IgE and T cell reactivity to a comprehensive panel of cockroach allergens in relation to disease

IgE sensitization to cockroach allergens is associated with development of allergic diseases, such as asthma. To understand the relevance of different cockroach allergens for diagnosis and immunotherapy, a comprehensive analysis of IgE antibody levels and T cell reactivity to an expanded set of cockroach allergens and their relationship to disease was performed in a cohort of USA cockroach sensitized patients. IgE antibody levels to recombinant chitinase and hemocyanin were measured for 23 subjects by custom-made ImmunoCAPs and compared with IgE levels to eight cockroach allergens we previously reported for the same cohort

Keeping Allergen Names Clear and Defined

The World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee was established in 1986 by leading allergists to standardize names given to proteins that cause IgE-mediated reactions in humans. The Sub-Committee’s objective is to assign unique names to allergens based on a critical analysis of confidentially submitted biochemical and clinical data from researchers, often prior to publication to preserve consistency. The Sub-Committee maintains and revises the database as the understanding of allergens evolves. This report summarizes recent developments that led to updates in classification of cockroach group 1 and 5 allergens to animal as well as environmental and occupational allergens. Interestingly, routes, doses, and frequency of exposure often affects allergenicity as does the biochemical properties of the proteins and similarity to self and other proteins. Information required by the Sub-Committee now is more extensive than previously as technology has improved. Identification of new allergens requires identification of the amino acid sequence and physical characteristics of the protein as well as demonstration of IgE binding from subjects verified by described clinical histories, proof of the presence of the protein in relevant exposure substances, and demonstration of biological activity (skin prick tests, activation of basophils, or mast cells). Names are assigned based on taxonomy with the abbreviation of genus and species and assignment of a number, which reflects the priority of discovery, but more often now, the relationships with homologous proteins in related species

Molecular Cloning and Expression of Cro s 1: an Occupational Allergen from Saffron Pollen (Crocus sativus)

Background: The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen. Methods: The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA. Results: The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1. Conclusion: We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb's-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron

Heterogeneity of magnitude, allergen immunodominance, and cytokine polarization of cockroach allergen-specific T cell responses in allergic sensitized children.

Background: Characterization of allergic responses to cockroach (CR), a common aeroallergen associated with asthma, has focused mainly on IgE reactivity, but little is known about T cell responses, particularly in children. We conducted a functional evaluation of CR allergen-specific T cell reactivity in a cohort of CR allergic children with asthma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 71 children, with mild-to-moderate asthma who were enrolled in a CR immunotherapy (IT) clinical trial, prior to treatment initiation. PBMC were stimulated with peptide pools derived from 11 CR allergens, and CD4+ T cell responses assessed by intracellular cytokine staining. Results: Highly heterogeneous responses in T cell reactivity were observed among participants, both in terms of the magnitude of cytokine response and allergen immunodominance. Reactivity against Bla g 9 and Bla g 5 was most frequent. The phenotype of the T cell response was dominated by IL-4 production and a Th2 polarized profile in 54.9% of participants, but IFNγ production and Th1 polarization was observed in 25.3% of the participants. The numbers of regulatory CD4+ T cells were also highly variable and the magnitude of effector responses and Th2 polarization were positively correlated with serum IgE levels specific to a clinical CR extract. Conclusions: Our results demonstrate that in children with mild-to-moderate asthma, CR-specific T cell responses display a wide range of magnitude, allergen dominance, and polarization. These results will enable examination of whether any of the variables measured are affected by IT and/or are predictive of clinical outcomes

Variability in German Cockroach Extract Composition Greatly Impacts T Cell Potency in Cockroach-Allergic Donors

