A. Weyer

Molecular characterization of human IgG monoclonal antibodies specific for the major birch pollen allergen Bet v 1. Anti‐allergen IgG can enhance the anaphylactic reaction

We report the molecular characterization of five human monoclonal antibodies, BAB1–5 (BAB1: IgG1; BAB4: IgG2; BAB2, 3, 5: IgG4), with specificity for the major birch pollen allergen, Bet v 1. BAB1–5 were obtained after immunotherapy and contained a high degree of somatic mutations indicative of an antigen‐driven affinity maturation process. While BAB1 inhibited the binding of patients IgE to Bet v 1, BAB2 increased IgE recognition of Bet v 1, and, even as Escherichia coli‐expressed Fab, augmented Bet v 1‐induced immediate type skin reactions. The demonstration that IgG antibodies can enhance allergen‐induced allergic reactions is likely to explain the unpredictability of specific immunotherapy.

Human serum IgE‐mediated mast cell degranulation shows poor correlation to allergen‐specific IgE content

Background:  Although allergen‐specific IgE content in serum can be determined immunochemically, little is known about the relationship between this parameter and the strength of the degranulation response upon allergen triggering.

Mapping of Dermatophagoides farinae mite allergens by two-dimensional immunoblotting

BACKGROUND Allergens from the house dust mite Dermatophagoides farinae are responsible for frequent respiratory allergic disorders, but only 3 groups of these allergens are well characterized. OBJECTIVE This study was performed to complete the repertoire of D farinae allergens using two-dimensional (2-D) electrophoresis. METHODS D farinae mite allergens, extracted from whole cultures in the presence of a mild detergent, were separated by 2-D electrophoresis with subsequent immunoblotting. IgE-binding proteins were detected with individual mite-sensitive patient sera and the anti-D pteronyssinus human serum pool. Allergens were identified by an inhibition immunoblot test, by means of specific mAbs, or by biochemical characterization. The internal peptides of 2 allergens were microsequenced. RESULTS 2-D immunoblotting with individual patient sera showed a marked heterogeneity in the isoelectric point of the allergens, as well as differences in the individual IgE-binding patterns. In addition to identification of allergens Der f 1, Der f 2, and Der f 3, new allergens have been characterized as Der f 4, Der f 5, and 2 high molecular mass allergens. Microsequencing of peptides from the latter allergens revealed significant homologies with allergen Mag 3 from D farinae and with a chitinase from prawn Penaeus japonicus. CONCLUSION 2-D electrophoresis with subsequent immunoblotting and protein microsequencing allowed characterization of a more complete repertoire of D farinae allergens and their multiple isoforms, and identification of six new allergens.

Specific signaling pathways in the regulation of TNF-α mRNA synthesis and TNF-α secretion in RBL-2H3 mast cells stimulated through the high affinity IgE receptor

Abstract.Objective: In the present study, we investigated signal transduction pathways involved in TNF-α gene expression and TNF-α secretion by mast cells stimulated through the high affinity IgE receptor (FcɛRI).¶Materials and Methods: TNF-α mRNA steady state levels and TNF-α secretion in the presence of specific pharmacological agents were monitored using rat basophilic leukemia cells (RBL-2H3) stimulated through FcɛRI. Relative amounts of TNF-α mRNA versus β-actin levels were quantified by RNase protection and RT-PCR assays. TNF-α secretion was measured by a current ELISA test.¶Results: We show that EGTA (5 mM) prevented TNF-α mRNA expression and TNF-α secretion in antigen-stimulated cells. The protein kinase C (PKC) inhibitor bisindolylmaleimide I substantially blocked TNF-α secretion at 2 μM but had only a marginal effect on TNF-α mRNA expression. The results were similar when PKC isoforms were depleted by long-term exposure to 100 nM phorbol ester (PMA). The PI 3-kinase inhibitor wortmannin blocked TNF-α secretion at low doses (EC50 = 13 nM), but only partially affected mRNA expression.¶Conclusions: Our results show that in FcɛRI-stimulated RBL-2H3 cells calcium mobilization, activation of PKC and PI 3-kinase are necessary for TNF-α secretion while for the increased TNF-α mRNA expression PKC activity is dispensable and PI 3-kinase activity only partially required.

Studies on Dermatophagoides pteronyssinus allergens: measurement of the relative potencies of D. pteronyssinus purified extracts by in vitro and in vivo methods.

