and risk factors. Int Arch Allergy Immunol 2008;146:156-161

BACKGROUND: Although development of clinical tolerance is the rule in allergy to cow's milk (CM), food challenges are required in order to reintroduce CM into the patient's diet. Less 'invasive' procedures able to predict tolerance would be useful as clinical tools. The purpose of this study was to identify potential risk factors for clinical reactivity in CM-allergic children assessed for CM reintroduction . METHODS: One hundred and sixteen open challenges performed in children 10-47 months old with IgE-mediated allergy to CM, in order to reintroduce CM into the diet, were retrospectively evaluated. Specific IgE (sIgE) levels assessed by the CAP System FEIA and skin prick tests (SPT) were obtained at diagnosis and prechallenge. Demographic parameters and measures of sIgE were evaluated as potential predictors of a positive challenge in univariate and multivariate logistic regression models . RESULTS: Twenty-four out of 116 challenges were positive, 9 of which required the use of adrenaline. In order of performance, prechallenge sIgE <3.94 kU/l, the combination of SPT and sIgE or an SPT wheal <4 mm could correctly predict a negative challenge outcome, whereas values of SPT >7.5 mm or sIgE >25.4 kU/l, or their combination, had a high positive predictive value. The presence of atopic dermatitis did not affect the predictive accuracy of these values . CONCLUSIONS: Milk sIgE level prechallenge is a useful predictor of challenge outcome in patients with milk allergy. SPT do not offer an additional predictive value, but can be used when sIgE is not available.

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[345] - Niggemann B, Celik-Bilgili S, Ziegert M, Reibel S, Sommerfeld C, Wahn U. Specific IgE levels do not indicate persistence or transience of food allergy in children with atopic dermatitis. J Investig Allergol Clin Immunol 2004;14:98-103

BACKGROUND: Food allergy in early childhood usually resolves with time; however, little is known about predictors for persistence or transience of food allergy in children with atopic dermatitis. The aim of the study was to evaluate whether specific IgE levels in serum could be a useful predictor of the outcome of oral re-challenges . METHODS: In 74 children, 99 oral food challenges were performed (cow milk n = 48, hen egg n = 37, and wheat n = 14) and repeated after a median time interval of 16 months. In 15 of the 74 children, a third challenge (n = 22) could be performed, with a median time interval from second challenge to third challenge of 15 months . RESULTS: There were 37 children with transient food allergy (positive first challenge and negative second challenge), while 62 children had persistent food allergy (positive first challenge and negative second challenge). Comparison of the two groups showed that specific IgE as well as total IgE in serum was significantly higher in the latter group. However, looking at the time course, specific IgE did not decrease significantly during elimination diet . CONCLUSION: Our results indicate that specific IgE in serum--although very helpful at the time of the first diagnosis--cannot predict whether a chid will become tolerant after a period of avoidance. Therefore, oral re-challenges remain mandatory.

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[346] - Gustafsson A, Kacskovics I, Breimer ME, Hammarstrom L, Holgersson J. Carbohydrate phenotyping of human and animal milk glycoproteins. Glycoconj J 2005;22:109-118

Breast-milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. The plethora of carbohydrate epitopes in breast-milk is known to differ between species, with human milk expressing the most complex one. We have investigated the expression of protein-bound carbohydrate epitopes in milk from man, cow, goat, sheep, pig, horse, dromedary and rabbit. Proteins were separated by SDS-PAGE and the presence of carbohydrate epitopes on milk proteins were analysed by Western blotting using different lectins and carbohydrate-specific antibodies. We show that ABH, Lewis (Le)x, sialyl-Lex, Lea, sialyl-Lea and Leb carbohydrate epitopes are expressed mainly on man, pig and horse milk proteins. The blood group precursor structure H type 1 is expressed in all species investigated, while only pig, dromedary and rabbit milk proteins carry H type 2 epitopes. These epitopes are receptors for Helicobacter pylori (Leb and sialyl-Lex), enteropathogenic (H type 1, Lea and Lex) and enterotoxic Escherichia coli (heat-stable toxin; H type 1 and 2), and Campylobacter jejuni (H type 2). Thus, milk from these animals or their genetically modified descendants could have a therapeutic effect by inhibiting pathogen colonization and infection.

