Enzymatic Treatment of Whey Proteins in Cow’s Milk Results in Differential Inhibition of IgE-Mediated Mast Cell Activation Compared to T-Cell Activation

Abstract

Background: Cow’s milk (CM) hydrolysates are frequently used as milk substitutes for children with CM allergy. In hydrolysates, allergenic epitopes within CM proteins are diminished by enzymatic treatment. The aim of this study was to examine the allergenic and immunogenic properties of whey proteins during hydrolysis. Methods: During hydrolysis, samples were obtained at 0, 10, 15, 30, 45, 60, 75 and 90 min. Degradation was checked by HPLC and SDS-PAGE. Allergenic potential was analyzed by IgE crosslinking capacity of human Fcε receptor type 1-transduced rat basophilic leukemia cells sensitized with serum of CM-allergic patients. Whey-sensitized C3H/HeOuJ mice were ear challenged intracutaneously with the hydrolysates. Immunogenicity was tested using whey-specific human T-cell clones and T-cell lines at the level of proliferation and release of IL-4, IL-10, IL-13 and IFN-γ. Results: After 15 min of hydrolysis, the majority of the proteins were degraded. Hydrolysis for 15 min resulted in 92% inhibition of mast cell degranulation and in 82% reduction of ear swelling in the mouse model. In contrast, T-cell-stimulatory capacity was less affected by hydrolysis: reduction of human T-cell proliferation was only 9%. This was further reduced to 57 and 74% after 30 and 45 min of hydrolysis, respectively. Cytokine production followed the pattern of T-cell proliferation. Conclusion: Via differential analysis of allergenic versus immunogenic properties of the time kinetics of hydrolysis of whey proteins, we have demonstrated specific hydrolysis conditions with reduced IgE-crosslinking responses but retained T-cell activating properties. This approach might be useful in better defining CM hydrolysates.

The degree of whey hydrolysis does not uniformly affect in vitro basophil and T cell responses of cow's milk‐allergic patients

“…While T cell activation is reduced more than 50% after 9 min of hydrolysis, it takes at least 20 min to reduce the basophil response to the same extent. This is in contrast to a previous study that showed a time-point with reduced allergenicity but retained immunogenicity [35]. Possible explanations for these contradictive results may be the different techniques and patient groups used in these studies.…”

Development of β-Lactoglobulin-Specific Chimeric Human IgEκ Monoclonal Antibodies for In Vitro Safety Assessment of Whey Hydrolysates

“…As shown in previous experiments in which whey protein degradation was followed during the hydrolysis process, a concurrent degradation of BLG and ALA was shown [23]. This study also showed that the capacity to cause IgE-mediated mast cell activation is size dependent.…”

“…Degranulation of RBL-huFcεRI cells with pooled serum from CMA patients was performed as described previously [23]. Confluent growing RBL-huFcεRI cells (1×10 5 /well) in 96-wells flat bottom culture plate were sensitized o/n with 5 µg/ml commercially available human purified IgE (Chemicon/Millipore) and stimulated with 10 µg/ml rabbit anti-human IgE antibodies (Dako) in Tyrode’s buffer at pH 7.4 with 0.1% HSA (Sigma-Aldrich, St. Louis, USA) for 1 hour.…”

Bovine Milk Allergens: A Comprehensive Review

“…Moreover, these peptide fragments significantly upregulated interferon (IFN)‐γ and interleukin (IL)‐10 production and downregulated IL‐4 secretion by murine splenocytes. A decreased allergenicity, confirmed by protein degradation, inhibition of mast cell degranulation, reduction of ear swelling, and reduction of T‐cell proliferation was also observed in whey protein concentrates after hydrolysis with a time‐dependent effect (Knipping and others ). Meulenbroek and others () stated that in some patients this time effect is not evident, indicating that the degree of hydrolysis is not decisive, but the presence and stability of IgE and T‐cell epitopes in the hydrolysates are recognized by individual patients.…”