In search of a tolerance-induction strategy for cow’s milk allergies: significant reduction of beta-lactoglobulin allergenicity via transglutaminase/cysteine polymerization

Abstract

OBJECTIVE: To explore the use of β-lactoglobulin polymerized using microbial transglutaminase and heating to identify whether protein polymerization could reduce in vivo allergenicity and maintain in vitro and ex vivo immunoreactivity for use in tolerance-induction protocols. METHODS: Based on previous protocols applied in mice and children, we performed in vivo challenges (using a skin prick test) with native and polymerized β-lactoglobulin in adult patients with an IgE-mediated allergy to β-lactoglobulin. In vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated immunoreactivity was analyzed using ex vivo challenges with native and polymerized β-lactoglobulin and monitored by leukocyte adherence inhibition tests. RESULTS: The skin tests demonstrated that there was a significant reduction in immediate cutaneous reactivity after polymerization. Polymerization did not decrease the immunoblotting detection of s-IgE specific to β-lactoglobulin. Cell-mediated immunoreactivity, as assessed by ex vivo challenges and leukocyte adherence inhibition tests, did not exhibit significant differences between leukocytes challenged with native versus polymerized β-lactoglobulin. CONCLUSIONS: The polymerization of β-lactoglobulin decreased in vivo allergenicity and did not decrease in vitro humoral or ex vivo cell-mediated immunoreactivity. Therefore, we conclude that inducing polymerization using transglutaminase represents a promising technique to produce suitable molecules for the purpose of designing oral/sublingual tolerance induction protocols for the treatment of allergies.

Microbial transglutaminase alters the immunogenic potential and cross-reactivity of horse and cow milk proteins

“…In contrast to cow, sheep, goat, and human breast milk, studies have reported the absence of a TG inhibitor in HM (de Jong et al, 2003); therefore, the effect of TG on the immunoreactivity of HM proteins seems to be worth studying. The effect of TG on CM has been previously studied (Olivier et al, 2012;. To the knowledge of the present authors, no studies investigating the effect of TG on the immunogenic potential of HM proteins have been conducted to date.…”

The Plasma Preincubation with Papain Before the Assay Suggests that a Gell and Coombs Type II Reaction is Been Demonstrated by the Leukocyte Adherence Inhibition Test

“…Whatsoever, the specificity and the prompt response to respond to challenging antigens suggests that an adaptive immune mechanism is involved with the presence of free and/ or membrane-bound specific antibodies. As we observed, it's enough just 30 minutes in contact with the specific antigen at an agitation device at physiologic temperature to generate the specific inhibition of the leukocyte adherence and/or the migration [29].…”

Role of specific IgE to β-lactoglobulin in the gastrointestinal phenotype of cow’s milk allergy

“…It is not known the cause why this happens. This could be argued because processing of dairy products could stimulate polymerization of BLG, which is the major whey protein of cow’s milk, in tetramers that probably changes spatial distribution of allergen epitopes [ 12 ] and decreasing allergenicity in children and adults with an IgE-mediated cow’s milk allergy [ 16 ]. Since there are differences between different products [ 12 ], it would also be necessary that manufacturers prove the presence or absence of BLG in natural form to unsure their labeling convinces the requirements to identify BLG in food products.…”