Subpollen particles are rich carriers of major short ragweed allergens and NADH dehydrogenases: quantitative proteomic and allergomic study.

BACKGROUND: Short ragweed (Ambrosia artemisiifolia) allergies affect more than 36 million people annually. Ragweed pollen grains release subpollen particles (SPP) of respirable size upon hydration or a change in air electrical conditions. The aim of this study was to characterize the proteomes and allergomes of short ragweed SPP and total pollen protein extract (TOT), and compare their effects with those of standard aqueous pollen protein extract (APE) using sera from short ragweed pollen-sensitized patients. METHODS: Quantitative 2D gel-based and shotgun proteomics, 1D and 2D immunoblotting, and quantitative ELISA were applied. Novel SPP extraction and preparation protocols enabled appropriate sample preparation and further downstream analysis by quantitative proteomics. RESULTS: The SPP fraction contained the highest proportion (94%) of the allergome, with the largest quantities of the minor Amb a 4 and major Amb a 1 allergens, and as unique, NADH dehydrogenases. APE was the richest in Amb a 6, Amb a 5 and Amb a 3, and TOT fraction was the richest in the Amb a 8 allergens (89% and 83% of allergome, respectively). Allergenic potency correlated well among the three fractions tested, with 1D immunoblots demonstrating a slight predominance of IgE reactivity to SPP compared to TOT and APE. However, the strongest IgE binding in ELISA was noted against APE. New allergenic candidates, phosphoglycerate mutase and phosphoglucomutase, were identified in all the three pollen fractions. Enolase, UTP-glucose-1-phosphate uridylyltransferase and polygalacturonase were observed in SPP and TOT fractions as novel allergens of the short ragweed pollen, as previously described. CONCLUSION AND CLINICAL RELEVANCE: We demonstrated that the complete major (Amb a 1 and 11) and almost all minor (Amb a 3, 4, 5, 6, 8 and 9) short ragweed pollen allergen repertoire as well as NADH oxidases are present in SPP, highlighting an important role for SPP in allergic sensitization to short ragweed.

Conjunctival provocation with airborne allergen in patients with atopic keratoconjunctivitis.

BACKGROUND: Atopic keratoconjunctivitis (AKC) is a chronic eye disease with periods of exacerbations. Many patients experience no obvious seasonal variation, although a majority of patients are allergic to common airborne allergens. OBJECTIVE: To investigate the allergic reaction, to conjunctival provocation with airborne allergens, in patients with AKC. METHODS: Eleven patients with AKC and birch and/or grass pollen allergy participated in the study, which was performed outside the pollen season. Five patients with seasonal allergic conjunctivitis (SAC) and five healthy subjects were included for validation purposes. The challenge was performed in one eye with the allergen, to which the patient was reactive, and with dilution buffer in the other eye. Signs and symptoms from both eyes were graded at baseline and at 10 min, 8 and 48 h after provocation. Tear fluid was collected from both eyes for cytokine analyses at baseline and at 8 and 48 h. RESULTS: A significant change in clinical symptoms and signs, (redness and chemosis) was evident 10 min after provocation compared with baseline (P = 0.005) and compared with the unprovoked eye (P = 0.005) in AKC subjects. These parameters were normalized after 8 and 48 h. A significant increase for IFN-γ (P = 0.021) and IL-6 (P = 0.015), and a near significant increase for IL-10 (P = 0.066) were seen in the tear fluid of the challenged eye at 48 h after provocation vs. baseline and vs. the control eye for IFN-γ (P = 0.005), IL-6 (P = 0.028) and IL-10 (P = 0.008) in AKC subjects. CONCLUSION AND CLINICAL RELEVANCE: In this single dose allergen provocation study, AKC patients responded with a typical IgE-mediated allergic reaction. An increase in cytokines at 48 h after the challenge was demonstrated and might, with further studies, give us a better understanding of the nature of inflammation in AKC.

IgE to peanut allergen components: relation to peanut symptoms and pollen sensitization in 8-year-olds.

