Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

House dust mites as potential carriers for IgE sensitization to bacterial antigens.

BACKGROUND: IgE reactivity to antigens from Gram-positive and Gram-negative bacteria is common in patients suffering from respiratory and skin manifestations of allergy, but the routes and mechanisms of sensitization are not fully understood. The analysis of the genome, transcriptome and microbiome of house dust mites (HDM) has shown that Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) species are abundant bacteria within the HDM microbiome. Therefore, our aim was to investigate whether HDM are carriers of bacterial antigens leading to IgE sensitization in patients suffering from atopic dermatitis. METHODS: Plasma samples from patients with AD (n = 179) were analysed for IgE reactivity to a comprehensive panel of microarrayed HDM allergen molecules and to S. aureus and E. coli by IgE immunoblotting. Antibodies specific for S. aureus and E. coli antigens were tested for reactivity to nitrocellulose-blotted extract from purified HDM bodies, and the IgE-reactive antigens were detected by IgE immunoblot inhibition experiments. IgE antibodies directed to bacterial antigens in HDM were quantified by IgE ImmunoCAP™ inhibition experiments. RESULTS: IgE reactivity to bacterial antigens was significantly more frequent in patients with AD sensitized to HDM than in AD patients without HDM sensitization. S. aureus and E. coli antigens were detected in immune-blotted HDM extract, and the presence of IgE-reactive antigens in HDM was demonstrated by qualitative and quantitative IgE inhibition experiments. CONCLUSION: House dust mites (HDM) may serve as carriers of bacteria responsible for the induction of IgE sensitization to microbial antigens.

Allergen-loaded strontium-doped hydroxyapatite spheres improve allergen-specific immunotherapy in mice.

BACKGROUND: Immunomodulatory interventions play a key role in the treatment of infections and cancer as well as allergic diseases. Adjuvants such as micro- and nanoparticles are often added to immunomodulatory therapies to enhance the triggered immune response. Here, we report the immunological assessment of novel and economically manufactured microparticle adjuvants, namely strontium-doped hydroxyapatite porous spheres (SHAS), which we suggest for the use as adjuvant and carrier in allergen-specific immunotherapy (ASIT). METHODS AND RESULTS: Scanning electron microscopy revealed that the synthesis procedure developed for the production of SHAS results in a highly homogeneous population of spheres. Strontium-doped hydroxyapatite porous spheres bound and released proteins such as ovalbumin (OVA) or the major cat allergen Fel d 1. SHAS-OVA were taken up by human monocyte-derived dendritic cells (mdDCs) and murine DCs and did not have any necrotic or apoptotic effects even at high densities. In a murine model of ASIT for allergic asthmatic inflammation, we found that OVA released from subcutaneously injected SHAS-OVA led to a sustained stimulation of both CD4+ and CD8+ T cells. Allergen-specific immunotherapy with SHAS-OVA as compared to soluble OVA resulted in similar humoral responses but in a higher efficacy as assessed by symptom scoring. CONCLUSION: We conclude that SHAS may constitute a suitable carrier and adjuvant for ASIT with great potential due to its unique protein-binding properties.

Dendritic cell-derived exosomes carry the major cat allergen Fel d 1 and induce an allergic immune response.

Exosomes are nano-sized membrane vesicles (50-120 nm), which are released from a wide variety of cells. Depending on their cellular origin, they can induce immune stimulatory-, inhibitory-, or tolerance-inducing effects. However, it is still unclear what role exosomes play during human inflammatory diseases. It has not been studied whether exosomes derived from human dendritic cells (DCs), the first cells to encounter allergens in the mucosa, can carry aeroallergens and contribute to allergic immune responses. We therefore explored whether DC-derived exosomes can present the major cat allergen Fel d 1 and whether they thereby contribute to the pathogenesis of allergic disease. Our results demonstrate that exosomes are able to present aeroallergens and thereby induce T-cell T(H)2-like cytokine production in allergic donors. Thus, these exosomes may be important immune-stimulatory factors in allergic immune responses and important targets or engineered tools in immunotherapy.

Differences in exosome populations in human breast milk in relation to allergic sensitization and lifestyle.

BACKGROUND: Breast-feeding has many beneficial effects on the developing immune system of the newborn. Breast milk contains immunoregulatory factors, such as nano-sized vesicles named exosomes. This study aimed at characterizing breast milk exosomes from human early milk and mature milk and to investigate whether allergic sensitization and an anthroposophic lifestyle could influence the exosome profile. METHODS: Breast milk was collected from 22 mothers at day 3-8 and from 61 mothers at 2 months postpartum, all part of the ALADDIN birth cohort. Isolated exosomes were captured on anti-MHC-class II- or anti-CD63 beads and analyzed by flow cytometry. Exosomal phenotype was related to lifestyle and allergic sensitization of the mothers, and sensitization of the child at 2 years of age. RESULTS: We found a higher content of exosomes in early milk compared with mature milk. Early milk exosomes were enriched in HLA-DR molecules and displayed significantly lower levels of HLA-ABC compared with those in mature milk. Phenotypically different subpopulations of exosomes were found in mature milk. Significantly lower levels of MUC1 were detected on CD63-enriched exosomes from sensitized mothers compared with nonsensitized. Furthermore, women with an anthroposophic lifestyle had significantly lower MUC1 expression on their HLA-DR-enriched milk exosomes and up-regulated levels of CD63 on CD63-enriched exosomes compared with nonanthroposophic mothers. Notably, mothers whose children developed sensitization had an increased amount of HLA-ABC on their milk exosomes enriched for CD63. CONCLUSIONS: The phenotype of exosomes in breast milk varies with maternal sensitization and lifestyle, which might influence allergy development in the child.

