ABSTRACT
BACKGROUND: Better understanding of the relationship between antibody response to peanut and clinical sensitivity might lead to more accurate prognostication. OBJECTIVE: We sought to investigate peanut-specific IgE and IgG4 epitope diversity in relation to challenge-defined clinical sensitivity to peanut in a group of peanut-sensitized children. METHODS: Clinical sensitivity was determined by means of double-blind, placebo-controlled peanut challenges in 24 sensitized children. Six atopic control subjects were included. Specific IgE and IgG4 binding to 419 overlapping 15-amino-acid peptides representing the sequence of recombinant Ara h 1, Ara h 2, and Ara h3 was analyzed by means of microarray immunoassay. RESULTS: Peanut-sensitized patient sera bound significantly more IgE and IgG4 epitopes than control sera. This patient group reacted to the same Ara h 1, Ara h 2, and Ara h 3 epitopes as reported previously. There was a positive correlation between IgE epitope diversity (ie, number of epitopes recognized) and clinical sensitivity (r = 0.6), such that patients with the greatest epitope diversity were significantly more sensitive than those with the lowest diversity (P = .021). No specific epitopes were associated with severe reactions to peanut. IgG4 binding was observed to largely similar epitopes but was less pronounced than IgE binding and did not relate to the clinical sensitivity to peanut. IgE and IgG4 epitope-recognition patterns were largely stable over a 20-month period. CONCLUSION: Clinical sensitivity, as determined by means of double-blind, placebo-controlled peanut challenge, is positively related to a more polyclonal IgE response, which remains stable over time.
Subject(s)
Allergens/immunology , Epitopes, B-Lymphocyte/immunology , Immunoglobulin E/immunology , Immunoglobulin G/immunology , Peanut Hypersensitivity/immunology , Adolescent , Antibody Specificity , Arachis/chemistry , Arachis/immunology , Child , Child, Preschool , Epitopes, B-Lymphocyte/chemistry , Humans , Immunoglobulin E/blood , Immunoglobulin E/chemistry , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Infant , Male , Peanut Hypersensitivity/bloodABSTRACT
BACKGROUND: Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping. OBJECTIVE: We designed a redundant peptide microarray for IgE and IgG4 epitope mapping of the previously characterized peanut allergen, Ara h 2. METHODS: Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies. RESULTS: By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG(4) signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length-7 of which (78%) agreed with previous mapping. CONCLUSION: Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map.
Subject(s)
Allergens/genetics , Allergens/immunology , Arachis/genetics , Arachis/immunology , Epitope Mapping/methods , Glycoproteins/genetics , Glycoproteins/immunology , Immunoglobulin E/metabolism , Immunoglobulin G/metabolism , Peanut Hypersensitivity/genetics , Peanut Hypersensitivity/immunology , Plant Proteins/genetics , Plant Proteins/immunology , 2S Albumins, Plant , Amino Acid Sequence , Antibody Specificity , Antigen-Antibody Reactions , Antigens, Plant , Case-Control Studies , Cluster Analysis , Epitope Mapping/statistics & numerical data , Humans , In Vitro Techniques , Molecular Sequence Data , Protein Array AnalysisABSTRACT
Agonists of the dopamine receptors have been demonstrated to have bronchodilatory properties in pathologically constricted airways. The mechanism by which these agonists induce bronchodilatation is thought to involve airway sensory nerves. In this study, the expression and function of dopamine D(2) receptor were examined in sensory ganglia supplying the airways. Neuronal dopamine D(2) receptor mRNA expression was demonstrated by single-cell RT-PCR following laser-assisted microdissection. The projection of the neurons to the airways was confirmed by retrograde neuronal labeling. In functional studies, dopamine D(2) receptor agonists (AR-C65116AB and ropinirole) inhibited intraneuronal calcium mobilization in rat capsaicin-sensitive primary sensory neurons and capsaicin-induced plasma extravasation in the rat trachea. Our results provide support to the hypothesis that dopamine D(2) receptor activation inhibits neurogenic inflammation and proinflammatory reflex responses.