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[347] - Holland JW, Deeth HC, Alewood PF. Analysis of O-glycosylation site occupancy in bovine kappa-casein glycoforms separated by two-dimensional gel electrophoresis. Proteomics 2005;5:990-1002

The ability of two-dimensional gel electrophoresis (2-DE) to separate glycoproteins was exploited to separate distinct glycoforms of kappa-casein that differed only in the number of O-glycans that were attached. To determine where the glycans were attached, the individual glycoforms were digested in-gel with pepsin and the released glycopeptides were identified from characteristic sugar ions in the tandem mass spectrometry (MS) spectra. The O-glycosylation sites were identified by tandem MS after replacement of the glycans with ammonia / aminoethanethiol. The results showed that glycans were not randomly distributed among the five potential glycosylation sites in kappa-casein. Rather, glycosylation of the monoglycoform could only be detected at a single site, T152. Similarly the diglycoform appeared to be modified exclusively at T152 and T163, while the triglycoform was modified at T152, T163 and T154. While low levels of glycosylation at other sites cannot be excluded the hierarchy of site occupation between glycoforms was clearly evident and argues for an ordered addition of glycans to the protein. Since all five potential O-glycosylation sites can be glycosylated in vivo, it would appear that certain sites remain latent until other sites are occupied. The determination of glycosylation site occupancy in individual glycoforms separated by 2-DE revealed a distinct pattern of in vivo glycosylation that has not been recognized previously.

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[348] - Gustafsson A, Kacskovics I, Breimer ME, Hammarstrom L, Holgersson J. Carbohydrate phenotyping of human and animal milk glycoproteins. Glycoconj J 2005;22:109-118

Breast-milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. The plethora of carbohydrate epitopes in breast-milk is known to differ between species, with human milk expressing the most complex one. We have investigated the expression of protein-bound carbohydrate epitopes in milk from man, cow, goat, sheep, pig, horse, dromedary and rabbit. Proteins were separated by SDS-PAGE and the presence of carbohydrate epitopes on milk proteins were analysed by Western blotting using different lectins and carbohydrate-specific antibodies. We show that ABH, Lewis (Le)x, sialyl-Lex, Lea, sialyl-Lea and Leb carbohydrate epitopes are expressed mainly on man, pig and horse milk proteins. The blood group precursor structure H type 1 is expressed in all species investigated, while only pig, dromedary and rabbit milk proteins carry H type 2 epitopes. These epitopes are receptors for Helicobacter pylori (Leb and sialyl-Lex), enteropathogenic (H type 1, Lea and Lex) and enterotoxic Escherichia coli (heat-stable toxin; H type 1 and 2), and Campylobacter jejuni (H type 2). Thus, milk from these animals or their genetically modified descendants could have a therapeutic effect by inhibiting pathogen colonization and infection.

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[349] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[350] - Pizzano R, Nicolai MA, Manzo C, Giannattasio M, Addeo F. Human IgE Binding to the Glycosidic Moiety of Bovine kappa-Casein. J Agric Food Chem 2005;53:7971-7975

IgE ability for recognizing milk proteins was assayed in the serum of an adult atopic patient who outgrew cow milk allergy in early childhood. A number of protein species included in casein from bovine milk were detected by human IgE in immunoblotting experiments. Comparing these results with those obtained from an analysis using antibody preparations specifically directed toward the different casein fractions, IgE-reactive bands were identified as isoforms of kappa-casein. IgE-reactive protein was not present in neither bovine cheese, regardless of cheese-making technology and time ripening, nor milk from any other dairy animal, such as ewe, goat, and water buffalo. Chemical deglycosylation of protein bands immobilized onto nitrocellulose proved that the glycosidic moiety of bovine kappa-casein was principally involved in IgE recognition.

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[351] - Commins SP, Satinover SM, Hosen J, Mozena J, Borish L, Lewis BD et al. Delayed anaphylaxis, angioedema, or urticaria after consumption of red meat in patients with IgE antibodies specific for galactose-alpha-1,3-galactose. J Allergy Clin Immunol 2009;123:426-433

Carbohydrate moieties are frequently encountered in food and can elicit IgE responses, the clinical significance of which has been unclear. Recent work, however, has shown that IgE antibodies to galactose-alpha-1,3-galactose (alpha-gal), a carbohydrate commonly expressed on nonprimate mammalian proteins, are capable of eliciting serious, even fatal, reactions. This study sought to determine whether IgE antibodies to alpha-gal are present in sera from patients who report anaphylaxis or urticaria after eating beef, pork, or lamb. Twenty-four patients with IgE antibodies to alpha-gal were identified. These patients described a similar history of anaphylaxis, angiodema or urticaria 3 to 6 hours after the ingestion of meat and reported fewer or no episodes when following an avoidance diet. SPTs to mammalian meat produced wheals of usually less than 4 mm, whereas intradermal or fresh-food SPTs provided larger and more consistent wheal responses. CAP-RAST testing revealed specific IgE antibodies to beef, pork, lamb, cow's milk, cat, and dog but not turkey, chicken, or fish. Absorption experiments indicated that this pattern of sensitivity was explained by an IgE antibody specific for alpha-gal.

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