BACKGROUND: Allergen-specific IgE testing is often performed with crude peanut extract, but the results may be difficult to interpret because of cross-reactions between peanut and other plant allergens. The aim was to investigate IgE reactivity to peanut allergen components in children from a birch-rich region in relation to pollen sensitization and peanut symptoms. METHODS: From a birth cohort, clinical parameters were obtained through questionnaires and IgE antibody levels to peanut and birch pollen were measured. Different peanut/birch sensitization phenotypes were defined among 200 selected children. IgE reactivity to peanut and pollen allergen components was analysed using microarray technique. RESULTS: Peanut symptoms were reported in 87% of the children with IgE reactivity to any of the peanut allergens Ara h 1, 2 or 3 but not to Ara h 8 (n = 46) vs 17% of children with IgE reactivity to Ara h 8 but not to Ara h 1, 2 or 3 (n = 23), P < 0.001. Furthermore, symptoms were more severe in children with Ara h 1, 2 or 3 reactivity. Children with IgE reactivity both to Ara h 2 and to Ara h 1 or 3 more often reported peanut symptoms than children with IgE only to Ara h 2 (97%vs 70%, P = 0.016), particularly respiratory symptoms (50%vs 9%, P = 0.002). CONCLUSIONS: IgE analysis to peanut allergen components may be used to distinguish between peanut-sensitized individuals at risk of severe symptoms and those likely to have milder or no symptoms to peanut if sensitized to pollen allergens and their peanut homologue allergens.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation.

BACKGROUND: Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. METHODS: The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. RESULTS: Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4(+) cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. CONCLUSIONS: Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.

Symptoms to pollen and fruits early in life and allergic disease at 4 years of age.

BACKGROUND: The predictive value of reported early symptoms to pollen or fruits on later allergic disease is unclear. Our aim is to evaluate if symptoms to pollen and/or to fruits early in life are associated with allergic disease and sensitization to pollen at 4 years. METHODS: The study included 3619 children from the Barn (Children), Allergy, Milieu, Stockholm, Epidemiology project (BAMSE) birth cohort. Reported symptoms of wheeze, sneeze or rash to birch, grass or weed, symptoms (vomiting, diarrhea, rash, facial edema, sneeze, or wheeze) to fruits including tree-nuts at 1 or 2 years of age, and definitions of asthma, rhinitis and eczema at 4 years were derived from questionnaire data. Sensitization to pollen allergens was defined as allergen-specific IgE-antibodies to any pollen (birch/timothy/mugwort) > or =0.35 kU(A)/l. RESULTS: At 1 or 2 years of age, 6% of the children were reported to have pollen-related symptoms, 6% had symptoms to fruits, and 1.4% to both pollen and fruits. Children with symptoms to both pollen and fruits at 1 or 2 years of age had an increased risk for sensitization to any pollen allergen at age 4 (OR(adj) = 4.4, 95% CI = 2.1-9.2). This group of children also had a substantially elevated risk for developing any allergic disease (asthma, rhinitis, or eczema) at 4 years irrespective of sensitization to pollen (OR(adj) = 8.6, 95% CI = 4.5-16.4). CONCLUSIONS: The prevalence of reported symptoms to pollen and fruits is very low in early childhood. However, children with early symptoms to both pollen and fruits appear to have a markedly elevated risk for allergic disease.

Sensitization to inhalant allergens between 4 and 8 years of age is a dynamic process: results from the BAMSE birth cohort.

BACKGROUND: There is limited knowledge of the development of IgE-antibody levels over time in childhood, with respect to persistency and co-sensitization to specific inhalant allergens. METHODS: Data from 2033 children participating in the BAMSE birth cohort was used. Background factors and clinical parameters were obtained and IgE antibody (ab) levels to eight common airborne allergens were measured (>or=0.35 kU(A)/L) when the children were 4 and 8 years of age. RESULTS: Between 4 and 8 years the proportion of children sensitized to any of the inhalant allergens tested increased from 15% to 25%. At 4 years IgE-ab to birch and cat dominated, whereas at the age of 8, there was a considerable increase in the proportion of sensitization to timothy and dog. Except for mites and moulds, IgE-ab levels to all aeroallergens increased significantly between 4 and 8 years among those already sensitized at 4. Transient sensitization to inhalant allergen was uncommon. Furthermore, sensitization to birch pollen at 4 years increased the risk for becoming sensitized to timothy, cat and dog later in life. Such an association was not observed among those sensitized primarily to animal dander. CONCLUSIONS: There is a prominent process of sensitization at pre-school age to inhalant allergens, and in Northern Europe sensitization to birch pollen early in life seems to be important for this process. Such a process has a probable impact on the development of allergic disease in the growing child.

Clinical effects of immunotherapy with genetically modified recombinant birch pollen Bet v 1 derivatives.

