Milk and egg allergies in children. Immunological changes and mechanisms underlying oral immunotherapys

Resum

Food allergy is a major health problem in the Western countries, notably in children, with an estimated prevalence of 2-8%, and is the most frequent reason for anaphylactic reactions at this age. Between the food allergies, immunoglobulin E (IgE) mediated cow's milk protein allergy (CMPA) and egg allergy are the most common in infants, affecting approximately 1-3% of children. Although these allergies are associated with a high rate of natural tolerance, the mechanisms of tolerance acquisition are not well understood. Moreover, about 50% of children do not overcome their allergy within the first years of life, which rekindle in the increasing incidence of the clinical disorder in adults. These food-allergic processes may endanger children's health causing reactions from mild atopic dermatitis to severe systemic anaphylaxis, which can be life-threatening. Unfortunately, at present, the only treatment for cow's milk (CM) and egg allergies is a complete dietary restriction of implicated foods. Because of milk and egg proteins are included in a wide range of cooked and manufactured foods, avoidance involves a wide dietary restriction, which leads to negative nutritional, social, psychological and economic consequences. However, and despite its importance, little is known about the specific immune mechanisms that could constitute differential factors for the development of allergy or for the maintenance of tolerance. Consequently, new studies to clarify these important issues and novel strategies for immune intervention are currently pursued. Given this scenario, the first research conducted in this thesis compiled an extensive analysis of immune cell subsets and cytokine secreting cells in infants with symptoms compatible with CMPA, which is the first allergy to appear in children. Samples were collected in the 1-4 days after the first adverse reaction, to decipher the immune alterations related with the establishment of this allergy. Interestingly, results revealed that children who developed CMPA had decreased regulatory T cell (Treg) counts and lower serum vitamin D levels. Furthermore, these parameters were statistically correlated and constituted good predictors to distinguish between healthy controls and CM allergic infants. Therefore, they could be crucial factors behind the onset of the allergic process in infants and a therapeutic target for the treatment of this food allergy. Because of oral immunotherapy (OIT) is nowadays one of the most promising approaches toward a treatment for food allergy, further studies to provide more scientific evidence about such treatment are demanded. OIT involves regular and gradually increased dosages of the allergenic protein under medical supervision. The goal is induce desensitization firstly, defined as an increase in the threshold for reactivity but requiring a continued consumption of the allergen to prevent the reappearance of reactivity; and a later induction of oral tolerance, which means a long-lasting unresponsiveness against the food allergen. Pilot studies have yielded promising results, with success ratios frequently higher to 70%. However, differences between protocols employed, and the lack of knowledge about the specific immune mechanisms responsible of the desensitization and tolerance acquisition, prevent from drawing robust conclusion and make difficult the improvement and further development of this therapeutic strategy. Moreover, there is no evidence enough in the biomarkers that reflect the success of the intervention yet, as well as which children could be good candidates for treatment and have a reduced risk of adverse reactions. In this Thesis, the clinical efficacy and immunologic changes associated with OIT for IgE-mediated CMPA and egg allergy in infants were evaluated. The basal immunologic status of the allergic children enrolled was assessed through comparison with those of a non-allergic group of the same age range and sex. Three different OIT schedules were evaluated: i) a rush protocol for egg desensitization based on a first 5-days rapid up dosing, with a rate of success (defined as the ability to eat one undercooked egg) of 93.8% of patients in 5 months of intervention; ii) a long-course regimen for egg desensitization, which allowed 60% children to eat the equivalent to a full egg (≥ 32 mL of pasteurized egg white) in an average period of 11.75 months; iii) a long-course OIT protocol for CM desensitization with 70% success (≥ 200 mL of CM) and, an average duration of 18.9 months. A distinct feature of the long protocols is the progressive introduction of egg or milk-containing foods into the patient’s diet. Moreover, the long-term efficacy of desensitization of these protocols was evaluated by the Allergy services in Hospital 24-48 months after being completed, reporting that 70-75% of the allergic children participants were consuming egg or CM as a part of their diet (data not published). Analysis of immunological outcomes underlying OIT treatments showed a decrease in serum allergen-specific IgE levels along the therapy, accompanied by a rise in the allergen-specific IgG4. Because the mechanisms by which OIT acts include modulation of T-cell responses, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and stimulated with ovalbumin (OVA) or β-casein (β-CN) for measuring T helper 2 (Th2), T helper 1 (Th1) and regulatory T (Treg) cells cytokine profile, as well as the expression of the master transcription factors of the corresponding T-cell differentiation (GATA3, T-bet and FoxP3). Results revealed a diminished allergic Th2 response in children successfully desensitized, with lower specific interleukin (IL)-13 and IL-5 production. Gene expression differences were not large enough to consider a significant change in either of the transcription factors studied. Higher baseline antigen-specific IgE levels are proposed predictors of a negative clinical response to OIT. In summary, main findings in this thesis highlighted that circulating Treg cells and serum vitamin D levels could be crucial factors behind the establishment of CMPA in infants. Oral rush immunotherapy protocols could be highly effective in inducing desensitization to egg proteins in few days with a long-term protection. Successful desensitization resulted in significant reductions in antigen-specific IgE with increases in antigen-specific IgG4 and a drop in Th2 cytokines associated with allergic processe.