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.
Background: Due to the lack of standardized laboratory procedures able to demonstrate specific non—IgE-mediated immune responses against latex allergens, these conditions are diagnosed mostly by clinical criteria based on empiric exclusion prescriptions monitored by in vivo challenge tests. Objective: To evaluate the opportunity of an ex vivo challenge immunoassay, the Leukocyte Adherence Inhibition (LAI) Test (LAIT), to discriminate non—IgE-mediated latex-specific immunoreactivity. Methods: Ex vivo challenge tests performed with Hevea Brasiliense’s latex extract, monitored by LAIT, were assayed in an asymptomatic control group and a group of patients with diverse respiratory and cutaneous non—IgE-mediated allergic conditions, clinically diagnosed by a certified allergologist. Results: The mean LAI of the control group was 8.3%. The mean LAI of the complete patients’ group was 41.1%. The non-parametric Wilcoxon-Mann-Whitney U (WMWU) test comparing the control group with the whole patient’s group showed significance with a p-value < 0.00001. The WMWU test comparing the control group with each other patient’s group showed significance with a p-value < α = 0.05 for all comparisons. The WMWU test comparing the patients’ groups between each other did not show any significant p-value. Conclusion: Several patients from the diverse non—IgE-mediated allergic phenotypes presented variable immunoreactivity against the latex extract, as demonstrated by the LAIT, which proved to be an easy, quick, and inexpensive ex vivo immunoassay with the potential to predict individual immunoreactivity against Hevea brasiliensis latex allergens in real-world patients with non—IgE-mediated allergies.
Background: The diagnosis of non-IgE mediated food allergies may be a complex puzzle when there is no start point to establish an elimination diet to allow a clear clinical field to initiate diagnostic Oral Food Challenges tests. Objective: To evaluate the opportunity of the tube titration of precipitins to select food allergens to proceed with elimination diets to assist the diagnosis and management of adult patients with Food Allergy manifested as Intrinsic Atopic Dermatitis (IAD). Methods: The tube titration of specific precipitins against anamnesis-chosen food allergens were performed in 64 IAD patients and their titers were associated with an Improvement Verbal Scale Rate (IVSR) of the patient’s perception of the benefits of the Precipitins-based Elimination Diet (PED) performed with these specific food allergens, as well correlated with their positive or negative perception of the impairment of symptoms after the reintroduction of the Symptom-Related Food Allergen (SRFA). Results: In most cases, the PED contributed to a significant clinical improvement that allowed the patients to evaluate the individual effect of the reintroduction of each food allergen on their diets. There was a significant positive correlation coefficient between the titers of the food-specific titration of precipitins and the percentage of positive SRFA (Pearson r = 0.91; p-value = 0.0004). Conclusion: The semiquantitative titration of specific precipitins against food allergens is a promising triage test to select food allergens to proceed with elimination diets to support the diagnosis and management of non-IgE mediated Food Allergy in patients with Intrinsic Atopic Dermatitis.
Background:The Leukocyte Adherence Inhibition Test (LAIT) is an ex vivo challenge test used to testimony specific immunoreactivity. Despite numerous studies had been performed to elucidate the interactions involved in the assay, there are yet several questions about its mechanisms and how to translate its results into the medical practice.Objectives: To perform an experiment with an antibody disassembler: the papain, to demonstrate the participation of specific antibodies in the reconnaissance phase of the assay performed with protein antigens.Methods: Paired side-by-side ex vivo allergen-specific challenges monitored by the LAIT, were performed with the plasma of allergic patients, preincubated (or not) with papain. Results:The mean of the differences (26.66%) of the two arms was considered significative by the paired t-test (p-value < 0.05). Conclusion:Our results suggests that the leukocyte adherence inhibition phenomenon and its inhibition is possibly mediated by an interaction of cells and specific antibodies and deserves further studies as an ex vivo challenge test tool for research of Gell & Coombs type II hypersensitivity reactions.
Background: Due to the lack of standardized laboratory procedures able to demonstrate specific immune responses against the culprit allergens, the non—IgE-mediated allergy syndromes are a group of conditions diagnosed mostly by clinical examination and exclusion criteria. Objective: To evaluate the opportunity of the Leukocyte Adherence Inhibition Test (LAIT) to discriminate specific immunoreactivity against Dermatophagoides pteronyssinus (Dp) in a group of patients with non–IgE-mediated chronic allergic conditions. Methods: Ex vivo challenge tests performed with Dp were monitored by LAIT in patients presenting diverse non–IgE-mediated allergic conditions: intrinsic Atopic Dermatitis (iAD), intrinsic Allergic Rhinitis (iAR), intrinsic Ocular Allergy (iOA), intrinsic Chronic Pharyngitis (iCP), and intrinsic Asthma (iAS). Results: The mean LAI of the control group was 7%; the mean LAI of the iAR group was 34%; the mean LAI of the iCP group was 44%; the mean LAI of the iAS group was 45%; the mean LAI of the iOA group was 47%; the mean LAI of the iAD group was 55%. The non-parametric Wilcoxon-Mann-Whitney U test comparing the control group with each other group showed significance with p-value < α = 0.05 for all groups. Conclusion: The Leukocyte Adherence Inhibition Test is an easy, quick, and inexpensive ex vivo immunoassay with the potential to predict individual immunoreactivity against HDM allergens in real-world patients with non–IgE-mediated allergies.
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