BACKGROUND: Birch pollen and pollen from related trees of the Fagales order are a major cause of allergic rhinitis, conjunctivitis, and asthma through the spring season in northern and central Europe. OBJECTIVE: To investigate the clinical effects of injection immunotherapy with genetically modified derivatives of major birch pollen allergen Bet v 1 on pollen-induced allergic symptoms. METHODS: A three-arm double-blind placebo-controlled immunotherapy study was conducted with one pre-seasonal course of treatment using two derivatives of Bet v 1, namely a recombinant Bet v 1 trimer and an equimolar mixture of two recombinant Bet v 1 fragments together representing the whole protein sequence. Analysis of local and systemic adverse events was performed for 124 patients who had received at least one dose of medication. Clinical efficacy was monitored by symptom medication scores and interval scoring in the per protocol-treated population (n=84). In addition, skin and nasal provocation responses and allergen-specific antibodies were assessed. RESULTS: There were trends towards improvement in the subjects' well-being and clinical symptoms (nasal scores), although comparisons with a placebo group did not show statistical significance in the main end-point, the combined symptom medication score. Reductions in skin and nasal sensitivity were observed for some subjects with a trend for the Bet v 1 trimer to be more effective than the fragments. Treatment induced strong IgG1 and IgG4 allergen-specific antibody responses. Local injection-site reactions were most frequent in the trimer group affecting 59.5% of patients as opposed to 37.8% and 30.6% in the fragment and placebo groups, respectively. Systemic reactions were elicited more frequently by fragments. A large proportion of adverse side-effects appeared hours following injections, and might be attributable to concurrent exposure to related pollens. CONCLUSION: Single courses of injection immunotherapy with Bet v 1 allergen derivatives showed trends towards improved well-being and reduced reactivity to specific allergen provocation, but did not yield significant improvement in the combined symptom medication score in this study.

Immune regulation by CD4+CD25+ T cells and interleukin-10 in birch pollen-allergic patients and non-allergic controls.

BACKGROUND: CD4+CD25+ regulatory T (Treg) cells and the cytokines IL-10 or TGF-beta play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non-infectious antigens such as allergens. OBJECTIVE: To investigate the regulation of immune responses to birch pollen allergen compared with influenza antigen by Treg cells obtained from birch pollen-allergic patients and non-allergic controls. METHODS: Peripheral blood was collected from 10 birch pollen-allergic patients and 10 non-allergic healthy controls. CD4+CD25+ and CD4+CD25- cells isolated by magnetic-activated cell sorting were co-cultured and stimulated with birch pollen extract or influenza vaccine in the absence or presence of anti-IL-10 or soluble TGF-betaRII. RESULTS: CD4+CD25+ cells from non-allergic controls were able to suppress influenza antigen and birch pollen stimulated effector cell proliferation, whereas CD4+CD25+ cells from allergic patients suppressed influenza antigen-, but not birch pollen-stimulated proliferation. The production of Th1 cytokines, but not Th2 cytokines, was suppressed by CD4+CD25+ cells from both allergic patients and controls, upon stimulation with birch pollen extract. Neutralization of IL-10 led to significantly increased production of IFN-gamma in cultures with CD4+CD25- T effector cells. In addition, six-fold higher concentrations of TNF-alpha were detected after neutralization of IL-10 in both CD4+CD25- and CD4+CD25+ cell cultures from allergic patients and controls. CONCLUSION: We demonstrate that the allergen-specific suppressive function of CD4+CD25+ cells from allergic patients is impaired compared with non-allergic controls. Moreover, neutralization of IL-10 enhances the production of TNF-alpha, suggesting counter-acting properties of IL-10 and TNF-alpha, where IL-10 promotes tolerance and suppression by Treg cells and TNF-alpha promotes inflammatory responses.

Sensitization to different pollens and allergic disease in 4-year-old Swedish children.

