Gabriele Gadermaier

Pollen-food syndromes associated with weed pollinosis: an update from the molecular point of view.

Pollinosis patients often display adverse reactions upon the ingestion of plant‐derived foods as a result of immunoglobulin E (IgE) cross‐reactive structures shared by pollen and food allergen sources. The symptoms of such pollen‐food syndromes (PFS) or class 2 food allergies range from local oral allergy syndrome to severe systemic anaphylaxis. Two clinical syndromes, the celery‐mugwort‐spice syndrome and the mugwort‐mustard‐allergy syndrome have been described in association with weed pollinosis. However, other associations between weed pollinosis and hypersensitivity to certain kinds of food have also been observed, like the mugwort–peach, the ragweed–melon–banana, the plantain–melon, the pellitory–pistachio, the goosefoot–fruit, the Russian thistle–saffron, and the hop–celery association. The number of allergen sources involved, the allergens, and influencing factors including geography, diet, and food preparation contribute to the high clinical complexity of PFS. So far, known causative cross‐reactive allergens include profilins, lipid transfer proteins, and high‐molecular weight allergens and/or glycoallergens. The current usage of nonstandardized allergen extracts poses additional problems for both diagnosis and therapy of PFS patients. Further identification and characterization of involved allergens is inescapable for better understanding of PFS and vaccine development. Panels of recombinant allergens and/or hypo‐allergens are promising tools to improve both PFS diagnostics and therapy.

The CREATE Project : development of certified reference materials for allergenic products and validation of methods for their quantification

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE‐binding potencies as their focus. Unfortunately, each company is using their own in‐house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

EAACI Molecular Allergology User's Guide

The availability of allergen molecules (‘components’) from several protein families has advanced our understanding of immunoglobulin E (IgE)‐mediated responses and enabled ‘component‐resolved diagnosis’ (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology Users Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low‐abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross‐reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE‐mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross‐reactive panallergens from plant (lipid transfer proteins, polcalcins, PR‐10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE‐mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

The Spectrum of Allergens in Ragweed and Mugwort Pollen

Ragweed and mugwort are important allergenic weeds belonging to the Asteraceae or Compositae plant family. Pollen of mugwort is one of the main causes of allergic reactions in late summer and autumn in Europe and affects about 10–14% of the patients suffering from pollinosis. Ragweed pollen represents the major source of allergenic protein in the United States, with a prevalence of about 50% in atopic individuals. In Europe, ragweed allergy is now rapidly increasing particularly in certain areas in France, Italy, Austria, Hungary, Croatia, and Bulgaria. Amb a 1 and Art v 1, the major allergens of ragweed and mugwort, respectively, are unrelated proteins. Amb a 1 is an acidic 38-kDa nonglycosylated protein. The natural protein undergoes proteolysis during purification and is cleaved into a 26-kDa alpha chain, which associates noncovalently with the beta chain of 12 kDa. The two-chain form seems to be immunologically indistinguishable from the full-length molecule. Art v 1 is a basic glycoprotein comprising two domains: an N-terminal cysteine-rich, defensin-like domain and a C-terminal proline/hydroxyproline-rich module. The proline/hydroxyproline-rich domain was recently shown to contain two types of glycosylation: (1) a large hydroxyproline-linked arabinogalactan composed of a short β1,6-galactan core substituted by a variable number (5–28) of α-arabinofuranose residues forming branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses, and (2) single and adjacent β-arabinofuranoses linked to hydroxyproline. As described for other pollen, ragweed and mugwort pollen also contain the pan-allergen profilin and calcium-binding proteins, which are responsible for extensive cross-reactivity among pollen-sensitized patients.

The role of lipid transfer proteins in allergic diseases.

Nonspecific lipid transfer proteins (LTPs) are important allergens in fruits, vegetables, nuts, pollen, and latex. Despite their wide distribution throughout the plant kingdom, their clinical relevance is largely confined to the Mediterranean area. As they can sensitize via the gastrointestinal tract, LPTs are considered true food allergens, and IgE reactivity to LTPs is often associated with severe systemic symptoms. Although Pru p 3 represents the predominant LTP in terms of patients’ IgE recognition, the contribution of pollen LTPs in primary sensitization cannot be ruled out. Due to structural homology, LTPs from different allergen sources are generally IgE cross-reactive. However, sensitization profiles among allergic patients are extremely heterogeneous, and individual cross-reactivity patterns can be restricted to a single LTP or encompass many different LTPs. Molecule-based approaches in allergy research and diagnosis are important for better understanding of LTP allergy and could assist clinicians with providing adequate patient-tailored advice.

Distinct Roles of Secreted HtrA Proteases from Gram-negative Pathogens in Cleaving the Junctional Protein and Tumor Suppressor E-cadherin

Background: The function of HtrA proteases in bacterial infections is widely unknown. Results: Secreted HtrA from various bacterial pathogens exhibits a conserved specificity for cleavage of E-cadherin. Conclusion: HtrA-mediated E-cadherin cleavage is a prevalent novel mechanism in bacterial pathogenesis. Significance: HtrA activity plays a direct role in the pathogenesis of different bacteria. The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple Gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections.

Array-based profiling of ragweed and mugwort pollen allergens.

Background:  Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons.

Artemisia and Ambrosia hypersensitivity: co-sensitization or co-recognition?

Background Ragweed and mugwort have nearly identical flowering periods. Clinical and serological studies showed that ragweed and mugwort sensitization are often associated and this poses relevant clinical problems in patients for whom specific immunotherapy is warranted.

Mutational Analysis of Amino Acid Positions Crucial for IgE-Binding Epitopes of the Major Apple (Malus domestica) Allergen, Mal d 1

Background: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1. Methods: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed. Results: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88–30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays. Conclusions: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.

A new allergen from ragweed (Ambrosia artemisiifolia) with homology to Art v 1 from mugwort

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29–31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more α-arabinofuranosyl residues with some β-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked β-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.