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Polymerized Beta-lactoglobulin Comparative Immunoreactivity (ImmuneBLG)

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ClinicalTrials.gov Identifier: NCT01589731
Recruitment Status : Completed
First Posted : May 2, 2012
Last Update Posted : May 2, 2012
Information provided by (Responsible Party):

Study Description
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.

Condition or disease

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 .

Study Design

Study Type : Observational
Actual Enrollment : 114 participants
Observational Model: Case Control
Time Perspective: Cross-Sectional
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 Start Date : July 2010
Primary Completion Date : July 2010
Study Completion Date : July 2011

Resource links provided by the National Library of Medicine

MedlinePlus related topics: Allergy
U.S. FDA Resources

Groups and Cohorts

Allergic group
The first group (group A) included 45 patients (17 males; mean age: 46.2 years, SD: 12.2 years) with convincing clinical histories of reproducible adverse reactions to bovine milk. All subjects presented βs-IgE that were detectable by SDS-PAGE immunoblotting.
Non Allergic group
The second group (group B) was used as a control for the immunoblotting analysis performed in the first group and included 20 individuals selected based on an evident tolerance to cow's milk, an absence of βs-IgE by ImmunoCAP assay and SPT non-reactivity to β-Lg or TgPolβ-Lg (6 males; mean age: 21.9 years, SD: 17.6 years).
Atopic group
The third group (group C) included 49 subjects with atopic respiratory and/or dermatological diseases (19 males; mean age: 28.7 years, SD: 20.6 years) regardless of βs-IgE status. This group was used to compare the ex vivo cell-mediated immunoreactivity between β-Lg and TgPolβ-Lg by comparing the mean ex vivo antigenic challenge results determined using the leukocyte adherence inhibition test (LAIT).

Outcome Measures

Primary Outcome Measures :
  1. Evaluation of beta-lactoglobulin immunoreactivity after polymerization [ Time Frame: two years ]
    The skin tests and ex vivo challenges are used to demonstrate if the polymerization can alter beta-lactoglobulin immunorreactivity.

Eligibility Criteria

Information from the National Library of Medicine

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Ages Eligible for Study:   18 Years to 70 Years   (Adult, Senior)
Sexes Eligible for Study:   All
Accepts Healthy Volunteers:   Yes
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.

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
Contacts and Locations

Information from the National Library of Medicine

To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.

Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT01589731

University of Campinas
Campinas, Sao Paulo, Brazil, 13083-887
Sponsors and Collaborators
University of Campinas, Brazil
Study Chair: Ricardo L Zollner, MD PhD University of Campinas
More Information

Additional Information:
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. Ped Allergy Immunol Pulmonol. 2012; 25(1)30-33.

Responsible Party: Celso Eduardo Olivier, Principal Investigator, University of Campinas, Brazil
ClinicalTrials.gov Identifier: NCT01589731     History of Changes
Other Study ID Numbers: UEC-2008
First Posted: May 2, 2012    Key Record Dates
Last Update Posted: May 2, 2012
Last Verified: April 2012

Keywords provided by Celso Eduardo Olivier, University of Campinas, Brazil:
Cow's milk allergy
Leukocyte adherence inhibition test

Additional relevant MeSH terms:
Milk Hypersensitivity
Immune System Diseases
Food Hypersensitivity
Hypersensitivity, Immediate