BACKGROUND: Although the relationship between sensitization to different inhalant allergens in adolescents and adults has been intensively studied, information concerning sensitization in children is scarce in particular to pollens. OBJECTIVES: In 4-year-old children to elucidate the pollen immunoglobulin (IgE) antibody profile (birch only, timothy only and combinations of three pollens (birch, timothy or mugwort) and to relate the results to other inhalant and food allergens, as well as the presence of allergic diseases. METHODS: A total of 2551 4-year-old children belonging to a prospective birth cohort, which has been followed longitudinally (BAMSE), were investigated with respect to IgE antibodies to pollen and other inhalant and food allergens, and expression of allergic disease, based on questionnaire data. RESULTS: Eleven percent (n=285) of the children were sensitized to pollen. Birch was the dominating cause of pollen sensitization (birch sensitization only, n=133); followed by timothy grass pollen (n=56) and a combination of two (n=64) or three (n=30) pollens. A remarkably high proportion of the children sensitized only to birch was also sensitized to other inhalant allergens. This was not seen for children sensitized only to timothy. The highest frequencies of IgE reactivity to food were found in the group of children sensitized to the combination of birch, timothy and mugwort pollen. Children sensitized to timothy only, exhibited symptoms of allergic disease significantly less frequently compared with children sensitized to birch only. Sensitization to birch pollen was found to be closely associated with rhinitis and eczema compared with asthma. The highest frequency of asthma and/or rhinitis and/or eczema was reported in children sensitized to at least two pollens. CONCLUSION: Our results demonstrate that birch is the dominating source of pollen sensitization at the age of four in Sweden. This might associate with the pattern of sensitization to other inhalant and food allergens as well as influence on the expression of allergic disease in this particular age group.

Allergic diseases and atopic sensitization in children related to farming and anthroposophic lifestyle--the PARSIFAL study.

BACKGROUND: The prevalence of allergic diseases has increased rapidly in recent decades, particularly in children. For adequate prevention it is important not only to identify risk factors, but also possible protective factors. The aim of this study was to compare the prevalence of allergic diseases and sensitization between farm children, children in anthroposophic families, and reference children, with the aim to identify factors that may protect against allergic disease. METHODS: The study was of cross-sectional design and included 14,893 children, aged 5-13 years, from farm families, anthroposophic families (recruited from Steiner schools) and reference children in Austria, Germany, The Netherlands, Sweden and Switzerland. A detailed questionnaire was completed and allergen-specific IgE was measured in blood. RESULTS: Growing up on a farm was found to have a protective effect against all outcomes studied, both self-reported, such as rhinoconjunctivitis, wheezing, atopic eczema and asthma and sensitization (allergen specific IgE > or = 0.35 kU/l). The adjusted odds ratio (OR) for current rhinoconjunctivitis symptoms was 0.50 (95% confidence interval (CI) 0.38-0.65) and for atopic sensitization 0.53 (95% CI 0.42-0.67) for the farm children compared to their references. The prevalence of allergic symptoms and sensitization was also lower among Steiner school children compared to reference children, but the difference was less pronounced and not as consistent between countries, adjusted OR for current rhinoconjunctivitis symptoms was 0.69 (95% CI 0.56-0.86) and for atopic sensitization 0.73 (95% CI 0.58-0.92). CONCLUSIONS: This study indicates that growing up on a farm, and to a lesser extent leading an anthroposophic life style may confer protection from both sensitization and allergic diseases in childhood.

Bacterial and fungal agents in house dust and wheeze in children: the PARSIFAL study.

BACKGROUND: Growing up on a farm and an anthroposophic lifestyle are associated with a lower prevalence of allergic diseases in childhood. This might be related to increased inhalatory exposure to microbial agents. OBJECTIVE: To assess the association between microbial agents in house dust and atopic wheeze in farm children, Steiner school children and reference children. METHODS: Levels of bacterial endotoxin, fungal beta(1,3)-glucans and fungal extracellular polysaccharides (EPS) in mattress and living room floor dust were measured in a population of 270 atopic (=Phadiatop-positive) children with self-reported wheezing, including 168 current atopic wheezers, and 441 non-atopic, non-symptomatic controls. These children were selected from a cross-sectional study in five European countries. RESULTS: In the study population as a whole, average levels of mattress dust endotoxin, EPS and glucans were slightly (1.1-1.2-fold; P<0.10) higher in control children than in atopic wheezers. Atopic wheeze was related to mattress levels of endotoxin, EPS and glucans in farm and farm-reference children. However, when adjusting for group (farm vs. farm-reference children), the associations became non-significant whereas the group effect remained. No associations between atopic wheeze and microbial agents were observed in Steiner and Steiner-reference children. For current atopic wheeze, the farm effect became non-significant after adjustment for microbial agent levels. CONCLUSION: Not only bacterial endotoxin but also mould components might offer some protection against atopic wheeze in children. However, the protective effect of being raised on a farm was largely unexplained by the mattress microbial agent levels measured in this study.