The New Perspective of Pathophysiology and Pathogenesis Cow Milk Allergy

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 (αs1-, αs2-, β-, and κ-casein; α-lactalbumin; and β-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. Cow’s milk is a significant cause of food allergy in early childhood. The mechanisms leading to persistent (and transient) CMA are unknown.

Cow’s milk is one of the most common causes of food allergy in the first years of life, with approximately 2% to 2.5% of newborns experiencing allergic reactions to cow’s milk during this time. Symptoms of cow’s milk allergy are non-specific; as a result, suspected cow’s milk allergy is far more common than proven allergy to cow’s milk. Cow’s milk allergy in infants is therefore most probably a fairly uncommon clinical picture; cow’s milk allergy is estimated to occur in less than one per cent of infants. The only valuable additional diagnostic tool is food challenge, preferably double blind. Therapy consists of a formula free of cow’s milk (preferably containing extensively hydrolysed whey protein) from the moment the mother ceases nursing her child until the age of 6-12 months. Solids can be introduced in the usual fashion; there is no scientific basis for introducing them in a step by step fashion. Prevention of cow’s milk allergy by using hypoallergenic formula (partially hydrolysed cow milk protein) in the first year of life has been shown to be unsuccessful, and can no longer be recommended. In the future, oral immunotherapy may be a promising new treatment for cow’s milk allergy.

Caseins account for about 80% of the total protein content in cow’s milk, whereas whey proteins comprise the rest. Casein includes 4 protein fractions, αs1-, αs2-, β-, and κ-casein, comprising 32%, 10%, 28%, and 10% of the total milk protein, respectively. In solution, different caseins form complexes and ordered aggregates (ie, micelles). These globular complexes are composed of a peripheral hydrophilic layer and a hydrophobic core. In the core, caseins are assembled by means of intermolecular interactions between the colloidal calcium phosphate and the phosphoserine groups of the αs1-, αs2-, and β-caseins, whereas the C-terminal polar fragment of the κ-casein and the polar domains of the other caseins are exposed at the periphery.  In addition, αs2– and κ-casein both contain one disulfide bridge per molecule. Whey fraction contains essentially globular proteins, α-lactalbumin and β-lactoglobulin, containing 4 and 2 disulfide bridges and comprising 5% and 10% of total milk protein, respectively.

The relative allergenicity of each cow’s milk protein remains unclear, although data from recent studies have emphasized the importance of the caseins as major milk allergens, and significant reactivity to the whey proteins (α-lactalbumin and β-lactoglobulin) was also noted. All these proteins share little primary structural homology.  Recently some study mapped the major IgE- and IgG-binding epitopes on αs1-, αs2-, β-, and κ-casein; α-lactalbumin; and β-lactoglobulin

Accurate diagnosis of CMA is challenging and essential. The determination of the immunoglobulin E (IgE)-mediated response to sequenced and characterized allergens may be more useful in predicting the presence and severity of clinical allergy than the currently used skin or blood tests performed with whole extracts. However, as component recognition pattern heterogeneity is observed in different areas, further clinical studies are essential to correlate useful molecular diagnostics and biological markers with disease and patient profiles. Until such markers are found and validated in different age groups, oral food challenge remains the reference standard for the diagnosis of CMA.

Pathogenesis of CMA: IgE and non–IgE mediated

CMA presents in 3 clusters of immune mechanisms. The IgE-mediated forms are characterized by acute onset and involve 1 or more target organs, such as the skin (urticaria and angioedema), the respiratory system (rhinoconjunctivitis and asthma), and the gastrointestinal tract (nausea, vomiting, and diarrhea). The cell-mediated, non-IgE forms are of delayed or chronic onset, with enterocolitis and proctocolitis as frequent clinical presentations. A “mixed” IgE and non-IgE setting, also with a delayed or chronic onset, might present as atopic dermatitis (AD) or as one of the eosinophilic gastroenteropathies (CMA phenotypes).

Immune mechanisms and Clinical manifestations of CMA

Gastrointestinal reactions

  • Oral allergy syndrome (rare in pediatric patients)
  • Lip swelling is a commonly observed manifestation during food challenge procedures.
Immediate gastrointestinal allergy
  • Vomiting (described in children both isolated and as part of allergic/anaphylactic reactions)
  • Diarrhea (usually in, but not limited to, delayed reactions)
CMA in short bowel syndrome
  • Greater than 50% of these patients are also allergic to cow’s milk, according to 1 case study.

