Study subjects

We evaluated IgE-mediated CMA children (aged 3 to 18 months) consecutively referred to our tertiary Pediatric Allergy Center for oral food challenge. Oral food challenge was requested to obtain a diagnosis because of recent evidence of CMA signs and symptoms (“active CMA patients”; group 1) or to investigate the occurrence of oral tolerance acquisition after dietary treatment with an extensively hydrolyzed casein formula containing the probiotic Lactobacillus rhamnosus GG (LGG, 1 × 10⁶/ml CFU) (“subjects who outgrew CMA with extensively hydrolyzed casein formula (EHCF) + LGG”; group 2) or with other formulas (“subjects who outgrew CMA with other formulas”; group 3). All patients underwent a double-blind placebo-controlled oral challenge (DBPCFC), as described previously [20]. All oral food challenges took place at our Center on two separate days with a 1-week interval. Parents of children taking antihistamine were advised to withhold these medications for at least 72 h before and during the challenge. Randomization and preparation of the challenges were performed by experienced food allergy dieticians not directly involved in the procedures. Briefly, every 20 min, successive doses (0.1, 0.3, 1, 3, 10, 30, and 100 ml) of fresh pasteurized cow’s milk containing 3.5 % fat (verum) or hypoallernic or soy formula that the child was already consuming (placebo) were administered. Full emergency equipment and medications (epinephrine, antihistamines, and steroids) were available. The results were assessed simultaneously by three experienced pediatric allergists. Study subjects were scored for nine items divided into four main categories: (i) general (lowered blood pressure plus tachycardia); (ii) skin (rash, urticaria/angioedema); (iii) gastrointestinal (nausea/repeated vomiting, crampy-like abdominal pain, diarrhea); and (iv) respiratory (sneezing/itching, nasal congestion/rhinorrhea, stridor deriving from upper airway obstruction or wheezing) on a 0- to 3-point scale (0, none; 1, light; 2, moderate; and 3, severe). If at least two of the three physicians independently scored any item at level 3, or 2 (or more) items at level 2, the test result was considered positive. Clinical symptoms occurring within 2 h of administering the highest dose were defined as “IgE-mediated reactions.” The infants were observed for 2 h after the final dose and then discharged. In the case of a positive DBPCFC, at any testing dose, the patient remained under observation until symptom resolution. If the patient did not show any symptom within the first 24 h, parents were advised to give one single feed of 100 ml of the tested formula (verum or placebo) everyday at home for 7 days. If any symptom occurred during this period, the patient returned to the outpatient clinic on the same day. After 7 days of verum or placebo administration, the patients were examined and the parents interviewed at the Center. To rule out a false-negative challenge result, parents were asked to contact the Center if any symptoms occurred in the 7 days after the DBPCFC procedures. The challenge was considered negative if the patient tolerated the entire challenge, including the observation period. Clinical tolerance acquisition was defined by the presence of a negative DBPCFC. Patients with a negative oral challenge (groups 2 and 3) were reassessed 4 weeks later to verify persistence of clinical tolerance. A venous blood sample (4 ml) was obtained from all patients after oral challenge.

Exclusion criteria were as follows: allergic disorders or food allergies other than CMA, eosinophilic disorders of the gastrointestinal tract, food protein-induced enterocolitis syndrome, concomitant chronic systemic diseases, congenital cardiac defects, active tuberculosis, autoimmune diseases, immunodeficiency, chronic inflammatory bowel diseases, celiac disease, cystic fibrosis, metabolic diseases, lactose intolerance, malignancy, chronic pulmonary diseases, and malformations of the gastrointestinal tract. During the same study period, consecutive healthy children, not at risk of atopic disorders (namely, those without a first-degree relative affected by an atopic disorder), attending our Center because of minimal surgical procedures served as a control group (group 4).

A venous blood sample (4 ml) was collected also from these healthy subjects. They were assessed for the presence of food allergy and other allergic diseases at enrollment and 6 months after blood sampling by pediatric allergists at our Center.

Total IgE and specific IgE against proteins and epitopes of cow’s milk

Serum was obtained by centrifugation for 10 to 15 min. Serum was flash frozen and stored at −80 °C until analysis. Serum total IgE and specific IgE against epitopes of cow’s milk (alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, casein, lactoferrin) were analyzed by enzymatic immunoassay (Phadia 100 ThermoFisher Scientific CAP system, Rodano Milano, Italy). Results were expressed as kilounits per liter (kU/l).

DNA methylation and mRNA expression

Peripheral blood mononuclear cells were isolated from whole blood samples using the Ficoll-Paque (Sigma-Aldrich, St. Louis, MO, USA) method, as described previously [2]. The primers used for DNA methylation analysis of FoxP3 TSDR are reported elsewhere [15]. High-resolution melting real-time PCR for methylation analysis was performed as described previously [2]. The results of methylation analysis were verified by direct sequencing (using the Sanger method modified as follows: ddNTPs labeled with four different fluorophores) and analyzed by capillary electrophoresis (the analytical specificity and sensitivity of the test was >99 %). Real-time PCR was performed with the LightCycler® 480 instrument (Roche Applied Science, Penzberg, Germany) using 96-well plates (Roche Applied Science). Briefly, RNA was extracted from the PBMCs of the four study groups using the Trizol protocol (Invitrogen, Life Technologies Europe BV, Monza, Italy), as previously described [2]. The concentration and purity of RNA samples were measured and verified by NanoDrop 1000 spectrophotometry (Thermo Scientific, Wilmington, DE, USA). For complementary (cDNA) synthesis, 1 μg total RNA was transcribed with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The 10-μl reaction volumes contained 1 μl template, 10 μl SYBR Green (Applied Biosystems), and 5 μM primers (FoxP3 forward primer 5′-AGCTGGAGTTCCGCAAGAAAC-3′; FoxP3 reverse primer 5′-TGTTCGTCCATCCTCCTTTCC-3′; GenBank Accession number NC_000023.11). Quantitative real-time amplifications were performed in triplicate with an initial incubation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s, using a Light Cycler 79 HT (Applied Biosystems). The quantitative gene expression was calculated with the comparative Ct method and normalized against the Ct of glucuronidase (GUS) messenger as reference gene.

Statistical analysis

The Kolmogorov-Smirnov test was used to determine whether variables were normally distributed. The χ ² test and Fisher’s exact test were used for categorical variables. We used the t test and one-way ANOVA to evaluate differences among continuous variables. To determine which groups in the sample differ, the Bonferroni correction was performed. Pearson’s correlation coefficient “r” was used to evaluate the correlation between continuous variables. The level of significance for all statistical tests was two-sided, P < 0.05. All analyses were conducted by a statistician blinded to patient group assignment, using SPSS, version 19.0 for Windows (SPSS Inc., Chicago, IL, USA).