To standardize the Dermatophagoides pteronyssinus extracts used in clinical allergy practice, the relative potencies of several purified extracts were estimated by radioallergosorbent test (RAST) inhibition, histamine release, and intracutaneous titration testing, and the results were compared. The potencies of nine independently prepared D. pteronyssinus extracts were assayed with respect to an extract adopted as a reference. In vivo the cutaneous wheal surface after intradermal injections was measured on a population of D. pteronyssinus-sensitive subjects. In vitro RAST inhibition was performed with the reference extract as allergosorbent and with a pool of human IgE-rich sera from patients sensitized to D. pteronyssinus but untreated. For histamine release human basophils from patients allergic to D. pteronyssinus were used. In the three method dose-response curves were plotted for the reference extract and the other extracts. A comparison of the measurement of in vitro and in vivo potencies is reported. Simialr results were obtained with the three methods: all the extracts but one were as potent as the reference preparation. However, for routine purposes RAST inhibition appears to be the most convenient and reliable procedure.

Identification of allergenic epitopes on Der ⨍ I, a major allergen of Dermatophagoides farinae, using monoclonal antibodies

The antigenic and allergenic structure of Der f I, a major allergen of the house dust mite Dermatophagoides farinae (Df) was investigated by means of a panel of 11 selected monoclonal antibodies (mAb) obtained from BALB/c mice immunized with purified Der f I. The species specificity of these mAb, tested with Der f I and Der p I--the homologous allergen from Dermatophagoides pteronyssinus--was generally restricted to Der f I since 10 out of 11 mAb reacted only with this allergen. Epitope specificity of the mAb was determined by both competitive inhibition and sandwich ELISA experiments. The results indicated the presence of at least four non-overlapping, non-repeated antigenic sites on Der f I, which were recognized by one or several mAb (sites A, B, C and D). Comparative epitope specificity studies between human IgE antibodies and mice mAb were performed, on sera and basophils of Df sensitive patients, using different inhibition assays (ELISA and histamine release experiments). The degree of inhibition varied between the patients and upon the assay design. Most of the mAb tested were found to significantly inhibit the binding of human IgE to Der f I (p less than 0.01) when compared with Der p I specific mAb as a control. The mAb reacting with site A was found to be the most potent inhibitor, presenting a mean inhibition of up to 56% in ELISA as well as in histamine release experiments. The results show that both human IgE antibodies and mAb can be directed against identical or closely related epitopes of Der f I. Therefore anti-Der f I mAb constitute immunologic probes in further allergenic epitope and peptide analysis of this major mite allergen.

Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

Expression of the Alpha Chain of Human FcεRI in Transfected Rat Basophilic Leukemia Cells: Functional Activation after Sensitization with Human Mite-Specific IgE

The actual dilemma in studying the binding and triggering capacity of IgE from allergic patients is the lack of cultured basophils or mast cell analogs of human origin. Human IgE binds with exquisite species specificity to the high affinity IgE receptor (Fc epsilon RI) expressed on the surface of these cells. In rodents this receptor has been characterized as a tetrameric plasma membrane protein composed of an IgE-binding alpha chain, a beta chain and two disulfide-linked gamma chains. In order to establish a cell line expressing the alpha chain of human Fc epsilon RI which can be triggered with IgE from human patients and specific allergen, we transfected the cDNA coding for the human alpha subunit into rat basophilic leukemia cells. The resulting transfectants express the human alpha chain on the cell surface in the form of a hybrid complex associated with endogenous rat gamma chains. After sensitization with human IgE from mite-specific patients, the transfectant produces a calcium response upon incubation with allergen. The established cell line can be used as a model system to study the mechanism of mast cell triggering through IgE from allergic patients.

Relationship between Specific Circulating IgE, Basophil Cell-Bound IgE and Histamine Release Induced by Purified Allergens of Dermatophagoides Farinae

In this study we analysed the presence and proportions of IgE specific for Df 6 and Df 11, two purified allergens of Dermatophagoides farinae. The characterisation of serum IgE and cell-bound IgE for 3 D. farinae-sensitive patients were performed by CRIE, RAST and PRIST assays. Furthermore the basophils from these same patients were studied by histamine release assays in the presence of Df 6 and Df 11. All the individual patients cell and serum IgE samples displayed the presence of IgE antibodies specific for Df 6 and Df 11, but the relative quantities of the IgE for these two specificities were characteristic of each patient. The ratios (Specific IgE for Df 11) : (Specific IgE for Df 6) (ratio 11 : 6) were similar in the serum and on the cells for an individual patient. As judged by histamine release assays, the basophil sensitivities towards Df 6 and Df 11 were very different from one atopic patient to another. Moreover cell sensitivities reflected the proportion of Df 6- and Df 11-specific IgE antibodies found in the serum and in the cell eluate.

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation, thermal or oxidative stress

Background: Rat basophilic leukemia (RBL‐2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell‐dependent reactivity, we examined the adaptive responses of RBL‐2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke.