M van Hage-Hamsten’s research while affiliated with Karolinska Institutet and other places

Allergen-specific immunotherapy (SIT) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). Drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. An alternative carrier is 2 μm carbohydrate-based particles (CBPs). In mouse, allergen-coupled CBPs have been demonstrated to skew the allergen-specific immune response towards a Th1-like activity (Grönlund et al. Immunology, 2002). We here coupled the recombinant major cat allergen Fel d 1 to CBPs (CBP-Fel d 1) by cyanogen-bromide activation, resulting in covalent binding. The effect of CBP-Fel d 1 on monocyte-derived dendritic cells (MDDCs) from healthy human blood donors was studied. We found that the majority of the CD1a+ MDDCs were capable of taking up FITC-labelled CBP-Fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. Furthermore, incubation with CBP-Fel d 1 resulted in an upregulation of the costimulatory molecule CD86 on the MDDCs, which was not observed with Fel d 1 or CBPs alone. Finally, CBP-Fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α and a fourfold increase in the release of the chemokine interleukin-8 from MDDCs. Taken together, the effects CBPs possess make them interesting as novel allergen carriers for SIT.

We demonstrated the presence of IgE(+) plasma cells in the adenoids of atopic children. Our data suggest that adenoids are capable of local production of IgE and support the role of adenoids as an inductive site for allergic reactions. We have previously demonstrated increased numbers of IgE(+) cells in the adenoids of atopic children and also found support for an IL-4-induced class switch to IgE production in adenoids. In search of further evidence of adenoids being involved in IgE-mediated sensitization, we investigated the distribution of plasma cells and macrophages positive for IgE in adenoids. Adenoid tissue from atopic and non-atopic children was examined using immunohistochemical markers for IgE, plasma cells (CD138) and macrophages (CD68). The distribution of positive cells was determined in the extrafollicular area and in the follicles of adenoids. Co-localization of IgE and CD138(+) plasma cells and CD68(+) macrophages was examined using immunohistochemical double-staining methods. Non-atopic adenoids contained no or very few IgE(+) cells. In contrast, IgE(+) cells were found in high numbers in atopic adenoids, mainly in the extrafollicular area. Higher numbers of IgE(+) plasma cells and IgE(+) macrophages were also found in the adenoids of atopic children.

It is well established that early diagnosis of allergic disease is warranted. In a prospective birth cohort study (BAMSE) 3743 children at 4 years of age were included. Children were classified as having any allergic disease, e.g. asthma, suspected allergic rhinitis (suspAR), eczema or oro-gastro-intestinal symptoms with questionnaire. Blood was obtained from 2612 of these children and analysed for IgE antibodies (ab) towards 14 common food and airborne allergens. Positive IgE ab results were found in 38% of the children with any allergic disease, whereas such IgE ab results were found in 17% among those without any allergic disease. Furthermore, among children with any allergic disease the median summated IgE ab levels were 10.7 kU(A)/l compared with 1.5 kU(A)/l among those without such symptoms. The highest IgE ab levels were found to birch, peanut, cat and horse. When the sum of the IgE-ab levels towards the selected allergens was at least 34 kU(A)/l, or, alternatively, more than four allergen tests were positive, there was a 75% likelihood of identifying the individual with any allergic disease. To identify those with asthma, as well as those with suspAR, a significant interaction was found for the combination of the sum of IgE-ab levels and number of allergens positive at test. For eczema only, the number of positive allergens at test was associated to the likelihood of such disease. In children, 4 years of age, allergic disease was frequently not associated with the presence of single positive IgE antibody results, whereas increased IgE ab levels were significantly more prevalent among those with allergic disease. Thus, testing a certain profile of airborne and food allergens, and utilizing the sum of the IgE-ab levels in combination with the number of allergens positive at tests, may represent a more efficient diagnostic tool then to use just single positive IgE-ab results.

Allergen-specific immunotherapy is the only treatment for allergic disease providing long-lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy. The aim of this study was to provide a rational design of hypoallergen candidates for immunotherapy by using structural information and knowledge of B and T cell epitopes of an allergen. The three-dimensional structure of the major cat allergen Fel d 1 was systematically altered by duplication of selected T cell epitopes and disruption of disulphide bonds. Seven Fel d 1 derivatives were generated and screened for allergenic reactivity in comparison with recombinant Fel d 1 in competition-ELISA. The allergenicity was further evaluated in basophil activation experiments and T cell reactivity was assessed in a lymphoproliferation assay. Three out of seven Fel d 1 derivatives, with two duplicated T cell epitopes and one or two disulphide bonds disrupted, were carefully evaluated. The three derivatives displayed a strong reduction in allergenicity with 400-900 times lower IgE-binding capacity than recombinant Fel d 1. In addition, they induced a lower degree of basophil activation and similar or stronger T cell proliferation than recombinant Fel d 1. By a rational approach, we have constructed three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell reactivities. This strategy may be applied to any well-characterized allergen to improve immunotherapy for allergic patients.

