This is a 12-month, prospective, randomized interventional study designed to evaluate immunological tolerance development in young children with confirmed cow's milk or egg allergy. The primary endpoint is the acquisition of clinical tolerance to raw or minimally processed forms of the allergen after structured dietary exposure to baked forms. The study integrates immunologic, clinical, and laboratory evaluations to assess the therapeutic potential and immunomodulatory effects of gradual allergen introduction.
Study Population Inclusion Criteria Age: 12 to 72 months (1 to 6 years). Milk Allergy Subgroup: Confirmed diagnosis based on at least one of the following: suggestive clinical history, positive skin prick test (SPT) ≥3 mm over negative control and/or specific IgE ≥0.35 kU/L to cow's milk or casein.
Egg Allergy Subgroup: Either: suggestive clinical history and sensitization (SPT or sIgE ≥0.35 kU/L to egg white or ovomucoid).
Exclusion Criteria Inability to obtain written informed consent. Any contraindication to epinephrine use (e.g., uncorrected congenital heart disease).
Ongoing consumption of baked milk or egg ≥3 times/week within the last 3 months.
Baseline Procedures and Immune Profiling (T0)
All participants will undergo:
Supervised Oral Food Challenge (OFC) with a standardized baked milk or egg product (e.g., muffin containing measured protein quantity), carried out under hospital supervision with immediate access to emergency care.
Blood Sampling for:
Quantitative allergen-specific IgE, IgG4, IgA to milk/egg and their major components (e.g., casein, ovomucoid).
Flow cytometry for T-cell phenotyping (CD4⁺CD25⁺FoxP3⁺ regulatory T cells). Cytokine profiling via ELISA or multiplex immunoassay: IL-4, IL-5, IL-10, IL-13, TGF-β.
Basophil Activation Test (BAT) using CD63/CD203c expression upon allergen stimulation.
Skin Prick Testing with commercial milk/egg extracts and baked food extracts, if available.
Randomization and Treatment Arms
Participants are stratified based on baked food OFC outcome:
A. OFC-Positive to Baked Product
Randomized 1:1 to:
Baked Group 1 (Low-dose exposure): Daily ingestion of baked product containing fixed dose (e.g., 0.0375 g milk protein or 0.11 g egg protein).
Control Group 1 (Strict avoidance): Elimination of all milk/egg and derivatives.
Non-tolerant to minimum baked dose: Excluded from active intervention; followed as observational cohort with repeated testing at 12 months.
B. OFC-Negative to Baked Product Progress to OFC with pasteurized milk or cooked egg. If tolerant → study exit.
If reactive, randomization into:
Baked Group 2 (High-dose exposure): Daily ingestion of baked food containing higher protein load (e.g., 0.55 g milk protein or 1.1 g egg protein).
Control Group 2 (Strict avoidance).
All OFCs use a standardized 6-step titration protocol with cumulative dosing and observation for ≥2 hours post-ingestion.
Dose Escalation and Monitoring After 6 months: Hospital-based supervised dose escalation (doubling daily intake) for Baked Groups 1 and 2.
Final Assessment (T1 - 12 months) Repeat OFC with unheated (raw) milk or cooked/raw egg. Repeat blood testing, SPT, and BAT.
Comparative analysis between baseline and T1 to determine:
Acquisition of tolerance (negative OFC). Immune modulation (e.g., shifts in IgE/IgG4 ratios, Treg expansion, cytokine profile normalization).
Predictive value of baseline biomarkers for outcome stratification.
This detailed protocol structure enables reproducibility, comparability, and the possibility of identifying immunological biomarkers that correlate with successful oral immunotolerance induction through gradual dietary exposure to baked allergens.
