Lothar Vogel

Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system

Background:  Biochemical and immunochemical methods used for batch control of allergen extracts rely on the binding of IgE molecules to allergens. They do not measure the ability of a protein to induce type I allergic reactions. Therefore, a biological assay was established that is based on the cellular mechanisms of allergies in order to assess the cross‐linking capacity of allergens.

Nitration enhances the allergenic potential of proteins

Background: Recent investigations have shown that proteins, including Bet v 1a, are nitrated by exposure to polluted urban air. We have investigated immunogenic and allergenic properties of in vitro nitrated allergens in in vivo models. Methods: Untreated and nitrated samples of ovalbumin or Bet v 1a were compared for their ability to stimulate proliferation and cytokine secretion in splenocytes from DO11.10 or from sensitized BALB/c mice, and for their ability to induce specific immunoglobulin (Ig)G1, IgG2a and IgE in sensitized mice. Additionally, sera from birch pollen-allergic individuals were analysed for IgE and IgG specific for nitrated Bet v 1a. Results: Upon splenocyte stimulation with nitrated as compared with unmodified allergens, proliferation as well as interleukin 5 and interferon-γ production were enhanced. Sera of mice sensitized with nitrated allergens showed elevated levels of specific IgE, IgG1 and IgG2a, compared with sera from mice sensitized with unmodified allergens. Moreover, cross-reactivity of antibodies against unrelated, nitrated allergens was observed in mice. We also found higher amounts of functional, specific IgE against nitrated than against untreated Bet v 1a in sera from birch pollen-allergic patients. Conclusions: Our findings suggest that nitration enhances allergic responses, which may contribute to an increased prevalence of allergic diseases in polluted urban environments.

Identification of enolases and aldolases as important fish allergens in cod, salmon and tuna: component resolved diagnosis using parvalbumin and the new allergens.

The majority of fish‐allergic patients are sensitized to parvalbumin, known to be the cause of important IgE cross‐reactivity among fish species. Little is known about the importance of fish allergens other than parvalbumin.

Mediator release assays based on human or murine immunoglobulin E in allergen standardization

Background The biological potency of allergens can be measured by provoking mediator release from effector cells. As established immunochemical methods in allergen standardization only determine inhibition potency or major allergen content, routine tests for biological potency may enhance standardization and batch control of allergen products.

Kiwifruit allergy across Europe: Clinical manifestation and IgE recognition patterns to kiwifruit allergens

BACKGROUND Kiwifruit is a common cause of food allergy. Symptoms range from mild to anaphylactic reactions. OBJECTIVE We sought to elucidate geographic differences across Europe regarding clinical patterns and sensitization to kiwifruit allergens. Factors associated with the severity of kiwifruit allergy were identified, and the diagnostic performance of specific kiwifruit allergens was investigated. METHODS This study was part of EuroPrevall, a multicenter European study investigating several aspects of food allergy. Three hundred eleven patients with kiwifruit allergy from 12 countries representing 4 climatic regions were included. Specific IgE to 6 allergens (Act d 1, Act d 2, Act d 5, Act d 8, Act d 9, and Act d 10) and kiwifruit extract were tested by using ImmunoCAP. RESULTS Patients from Iceland were mainly sensitized to Act d 1 (32%), those from western/central and eastern Europe were mainly sensitized to Act d 8 (pathogenesis-related class 10 protein, 58% and 44%, respectively), and those from southern Europe were mainly sensitized to Act d 9 (profilin, 31%) and Act d 10 (nonspecific lipid transfer protein, 22%). Sensitization to Act d 1 and living in Iceland were independently and significantly associated with severe kiwifruit allergy (odds ratio, 3.98 [P = .003] and 5.60 [P < .001], respectively). Using a panel of 6 kiwifruit allergens in ImmunoCAP increased the diagnostic sensitivity to 65% compared with 20% for skin prick tests and 46% ImmunoCAP using kiwi extract. CONCLUSION Kiwifruit allergen sensitization patterns differ across Europe. The use of specific kiwifruit allergens improved the diagnostic performance compared with kiwifruit extract. Sensitization to Act d 1 and living in Iceland are strong risk factors for severe kiwifruit allergy.

Structural, immunological and functional properties of natural recombinant Pen a 1, the major allergen of Brown Shrimp, Penaeus aztecus

Background Recombinant allergens are considered the basis for new diagnostic approaches and development of novel strategies of allergen‐specific immunotherapy. As Pen a 1 from brown shrimp Penaeus aztecus is the only major allergen of shrimp and binds up to 75% of all shrimp‐specific IgE antibodies this molecule may be an excellent model for the usage of allergens with reduced IgE antibody‐binding capacity for specific immunotherapy.

Birch pollen‐related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1

Background Recently allergic reactions to legumes mediated by Bet v 1‐homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1‐homologous allergen Vig r 1.

IgE recognition patterns in peanut allergy are age dependent: perspectives of the EuroPrevall study

We tested the hypothesis that specific molecular sensitization patterns correlate with the clinical data/manifestation in a European peanut‐allergic population characterized under a common protocol.

Standardization of allergen extracts for immunotherapy: where do we stand?

Purpose of review The current state of the art in allergen standardization and recent progress in the field is summarized, and future developments are discussed. Recent findings The main focus of recent research in allergen standardization was on sandwich enzyme-linked immunosorbent assays or competitive tests for the quantification of individual allergens in extracts. New assays for quantifying major or minor allergens have been developed for tree and weed pollens from the Mediterranean area, grass pollens, and foods such as peanut and apple. In several cases, a good correlation with allergenic activity, measured by inhibition tests, was obtained. In addition, the potential of cellular mediator release assays in allergen standardization was evaluated in one study. Summary Several new tests have been developed to make more major and minor allergens from various allergen sources accessible to allergen standardization programmes such as the CREATE project. It is expected that assays to determine the majority of all clinically relevant major allergens from aeroallergen sources will be available in the near future. Standardized and validated mediator release assays may be a complementary tool for evaluating the biological potency of reference allergens and for correlating allergen concentrations to biological potency.

Isoform identification and characterization of Art v 3, the lipid-transfer protein of mugwort pollen.

Art v 3, the lipid-transfer protein (LTP) of Artemisia vulgaris pollen is a relevant allergen showing frequent cross-reactivity with homologues in other plants. Here we report the identification of four full-length Art v 3 sequences obtained by cDNA cloning using mass spectrometry-based sequencing. Two isoforms, Art v 3.0201 and Art v 3.0301 were expressed as soluble proteins in Escherichia coli Rosetta-gami B(DE3) pLysS using different expression systems. Purified natural and recombinant Art v 3 demonstrated similar secondary structures in circular dichroism analysis. All preparations showed high thermal stability but low resistance to gastric digestion with pepsin. Patient-specific IgE reactivity patterns to natural or recombinant isoallergens were observed among Art v 3-sensitized subjects. Using Immuno Solid-phase Allergen Chip (ISAC) assays, frequent cross-reactivity of Art v 3 with LTPs from peach and hazelnut was shown. The biological activity of both isoforms was comparable to the natural allergen in basophil release assays. The newly identified sequences provide the basis for recombinant mugwort LTP production enabling batch-to-batch reproducibility and thus ensuring high-quality products for diagnosis and therapy.