Polymerized Beta-lactoglobulin Comparative Immunoreactivity
Condition: COW'S MILK ALLERGY
MeSH terms: Hypersensitivity; Milk Hypersensitivity
Study type: Observational

Summary: 
      Bovine β-lactoglobulin (Bos d 5) is an allergen from cow's milk with relevance to human
      health. We employed β-lactoglobulin polymerized using microbial transglutaminase as a model
      of study to identify whether protein polymerization could reduce in vivo allergenicity and
      maintain in vitro and ex vivo immunoreactivity for the purpose of producing a suitable
      molecule for use in tolerance-induction protocols. 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 Bos d 5. In
      vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated
      immunoreactivity was analyzed using ex vivo challenges with native and polymerized
      β-lactoglobulin monitored by leukocyte adherence inhibition tests. The study hypothesis is to
      identify a decrease on beta-lactoglobulin immunoreactivity after polymerization.
    

Detailed description: 
      Cow's milk allergy is a debilitating condition of difficult diagnosis and, until the moment,
      without a definitive solution that can be presented to the patient by the medical attendant.
      Amongst the dozens of proteins of the cow's milk, the beta-lactoglobulin (Bos d 5) is one of
      most allergenic for the fact that it is not produced by the human being and because of its
      hard digestibility. The allergenicity of beta-lactoglobulin in human beings is a subject well
      studied but little is known about the allergenicity of bioprocessed proteins as the
      polymerized beta-lactoglobulin.

      The objective of the present work is to study the immunoreactivity of the polymerized
      beta-lactoglobulin and to compare it with the immunoreactivity of the native
      beta-lactoglobulin in patients with and without clinical diagnosis of
      hypersensitivity/intolerance to cow's milk. It was used thermically induced polymerization
      and polymerization induced by transglutaminase in presence of cystein.

      Five groups of patients (three symptomatic and two controls) proceeding from two clinics of
      distinct characteristics had been studied according to presentation of the symptoms and
      submitted to allergic cutaneous tests and immunoassays for research of specific-IgE against
      beta-lactoglobulin, as well evaluation of cell-mediated immunoreactivity by challenge tests
      monitored by Leukocyte Adherence Inhibition Test.

      Side to side cutaneous tests with native and polymerized beta-lactoglobulin in 56 adults with
      problematic hypersensibility to cow's milk previously diagnosed as intolerants to the lactose
      had been carried through, as well research of specific-IgE against beta-lactoglobulin by
      ImmunoCAP and immunoblot. A control group of 20 subjects tolerant to cow's milk people with
      absence of specific-IgE against beta-lactoglobulin (detectable by ImmunoCAP or by allergic
      skin tests) had been selected for control of the technique of immunoblot. The results had
      shown that the analytical detection by immunoblot of specific-IgE is significantly more
      sensible when the polymerized beta-lactoglobulin is used in the immunoassay instead of the
      beta-lactoglobulin monomer. The results had shown that the research of specific-IgE by
      immunoCAP below of the detection limits, or the absence of cutaneous reactivity does not
      discard the possibility of IgE-mediated hypersensitivity as demonstrated by immunoblot.

      Side to side cutaneous tests with native and polymerized beta-lactoglobulin had been carried
      through in 22 symptomatic children, with confirmed by ImmunoCAP diagnosis of IgE-mediated
      allergy to beta-lactoglobulin. A matched control group of 22 asymptomatic children with not
      detectable specific-IgE for beta-lactoglobulin by ImmunoCAP was established for comparison of
      the technique of the skin tests. The results had shown that the cutaneous reactions carried
      through with the polymerized beta-lactoglobulin had been significantly lesser that the
      reactions of the cutaneous reactions carried through with the native beta-lactoglobulin.

      The cell-mediated immunoreactivity was studied on 49 atopic subjects by paired ex vivo
      allergen challenges monitored by the leukocyte adherence inhibition test. The results did not
      shown significant difference between the immunoreactivity against native versus polymerized
      beta-lactoglobulin.

      The polymerization of proteic antigens is a promising study model to be further investigated
      as a potential tool for the therapeutical induction of immunotolerance to alimentary
      proteins, because it decreases the in vivo immunoreactivity and does not destroy the
      allergenic epitopes as demonstrated by the in vitro and ex vivo assays .
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        Patients with convincing clinical histories of reproducible adverse reactions to bovine
        milk. All subjects presented βs-IgE that were detectable by SDS-PAGE immunoblotting.
      
Keywords: Allergenicity; Cow's milk allergy; Leukocyte adherence inhibition test; Polymerization