The present study shows that CMA infants possess specific IgE antibodies that recognize endogenous human milk proteins. We show that the presence of IgE specific to endogenous human milk proteins could be due to a high degree of sequence homology between the known bovine milk epitopes and the corresponding amino acid sequence on human milk proteins. These human milk-specific IgE antibodies were capable of mediator release in a functional assay in infants with high cow’s milk-specific IgE levels. Such functional studies are necessary, although not sufficient, to support the presence of clinical reactivity. There was only a trend towards detection of a polyclonal IgE response to human milk peptides in those infants who continued to have symptoms despite a maternal cow’s milk avoidance diet. However, a few such infants exhibited remarkably strong IgE binding to human milk peptides together with evidence that such autoreactive antibodies had functional properties in vitro. Our findings suggest that these antibodies could be clinically relevant in breastfed infants whose symptoms (eczema) cleared only after weaning. Similar antibodies were incapable of inducing mediator release (suggesting low-affinity antibodies) in infants who successfully responded to maternal milk restriction. Consequently, the role of these IgE antibodies in infants responding to maternal restriction and in older children with CMA remains unclear.
Significant clinical cross-reactivity exists between milk from ruminants such as cow, sheep, and goat, and to lesser extent between cow’s milk and mare’s or donkey’s milk, which is based on a high degree of amino acid sequence homology between these proteins [4
]. Similarly, based on the homology between certain cow’s milk and human milk proteins, previous research has suggested that cross-reactivity could exist [16
]. Clinical reactivity to human milk proteins was demonstrated in a case report describing an IgE-mediated allergic reaction in a male to human milk, but not cow’s milk, which was found to be due to specific IgE antibodies to human, but not bovine ALA [20
]. This case raises the possibility of a primary sensitization to human milk proteins. In a study by Schulmeister et al. [21
], CMA subjects demonstrated IgE reactivity to cross-reactive as well as non-cross-reactive human milk proteins, and immediate type skin reactions were elicited using SPT with both native and boiled human milk. However, the clinical significance of these findings remains unclear. In the present study, we found that while some infants had specific IgE antibodies that recognize both bovine milk and the corresponding human milk peptides, demonstrating cross-reactivity or co-sensitization, some infants’ IgE only recognized the endogenous human milk peptides suggesting primary sensitization to human milk proteins. On the other hand, binding to human milk peptides seemed equally inhibited in part by pre-incubation with either bovine or human ALA. This may indicate that the majority of IgE autoantibodies to human ALA were due to cross-reactivity with bovine ALA. It is also possible that full inhibition was not seen due to an insufficient amount of protein used in the inhibition assay. However, the R&A procedure poses limits to the maximum concentration of protein that can be processed. Unfortunately, due to small volumes of serum attained from infants, inhibition studies using their sera were not possible.
Prior studies have demonstrated that some patients with atopic dermatitis have IgE auto-antibodies to human proteins, such as manganese superoxide dismutase and calcium-binding proteins, suggesting auto-reactivity as a pathogenic mechanism in atopic dermatitis [22
]. About 25% of adults with atopic dermatitis have IgE antibodies against self proteins [25
]. IgE auto-antibodies can also be detected in patients with atopic dermatitis by their first birthday. These infants had higher total IgE levels and frequently were sensitized to food allergens [27
]. It has been postulated that these IgE auto-antibodies may also be involved in the persistence of symptoms even when the initial inciting environmental allergens are absent [22
]. Many of the patients with CMA in our study manifested symptoms of atopic dermatitis, and our findings raise the possibility that human ALA may represent a novel auto-antigen in infants with atopic dermatitis and CMA. Similar to the IgE auto-antibodies previously found to be associated with atopic dermatitis, human ALA may be responsible for the persistence of atopic dermatitis in milk-allergic infants who continue to have symptoms despite maternal avoidance of exogenous cow’s milk antigens. Further studies are needed to confirm this possibility.
The mechanism(s) responsible for the generation of such IgE auto-antibodies is unclear. Medullary thymic epithelial cells (mTECs) express a highly diverse set of genes representing all tissues of the body (tissue-restricted antigens or TRAs) termed promiscuous gene expression (pGE) [28
]. During the development of central tolerance, these mTECs are responsible for the negative selection of auto-reactive T cells, the development of tolerance to TRAs, and thus the prevention of auto-immune reactions. This development of self-tolerance occurs early during fetal development. Antigens expressed by the mTECs would result in deletion of thymocyte-specific antigen and tolerance of these self proteins. Human ALA is not expressed in the mTECs, thereby allowing the development/presence of human ALA-specific thymocytes potentially leading to auto-reactivity in infants exposed to human ALA via breast milk [28
]. On the other hand, β-casein is expressed in the thymus during mammary gland development, inducing negative selection (deletion) of autoreactive β-casein-specific thymocytes [28
]. However, the developmental delay in T regulatory cells maturation, which has been associated with CMA, may result in auto-reactivity despite adequate deletion of auto-reactive β-casein-specific thymocytes.
One limitation of our study is that we did not perform clinical studies to verify reactivity to human milk in our subjects utilizing either skin testing with human ALA, as reported by Schulmeister et al. [21
], or oral challenges with human milk after feeding with tolerated formula. However, the fact that symptoms subsided only after breastfeeding was discontinued in all six patients who failed to respond to maternal milk elimination further suggests that endogenous milk proteins may have played a role in the persistence of symptoms. Another possible limitation of the study is that the affinity or avidity of monoclonal or polyclonal antibodies to isolated and purified peptides may not reflect the strength of their interaction with the intact proteins.
Strengths of the study include a prospective format and sampling of a population of otherwise rarely encountered young subjects with severe CMA at an early age.
While breastfeeding has many benefits for both infants, in some cases in which the infant continues to display symptoms of CMA despite maternal dietary restrictions, it may be recommended that the mother stops breastfeeding. In rare cases, the continuation of symptoms in these infants may be due to continued passage of maternal dietary allergens into her breast milk or the presence of infant sensitization to endogenous human milk proteins. Further studies with larger sample sizes are needed to confirm these findings.