Clinical trial NCT01589731: Polymerized Beta-lactoglobulin Comparative Immunoreactivity
Official title: Study of the Immunoreactivity of Native and Polymerized Beta-lactoglobulin in Children and Adults With Cow's Milk Allergy and/or Intolerance
Study type: OBSERVATIONAL
Overall status: COMPLETED
Conditions: COW'S MILK ALLERGY
Keywords: Allergenicity
Cow's milk allergy
Leukocyte adherence inhibition test
Polymerization
Lead sponsor: University of Campinas, Brazil
Sponsor class: OTHER
First posted: 2012-05-02
Enrollment: 114
Sex: ALL
Minimum age (years): 18
Maximum age (years): 70
Standard ages: ADULT
OLDER_ADULT
Accepts healthy volunteers: yes
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.

Brief 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 .

Eligibility criteria: Inclusion Criteria:

* The presence of clinical cow's milk allergy and specific IgE against beta-lactoglobulin

Exclusion Criteria:

* The absence of clinical cow's milk allergy and specific IgE against beta-lactoglobulin

Primary outcomes:
measure: Evaluation of beta-lactoglobulin immunoreactivity after polymerization
timeFrame: two years
Secondary outcomes:
Other outcomes:
Locations:
city: Campinas
country: Brazil
facility: University of Campinas
state: Sao Paulo
Study officials:
affiliation: University of Campinas
name: Ricardo L Zollner, MD PhD
role: STUDY_CHAIR
References:
citation: Olivier CE, Villas-Boas MB, Netto FM, Zollner RL. Allergenicity of Bos d 5 in Children with Cow's Milk Allergy is Reduced by Transglutaminase Polymerization. Pediatr Allergy Immunol Pulmonol. 2012 Mar;25(1):30-33. doi: 10.1089/ped.2011.0101. Epub 2011 Dec 28.