German cockroach extract is used clinically to evaluate allergen-specific sensitization and for subcutaneous allergen-specific immunotherapy, though there are no guidelines for standardization in its manufacture. We performed an immunological evaluation of 12 different cockroach extracts prepared from different sources and their potency to induce allergen-specific T cell reactivity. PBMC from 13 cockroach allergic donors were expanded in vitro with 12 different German cockroach extracts. After culture expansion, cells were re-stimulated with the different extracts and T cell responses were assessed by FluoroSpot (IL-5, IFNγ and IL-10 production). In parallel to the extracts, single allergen peptide pools for allergens from groups 1, 2, 4, 5, and 11 were tested to determine allergen immunodominance. Furthermore, to assess allergy specificity, PBMC from 13 non-allergic donors were also tested with the most potent extract and T cell responses were compared to the allergic cohort. Dramatic variations in T cell reactivity were observed to the different cockroach extract batches. Response magnitudes varied over 3 logs within a single donor. IL-5 production in the allergic cohort was significantly higher compared to the non-allergic cohort (p=0.004). Allergen content determination by ELISA detected much lower concentrations of Bla g 5 compared to Bla g 1 and 2. Mass spectrometric analysis revealed that Bla g 5 was present in similar amounts to Bla g 1 and 2 in extracts made from whole body, whereas it was not detected in extracts made from fecal matter, suggesting that Bla g 5 is not excreted into feces. Different donors exhibit different response patterns to different extracts, potentially dependent on the donor-specific T cell allergen immunodominance pattern and the allergen content of the extract tested. These findings have dramatic implications for the selection of potent extracts used for diagnostic purposes or allergen-specific immunotherapy

Relevant B Cell Epitopes in Allergic Disease

The 3-dimensional structure of an allergen defines the accessible parts on the surface of the molecule or epitopes that interact with antibodies. Mapping the antigenic determinants for IgE antibody binding has been pursued through strategies based on the use of overlapping synthetic peptides, recombinant allergenic fragments or unfolded allergens. These approaches led to the identification of mostly linear epitopes and are useful for food allergens that undergo digestion or food processing. For inhaled allergens, conformational epitopes appear to be the primary targets of IgE responses. Knowledge of the molecular structure of allergens alone and in complex with antibodies that interfere with IgE antibody binding is important to understand the immune recognition of B cell-antigenic determinants on allergens and the design of recombinant allergens for immunotherapy. Starting with the molecular cloning and expression of allergens, and with the advent of X-ray crystallography and nuclear magnetic resonance techniques, we have been able to visualize conformational epitopes on allergens

Disruption of GABA-dependent chloride flux by cyclodienes and hexachlorocyclohexanes in primary cultures of cortical neurons

The effect of convulsant and nonconvulsant hexachlorocyclohexane (HCH) isomers and cyclodienes on GABA-induced Cl- flux was studied in primary cultures of neocortical neurons by measuring the GABA-stimulated 36Cl- uptake. GABA induced a dose-dependent chloride uptake. The convulsant agents gamma-HCH and cyclodienes alpha-endosulfan, dieldrin and aldrin blocked this 36Cl- uptake. A total or partial inhibition of GABA-induced 36Cl- uptake was produced by the noncompetitive GABAA antagonists picrotoxinin (PTX) and pentylenetetrazol, respectively. The inhibitory potencies of 36Cl- uptake by the organochlorine compounds (alpha-endosulfan > dieldrin > gamma-HCH > aldrin) were well correlated with their inhibitory potencies of [35S]TBPS binding. Positive modulators of GABAergic function (flunitrazepam and phenobarbital) prevented the blocking of GABA-induced chloride uptake by PTX but not that induced by alpha-endosulfan. The depressant beta- and delta-HCH isomers produced a biphasic response, increasing or decreasing the GABA-stimulated chloride uptake, depending on the HCH isomer and GABA concentrations used. The present results support the idea of cyclodienes and gamma-HCH action at the GABAA receptor by interacting with the TBPS binding site. A different interaction of PTX and alpha-endosulfan in the same recognition site is also suggested. An increase of GABA-induced 36Cl- flux by beta- and delta-HCH can account for the depressant activity of these compounds. This work also demonstrates the usefulness of primary neuronal cultures to perform functional studies of the GABAA receptor, taking into account allosteric interactions between the different recognition sites of the GABAA receptor