IgE-mediated respiratory reactions

  • Rhinitis occurs in ±70% of patients during oral cow’s milk challenge, and asthma occurs in less than 8%.
  • Reactions rarely occur in isolation
  • Reactions correlate with severe CMA.
  • Asthma makes for the worst prognosis in children with anaphylaxis.
  • Asthma in patients with CMA is of particular severity.
  • Respiratory symptoms in patients with CMA can progress to respiratory allergy.
  • Inhalation of milk vapor has been associated with severe respiratory tract reactions.

IgE-mediated skin reactions

Acute urticaria or angioedema
  • Urticaria is a feature of most anaphylactic reactions to cow’s milk.
  • Urticaria with inhalation63 or accidental skin contactis often severe.
Contact urticaria
  • Pattern varies from irritant to allergic contact dermatitis.
  • Generalized eczematous rash (systemic contact dermatitis) is present.
  • Contact reactions are frequent in patients with AD.

Late-onset reactions

  • Symptoms not IgE mediated
  • Mostly localized in the gastrointestinal tract
  • Typically develop 1 to several hours or even days after ingestion
  • No reliable laboratory tests to diagnose late-onset CMA: IgE test results are negative

B-cell epitopes and persistent cow’s milk allergy

The presence of IgE antibodies against certain sequential epitopes on various milk proteins is associated with persistence of CMA. Combination of these epitopes might be used in identifying the patients who will have life-long CMA.

Previous studies looking for a marker to predict whether children will remain allergic or outgrow CMA have investigated IgE and IgG antibody levels against whole cow’s milk and cow’s milk protein fractions. These studies have suggested that children with long-lasting CMA possess higher levels of total and cow’s milk-specific IgE antibodies,  than those who became tolerant. In contrast, James and Sampson found that initial milk-specific IgE antibody concentrations were not significantly lower in the patients ultimately becoming tolerant, but final casein and β-lactoglobulin-specific IgE levels were significantly lower. Significantly higher levels of IgG antibodies against certain milk proteins have been reported in children with persistent CMA and adult milk-intolerant patients. Finally, James and Sampson observed that casein- and β-lactoglobulin-specific IgE/IgG ratios were significantly lower in children who lost milk reactivity, suggesting that monitoring those parameters might help in predicting the outcome of CMA. At this time there are no specific IgE levels that have been demonstrated to be highly predictive of the development of clinical tolerance in children with milk allergy. In the present study we identified IgE specificity to informative allergenic B-cell epitopes as a screening instrument for persistent CMA. It should be noted, however, that the quantity of milk-specific IgE antibody correlated with OD measurements of binding to the informative epitopes (Spearman rank correlation test). This is in part explained by the fact that the epitopes used as informative epitopes had to be recognized by greater than 50% of the persistent patients and 0% of the patients outgrowing CMA. Our data suggest that measurement of epitope-specific IgE might provide a more accurate tool for predicting the outcome of CMA, but further studies in bigger populations will be needed to confirm the present data

Caseins are not significantly affected by heating but are very susceptible to many proteinases and exopeptidases, resulting in a great deal of modification after ingestion. In comparison, β-lactoglobulin is relatively resistant to acidic pH values and to proteolytic enzymes, leaving its structure relatively unchanged during digestion and possibly allowing passage of intact protein into the circulation. Consistent with this, the major IgE-binding regions, when projected onto the 3-dimensional structures of β-lactoglobulin, turned out to be located on the surface of the molecule, suggesting that the major IgE-binding sites of β-lactoglobulin are mainly conformational structures. The same applied to α-lactalbumin. However, IgE-binding epitopes of the whey proteins on the basis of sera from children with long-lasting CMA did not differ from those detected with sera of patients with transient CMA, indicating that only the sequential epitopes of the caseins are associated with the persistence of CMA. The explanation for this phenomenon was not investigated in the present study but can be speculated about on the basis of the more spatial structure of epitopes of whey proteins. The immaturity of the newborn intestine allows the highest degree of absorption of intact α-lactalbumin and β-lactoglobulin immediately after birth, significantly declining over the next several months of life as the gut permeability decreases. Therefore it might be hypothesized that hypersensitivity to whey proteins, which have mainly conformational epitopes, might attenuate during early childhood and might therefore not play a significant role in persistent CMA.

