Sensitization to grass pollen allergen molecules in a birth cohort-natural Phl p 4 as an early indicator of grass pollen allergy.

BACKGROUND: Grass pollen allergy is one of the most common allergies worldwide. OBJECTIVE: The aim of this study was to evaluate the usefulness of grass pollen allergen molecules for prediction of grass pollen allergy during childhood and up to adolescence. METHOD: Questionnaire data and sera obtained from the study subjects at the ages of 4, 8, and 16 years from the population-based Barn/Children Allergy Milieu Stockholm Epidemiology birth cohort were used. Sera from 763 representative subjects with serum samples available at all 3 ages were analyzed for IgE reactivity to 8 Phleum pratense (Phl p) allergens (MeDALL [Mechanisms for the Development of Allergies] chip) and to timothy grass extract (ImmunoCAP). Allergic rhinitis to grass pollen (ARg) was defined as upper airway symptoms during grass pollen exposure. RESULTS: The prevalence of sensitization to any Phl p molecule was higher compared with that to timothy extract at all 3 ages: at the age of 4 years, 9.7% versus 6.8%; at the age of 8 years, 28.4% versus 15.3%; and at the age of 16 years, 37.1% versus 27.1%. General estimating equations analyses revealed that among children sensitized at the age of 4 years, the overall odds ratio (OR) of later ARg (up to 16 years) was increased only for IgE reactivity to Phl p 1 (OR = 4.9) and natural Phl p 4 (OR = 6.9). The likelihood of later symptoms increased with the number of allergen molecules; at the age of 4 years, 2 or more molecules predicted ARg to 78% and 3 or more molecules predicted ARg to 95%. A positive test result for timothy extract predicted ARg to 70%. CONCLUSIONS: Natural Phl p 4 is a hitherto unrecognized early indicator of grass pollen allergy, in addition to Phl p 1. To identify grass pollen sensitization and predict later ARg, allergen molecules are of added value to timothy extract alone and may help clinicians improve prediction of grass pollen allergy.

Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites.

BACKGROUND: N-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity. OBJECTIVES: To engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE. METHODS: One or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-Five™ insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes. RESULTS: HHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2. CONCLUSION: Defined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity.

Genetic Variants in CHIA and CHI3L1 Are Associated with the IgE Response to the Ascaris Resistance Marker ABA-1 and the Birch Pollen Allergen Bet v 1.

Helminth infections and allergic diseases are associated with IgE hyperresponsiveness but the genetics of this phenotype remain to be defined. Susceptibility to Ascaris lumbricoides infection and antibody levels to this helminth are associated with polymorphisms in locus 13q33-34. We aimed to explore this and other genomic regions to identify genetic variants associated with the IgE responsiveness in humans. Forty-eight subjects from Cartagena, Colombia, with extreme values of specific IgE to Ascaris and ABA-1, a resistance marker of this nematode, were selected for targeted resequencing. Burden analyses were done comparing extreme groups for IgE values. One-hundred one SNPs were genotyped in 1258 individuals of two well-characterized populations from Colombia and Sweden. Two low-frequency coding variants in the gene encoding the Acidic Mammalian Chitinase (CHIA rs79500525, rs139812869, tagged by rs10494133) were found enriched in high IgE responders to ABA-1 and confirmed by genetic association analyses. The SNP rs4950928 in the Chitinase 3 Like 1 gene (CHI3L1) was associated with high IgE to ABA-1 in Colombians and with high IgE to Bet v 1 in the Swedish population. CHIA rs10494133 and ABDH13 rs3783118 were associated with IgE responses to Ascaris. SNPs in the Tumor Necrosis Factor Superfamily Member 13b gene (TNFSF13B) encoding the cytokine B cell activating Factor were associated with high levels of total IgE in both populations. This is the first report on the association between low-frequency and common variants in the chitinases-related genes CHIA and CHI3L1 with the intensity of specific IgE to ABA-1 in a population naturally exposed to Ascaris and with Bet v 1 in a Swedish population. Our results add new information about the genetic influences of human IgE responsiveness; since the genes encode for enzymes involved in the immune response to parasitic infections, they could be helpful for understanding helminth immunity and allergic responses. We also confirmed that TNFSF13B has an important and conserved role in the regulation of total IgE levels, which supports potential evolutionary links between helminth immunity and allergic response.

Recombinant allergen-based IgE testing to distinguish bee and wasp allergy.

BACKGROUND: The identification of the disease-causing insect in venom allergy is often difficult. OBJECTIVE: To establish recombinant allergen-based IgE tests to diagnose bee and yellow jacket wasp allergy. METHODS: Sera from patients with bee and/or wasp allergy (n = 43) and patients with pollen allergy with false-positive IgE serology to venom extracts were tested for IgE reactivity in allergen extract-based tests or with purified allergens, including nonglycosylated Escherichia coli-expressed recombinant (r) Api m 1, rApi m 2, rVes v 5, and insect cell-expressed, glycosylated rApi m 2 as well as 2 natural plant glycoproteins (Phl p 4, bromelain). RESULTS: The patients with venom allergy could be diagnosed with a combination of E coli-expressed rApi m 1, rApi m 2, and rVes v 5 whereas patients with pollen allergy remained negative. For a group of 29 patients for whom the sensitizing venom could not be identified with natural allergen extracts, testing with nonglycosylated allergens allowed identification of the sensitizing venom. Recombinant nonglycosylated allergens also allowed definition of the sensitizing venom for those 14 patients who had reacted either with bee or wasp venom extracts. By IgE inhibition studies, it is shown that glycosylated Api m 2 contains carbohydrate epitopes that cross-react with natural Api m 1, Ves v 2, natural Phl p 4, and bromelain, thus identifying cross-reactive structures responsible for serologic false-positive test results or double-positivity to bee and wasp extracts. CONCLUSION: Nonglycosylated recombinant bee and wasp venom allergens allow the identification of patients with bee and wasp allergy and should facilitate accurate prescription of venom immunotherapy.

Identification of a villin-related tobacco protein as a novel cross-reactive plant allergen.

In a paradigmatic approach we identified cross-reactive plant allergens for allergy diagnosis and treatment by screening of a tobacco leaf complementary DNA (cDNA) library with serum IgE from a polysensitized allergic patient. Two IgE-reactive cDNA clones were isolated which code for proteins with significant sequence similarity to the actin-binding protein, villin. Northern- and Western-blotting demonstrate expression of the villin-related allergens in pollen and somatic plant tissues. In addition, villin-related proteins were detected in several plant allergen sources (tree-, grass-, weed pollen, fruits, vegetables, nuts). A recombinant C-terminal fragment of the villin-related protein was expressed in Escherichia coli, purified and shown to react specifically with allergic patients IgE. After profilin, villin-related proteins represent another family of cytoskeletal proteins, which has been identified as cross-reactive plant allergens. They may be used for the diagnosis and treatment of patients suffering from multivalent plant allergies.

MAP kinase phosphorylation of plant profilin.

Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P-X-T-P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45(Ntf4) and SIPK, when activated by the tobacco MAP kinase kinase NtMEK2, can phosphorylate the tobacco profilin NtProf2. Mutagenesis of the threonine residue in this motif identified it as the site of MAP kinase phosphorylation. Fractionation of tobacco pollen extracts showed that p45(Ntf4) is found exclusively in the high-speed pellet fraction while SIPK and profilin are predominantly cytosolic. These data identify one of the first substrates to be directly phosphorylated by MAP kinases in plants.