Allergic diseases are influenced by both genes and environment. A 70-kb haplotype block in the G protein-coupled receptor for asthma susceptibility gene (GPR154; alias GPRA) on chromosome 7p was recently identified to influence susceptibility to asthma and elevated total serum IgE levels in adults. To assess the impact of GPR154 on childhood allergic disease, including allergic sensitization, asthma, and rhinoconjunctivitis, in study populations with diverse environmental backgrounds. We studied farm children, Steiner school children, and two reference groups from five Western European countries in the cross-sectional PARSIFAL (Prevention of Allergy Risk factors for Sensitization In children related to Farming and Anthroposophic Lifestyle) study and a sample of children from the Swedish birth cohort study BAMSE. DNA samples from 3,113 PARSIFAL and 800 BAMSE children were genotyped for 7 GPR154 polymorphisms and haplotypes were inferred. The proportions of alleles and haplotypes (H1-H7) were compared in affected children with their healthy counterparts. Data indicate a global association of the haplotype block to sensitization (allergen-specific serum IgE > or = 0.35 kU/L, p = 0.022), with significant haplotype-specific associations for H1, H5, and H6. Haplotypes H1 and H5 were also significantly associated with childhood allergic asthma (p = 0.045 and p = 0.023, respectively), and H5 to asthma regardless of sensitization. A broader involvement of GPR154 in allergic diseases was further supported in allergic rhinoconjunctivitis (H3: p = 0.046). The associated haplotypes could be allocated into risk (H5/H6) and nonrisk (H1/H3) groups, a pattern supported by allelic association of single nucleotide polymorphisms (SNPs) rs324384 and rs324396. Our results indicate that polymorphisms and haplotypes in the haplotype block of GPR154 are associated with asthma, rhinoconjunctivitis, and sensitization in European children.

Predatory mites are used as biological pesticides worldwide for control of spider mites and other pests in greenhouses. The aim of this study was to evaluate the impact of occupational exposure to Phytoseiulus persimilis and Hypoaspis miles on IgE sensitization among a large group of Swedish greenhouse workers and to examine the relationship between exposure and allergic asthma and rhinoconjunctivitis. A total of 96 greenhouse workers from the southern part of Sweden, who were using the predatory mites for control of pests, were investigated with a questionnaire and a medical examination including lung function test. Blood samples were taken to test for allergen-specific IgE antibodies to Phytoseiulus persimilis and Hypoaspis miles as well as to Tetranychus urticae, Dermatophagoides pteronyssinus/farinae and Tyrophagus putrescentiae. Seventeen of the 96 workers were positive in ImmunoCAP to predatory mites: 17 to P. persimilis (17.7%) and 14 to H. miles (14.6%). Subjects sensitized to predatory mites were significantly more often atopic (13/17), defined as a positive Phadiatop, than those who lacked IgE against these mite species (17/79) (P <0.01). IgE antibodies to the red spider mite T. urticae were present among 23 subjects. Thirty-five of the investigated subjects displayed a positive ImmunoCAP to at least one of the investigated mite species. Furthermore, sensitization to any of the mites tested was significantly associated with asthma (OR=9.3) and rhinoconjunctivitis (OR=4.3). IgE sensitization to predatory mites, P. persimilis and H. miles, is common among greenhouse workers. The findings stress the importance of improved allergen avoidance in greenhouse environments.

To compare the prevalence of self-reported food allergy and IgE antibodies to food allergens in wheezing and non-wheezing Estonian and Swedish schoolchildren, in the light of the disparities in the standard of living, food consumption and prevalence of respiratory allergies that still exist between Estonia and the Scandinavian countries. As a part of the ISAAC Phase II study, children from a random sample of schools in Tallinn in Estonia and Linköping and Ostersund in Sweden participated in skin prick tests to inhalant allergens and the parents replied to questionnaires. IgE antibodies against a panel of food allergens (egg white, milk, soy bean, fish, wheat and peanut) were taken from children with questionnaire-reported wheezing and a random sample of nonwheezing children. Children aged 10-11 y. The prevalence of self-reported food allergy was similar in Estonia and Sweden and about twice as high in wheezing children than in nonwheezing children. In Estonia, however, 3% of the children with perceived food allergy reported reactions from at least four different foods, as compared to 31% in Sweden. The prevalence of sensitisation to food allergens was similar in wheezing and nonwheezing children in Estonia (8%) while, in Swedish children, IgE antibodies to food allergens were more likely among wheezing children (Linköping 38 vs 11%, crude OR 5.1, 95% CI 2.2-11.6, and Ostersund 24 vs 7%, crude OR 4.1, 95% CI 1.9-8.5). Our study suggests that IgE-mediated food reactions were less likely in Estonian schoolchildren. Moreover, the perception of food allergy and thereby the meaning of self-reported food allergy appears to be different in the two countries.

The domestic cat (Felis domesticus) is one of the most important causes of allergic disease worldwide. A homologue of the major allergen Fel d 1 was created by linking the two chains that compose the protein. Fel d 1 (1+2) was expressed in Escherichia coli and subsequently purified using three chromatographic steps. Crystals of Fel d 1 (1+2) were obtained using the hanging-drop vapour-diffusion method in 22.5% PEG 3350, 0.5 M CaCl2. The crystals belong to space group P1, with unit-cell parameters a = 38.5, b = 42.9, c = 49.0 A, alpha = 70.7, beta = 80.5, gamma = 81.5 degrees , and diffract to 1.6 A resolution.