Performance of a component‐based allergen‐microarray in the diagnosis of cow's milk and hen's egg allergy

Background The food challenge test (FCT) is the gold standard for the diagnosis of food allergy. This procedure is time consuming, costly and can induce potentially severe symptoms. An ideal in vitro test should allow to avoid the FCT. Objective To assess the clinical performance of microarray for specific IgE (sIgE) detection in children with challenge‐proven/excluded cow's milk (CM) or hen's egg (HE) allergy. Methods One‐hundred and four children with suspected IgE‐mediated hypersensitivity to CM or HE were studied. In all patients, skin prick test, ImmunoCAP, microarray and FCT were performed. Results The microarray components Bos d 8 for CM (27/58 patients) and Gal d 1 (20/46 patients) and Gal d 2 (24/46) for HE were the most frequently recognized allergens. Using the FCT results as the reference parameter, sIgE to Bos d 8 and Gal d 1 had the highest area under the curves. These were not significantly different from those obtained using the ImmunoCAP. Use of 95% clinical decision points (CDP) for sIgE to Bos d 8 and Gal d 1 resulted in higher negative predictive values (78% and 79%, respectively) than those obtained with the ImmunoCAP (57% and 59%). Conclusions Our results show that in children with suspected CM or HE allergy, the microarray has a good ability to predict the FCT results. In a clinical application perspective, the microarray could be used as a second‐level assay, if the ImmunoCAP sIgE is <95% CDP. This approach would lead to a decrease in the number of the FCT to be performed, as well as of positive FCTs with a subsequent decrease in severe reaction risk. Cite this as: L. E. D'Urbano, K. Pellegrino, M. C. Artesani, S. Donnanno, R. Luciano, C. Riccardi, A. E. Tozzi, L. Ravà, F. De Benedetti and G. Cavagni, Clinical & Experimental Allergy, 2010 (40) 1561–1570.     

Impact of dietary regimen on the duration of cow's milk allergy: a random allocation study

Background The impact of diet on cow's milk allergy (CMA) duration and whether exposure to residual amounts of cow's milk protein influences the onset of tolerance are unknown. Objective To prospectively assess the dietary factors influencing disease duration in a randomized cohort. Methods We randomly switched the formula of symptomatic patients from the Milan Cow's Milk Allergy Cohort to one of three treatment groups according to the quarterly rotation of rice hydrolysate formula, extensively hydrolysed cow's milk formula and soy‐based formula. In this intention‐to‐treat, randomized analysis, a hazard ratio (HR) estimation model was used to analyse dietary impact on disease duration. Results Seventy‐two children aged a mean of 14.1±8.6 months at diagnosis were followed up for a median of 26 months. Fifty‐one reached tolerance at a mean of 34.1±15.2 months. The mean duration of disease was 40.2±4.8 months with milk hydrolysate, 24.3±3.6 months with rice and 24.3±2.6 months with soy. Dietary choice independently predicted shorter duration of disease [adjusted HRs 3.09 (P=0.007) for rice, 2.54 (P=0.02) for soy, both against milk hydrolysate]. In 50 children not co‐sensitized to soy, diet choice impacted the duration of disease more strongly [adjusted HRs 8.02 (P=0.006) for rice, 6.53 (P=0.015) for soy, both against milk hydrolysate]. Discussion Patients not exposed to cow's milk protein residue achieve cow's milk tolerance earlier than patients who follow an extensively hydrolysed cow's milk diet. This may be due to residual antigenicity in hydrolysed milks. As the effect of dietary intervention is stronger in patients not sensitized to soy, we infer that when atopic disease has progressed to multiple sensitizations, the elimination of allergenic exposure may not be sufficient to reduce the duration of CMA. Cite this as: L. Terracciano, G. R. Bouygue, T. Sarratud, F. Veglia, A. Martelli and A. Fiocchi, Clinical & Experimental Allergy, 2010 (40) 637–642.     

A protocol for oral desensitization in children with IgE-mediated cow's milk allergy

OBJECTIVES: To desensitize children with severe immunoglobulin (Ig)E-mediated cow's milk allergy in a period of 6 months by introducing increasing daily doses of cow's milk (CM) in order to enable the child to assume 200 ml of CM daily, or to induce tolerance of the highest possible CM dose. STUDY DESIGN: Twenty-one children at least 6 years old with severe IgE-mediated CM allergy were admitted to the study. A convincing history of IgE-mediated CM allergy or a positive double-blind placebo-controlled food challenge with CM confirmed the diagnosis. Oral desensitization was performed with increasing doses starting from 0.06 mg of CM proteins. RESULTS: Overall, 15 of 21 children (71.4%) achieved the daily intake of 200 ml during a 6-month period; three of 21 children (14.3%) tolerated 40-80 ml/day of undiluted CM; three of 21 children (14.3%) failed the desensitization because they presented allergic symptoms after ingesting minimal amounts of diluted CM. Conclusions: We successfully desensitized 15 of 21 children with severe IgE-mediated CM allergy in a period of 6 months. We stress the importance of the partial outcome in those three of 21 children who could not reach the maximum amount of 200 ml/day of whole CM, but were able to tolerate 40-80 ml/day of CM. In this way we dramatically reduced the risk of severe reactions after accidental or unnoticed introduction of low quantities of CM. We do not propose generalizing this method beyond trained staff.     

Biochemical markers of bone metabolism in children with cow's milk allergy

Introduction

Patients with cow''s milk allergy (CMA) and following a cow milk protein-free diet for a long time are potentially at risk of developing bone abnormalities. To assess the balance between bone formation and resorption processes, we determined serum concentrations of osteocalcin (OC), bone alkaline phosphatase (BALP), C-terminal telopeptide of type I collagen (CTX), fetuin-A, osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in children with CMA.

Material and methods

The study included 50 prepubertal children with diagnosed cow''s milk allergy, who were under systematic medical and nutritional care at the Institute of Mother and Child and 40 healthy counterparts as a control group. The concentrations of bone metabolism markers were determined by immunoenzymatic assays.

Results

The diets of all investigated children were correct in terms of phosphorus and magnesium contents but deficient in terms of calcium and vitamin D. Serum OC and CTX as well as fetuin-A concentrations were similar in both studied groups. The BALP activity was significantly (p < 0.05) higher in children with cow''s milk allergy than in the controls. Serum OPG concentration was comparable in both groups, but the RANKL level was higher (p < 0.05) in CMA children than in healthy ones. Hence, the ratio of OPG/RANKL was lower in children with CMA.

Conclusions

Our study demonstrates slight disturbances in the profile of bone metabolism markers in growing children with CMA. The increase in RANKL level and decrease in OPG/RANKL ratio may contribute to intensification of bone resorption in these patients.     

Total serum IgE level influences oral food challenge tests for IgE‐mediated food allergies

Probability curves predicting oral food challenge test (OFC) results based on specific IgE levels are widely used to prevent serious allergic reactions. Although several confounding factors are known to affect probability curves, the main factors that affect OFC outcomes are currently unclear. We hypothesized that an increased total IgE level would reduce allergic reactivity. Medical records of 337 and 266 patients who underwent OFCs for 3.5 g boiled hen's egg white and 3.1 ml raw cow's milk, respectively, were examined retrospectively. We subdivided the patients into three groups based on total IgE levels and age by percentile (<25th, 25–75th, and >75th percentiles), and logistic regression analyses were performed on each group. Patients with higher total IgE levels were significantly less responsive. In addition, age did not significantly affect the OFC results. Therefore, total IgE levels should be taken into account when predicting OFC results based on food‐specific IgE levels.     

Defective tumour necrosis factor-alpha production in mother's milk is related to cow's milk allergy in suckling infants

BACKGROUND: The precise role of leucocytes in human milk is still unresolved. OBJECTIVE: To assist in clarifying the immune mechanisms involved in the development of CMA in suckling infants, we studied the role of immunoregulatory leucocytes and their mediators in human breast milk. METHODS: The study population consisted of 43 lactating mothers and their infants, aged 0.25-8.0 months, followed-up prospectively from birth. Of these mothers, 27 had an infant with challenge-proven cow's milk allergy manifested with either skin (n = 23), gastrointestinal (n = 2) or skin and gastrointestinal symptoms (n = 3). Sixteen mothers with a healthy infant served as controls. We evaluated the spontaneous and mitogen-induced tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) production of human milk leucocytes and isolated peripheral blood lymphocytes in vitro with a commercial ELISA kit. RESULTS: TNFalpha production of breast milk leucocytes was significantly lower in the mothers with a cow's milk-allergic infant, whereas IFNgamma production of these cells was comparable in the two groups. CONCLUSION: Our results suggest that in the breast milk of mothers having an infant with cow's milk allergy, the number and function of TNFalpha-producing cells is defective. This might lead to a disturbance in the development of oral tolerance and thereby to the development of CMA in suckling infants. These novel results may help in clarifying the etiopathogenesis of CMA.     

Combined T regulatory cell and Th2 expression profile identifies children with cow's milk allergy

The role of T regulatory cells in spontaneous recovery from cow's milk allergy (CMA) is unclear. We investigated the mRNA expression of 12 T-cell markers and the protein expression of CD4, CD25, CD127, FoxP3 after in vitro β-lactoglobulin stimulation of peripheral blood mononuclear cells from children with persisting CMA (n = 16), early recovery (n = 20) or no atopy (n = 21). Artificial neural networks with exhaustive search for all marker combinations revealed that markers FoxP3, Nfat-C2, IL-16 and GATA-3 distinguished patients with persisting CMA most accurately from other study groups. FoxP3 mRNA expression following β-lactoglobulin stimulation was highest in children with persisting CMA. Also the FoxP3 intensity in CD4⁺ CD25^highCD127^low cells was higher in children with CMA compared with non-atopic children. The expression profile of both Th2- and T regulatory cell-related genes thus reflects the clinical activity of CMA. Tolerance, in contrast, is not characterized by activation of circulating T regulatory cells.