Understanding the mechanisms leading to persistent CMA is necessary to develop procedures that would interfere with this process. Immunotherapeutic interventions under investigation in our laboratory should be directed at those patients who will not outgrow their CMA, assuming this group of patients can be identified. We have evidence that the epitope-specific IgE antibodies are already present at an early age in patients with persistent CMA, suggesting that such a prediction could be made in the first years of life. Furthermore, longlasting food hypersensitivity is associated with subsequent allergic airway disease, and early differentiation of these patients might affect preventive approaches. Finally, knowledge of the likelihood of outgrowing CMA will be beneficial in counseling patients and directing their treatment because many children with milk and egg allergy are able to tolerate small amounts of these foods in a cooked, but not in a raw, form. During food processing and cooking, the native structure and many conformational epitopes of these proteins are modified or disrupted by heat, chemical treatments, or both, eliminating IgE binding to conformational epitopes and exposing sequential (linear) ones. Therefore a meticulous elimination diet and avoidance of cooked milk products might not be necessary in children who do not recognize sequential epitopes on cow’s milk proteins or who will outgrow their CMA irrespective of their diet.

Some IgE-binding sites on cow’s milk proteins that differentiated between patients with persistent CMA and those with transient CMA. The presence of IgE antibodies against at least 1 of 3 of these epitopes (AA 123-132 on αs1-casein, AA 171-180 on αs2-casein, and AA 155-164 on κ-casein) might be useful as a marker of persistent CMA. Because IgE antibodies to these informative epitopes appear to develop at the time of initial sensitization and seem not to depend on the IgE concentration, this approach provides the advantage of allowing screening at the time of initial diagnosis in infants. Studies are under way linking 3 informative epitopes (peptides) to a commercial matrix to conduct prospective studies in larger patient populations.

Allergen-specific responses of CD19(+)CD5(+)Foxp3(+) regulatory B cells (Bregs) and CD4(+)Foxp3(+) regulatory T cell (Tregs) in immune tolerance of cow milk allergy

Foxp3-expressing cells among CD19(+)CD5(+) B cells were identified as regulatory B cells. Cow Milk Allergy manifesting as late eczematous reactions is regarded as a non-IgE-mediated food allergy. The diagnosis for Cow milk allergy manifesting as late eczematous reactions was made on the basis of the findings obtained from a double-blind placebo-controlled food challenge in patients with atopic dermatitis.

Some patients with milk allergy and patients who could tolerate milk were selected. On casein stimulation, the CD19(+)CD5(+)Foxp3(+) B cell (Breg) fraction in CD5(+) B cells decreased  in the milk allergy group and increased in the milk-tolerant group. On the other hand, on allergen stimulation, the number of CD4(+)Foxp3(+) regulatory T cells (Tregs) in the milk allergy group and milk-tolerant group increased and respectively. Allergen-specific responses of Bregs, rather than those of Tregs, seem to influence the immune responses allergy or tolerance) to a cow milk allergen.

IgG-binding to linear epitopes on buffalo beta-lactoglobulin.

Buffalo milk safety was highlighted with the increase in dietary consumption, and a little information is available on buffalo milk allergy except for cross-reactivity between buffalo and cow milk.

Linear epitopes and critical amino acids of buffalo β-lactoglobulin were defined by 4 rabbit’s sera using SPOTTM peptide arrays approach based on the defined mimotope. The eight epitopes on buffalo β-lactoglobulin were located in the position of A6(21-30), A7(AA25-34), A8 (29-38), B4 (73-82), B5(77-86), C(87-96), F4(134-143) and F8(150-159), respectively. Among them, four epitopes (A7, A8, F4 and F8) were described as the most major epitopes and peptide (A6, B4, B5 and C) as the second major epitopes. Following single AA substitutions (Alanine or Glycine) at each position of the major epitopes, 2,3,2,3,5 and 3 of critical amino acids were identified on epitopes of A6, A8, B5, C , F4 and F8, respectively, which vary in distribution among the epitopes, such as in C terminal or N terminal and in continuous or discontinuous forms, characteristics including hydrophobicity, polar and charge, and existed frequency.


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