Protein biomarkers in vernix with potential to predict the development of atopic eczema in early childhood.

BACKGROUND: Atopic eczema (AE) is a chronic inflammatory skin disease, which has increased in prevalence. Evidence points toward lifestyle as a major risk factor. AE is often the first symptom early in life later followed by food allergy, asthma, and allergic rhinitis. Thus, there is a great need to find early, preferentially noninvasive, biomarkers to identify individuals that are predisposed to AE with the goal to prevent disease development. OBJECTIVE: To investigate whether the protein abundances in vernix can predict later development of AE. METHODS: Vernix collected at birth from 34 newborns within the Assessment of Lifestyle and Allergic Disease During INfancy (ALADDIN) birth cohort was included in the study. At 2 years of age, 18 children had developed AE. Vernix proteins were identified and quantified with liquid chromatography coupled to tandem mass spectrometry. RESULTS: We identified and quantified 203 proteins in all vernix samples. An orthogonal projections to latent structures-discriminant analysis (OPLS-DA) model was found with R(2) = 0.85, Q(2) = 0.39, and discrimination power between the AE and healthy group of 73.5%. Polyubiquitin-C and calmodulin-like protein 5 showed strong negative correlation to the AE group, with a correlation coefficient of 0.73 and 0.68, respectively, and a P-value of 8.2 E-7 and 1.8 E-5, respectively. For these two proteins, the OPLS-DA model showed a prediction accuracy of 91.2%. CONCLUSION: The protein abundances in vernix, and particularly that of polyubiquitin-C and calmodulin-like protein 5, are promising candidates as biomarkers for the identification of newborns predisposed to develop AE.

Efficient identification of Malassezia yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

BACKGROUND: Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in neonates, children and adults and can be life-saving for those patients. Ma-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique. OBJECTIVES: To create an extensive database of main mass spectra (MSPs) that will allow quick identification of Malassezia species by MALDI-TOF MS. METHODS: An in-house library of 113 MSPs was created from 48 reference strains from the CBS-KNAW yeast collection. The in-house library was challenged with two test sets of Malassezia strains, namely 165 reference strains from the CBS collection and 338 isolates collected in Greece, Italy, Sweden and Thailand. RESULTS: MALDI-TOF MS allowed correct identification of all 14 Malassezia spp. MALDI-TOF MS results were concordant with those of sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2 domains of the large subunit of the ribosomal DNA. CONCLUSIONS: Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks.

Sensitization to Malassezia in children with atopic dermatitis combined with food allergy.

BACKGROUND: The yeast Malassezia belongs to our normal cutaneous flora, but is capable of sensitizing individuals with atopic dermatitis (AD). Our objective was to investigate the prevalence of sensitization to Malassezia with a 10-yr follow-up among children suffering from AD combined with food allergy (FA) in relation to the extent of AD in infancy. METHODS: One hundred and eighty seven infants diagnosed with AD and milk/wheat allergy before 1 yr of age were included in the study. The area of AD was estimated from patient records of the first visit and measured with SCORAD at the 10-yr follow-up. Specific IgE against Malassezia was determined with ImmunoCAP™ at 11 yr of age. RESULTS: In infancy, 24 children (13%) were allergic to milk, 71 (38%) to wheat, and 92 (49%) to both milk and wheat, and 94 (50%) children had mild, 57 (30%) moderate and 36 (19%) severe AD. At the 10-yr follow-up visit, 19 (10%) of the children had ongoing milk and/or wheat allergy; 147 children (79%) had mild AD and 30 (16%) had SCORAD index of 0. Specific IgE against Malassezia mix was positive (≥0.35 kU/l) in 27% and specific IgE against M. sympodialis in 20% of the 187 children. The area of AD in infancy was associated with a greater risk of having allergen-specific IgE to Malassezia at the 10-yr follow-up. The risk ratio for FA was 3.11 (95% CI: 2.05-4.72; p < 0.001) if specific IgE to Malassezia was positive. CONCLUSIONS: Infants with severe AD and FA seem to have a greater risk of becoming sensitized to Malassezia during a 10-yr follow-up.

Environmental bacteria and childhood asthma.

BACKGROUND: We have previously found an inverse association of bacterial diversity with childhood asthma. It remains unclear whether certain bacteria account for the protective effect. METHODS: The high variability of the bacterial 16S rRNA gene allows assessing diversity and specificity of bacterial communities by single-strand configuration polymorphism (SSCP). DNA was extracted from mattress dust samples of 489 school-age children from rural and suburban regions in Germany. A fragment of the bacteria-specific 16S rRNA gene was amplified by PCR, digested to single-strand DNA, and subjected to electrophoresis. The resulting band patterns reflect the underlying DNA sequences. The individual bands were tested for associations with asthma, hay fever, and atopy in quantitative and qualitative multivariable analyses. Significantly associated bands were isolated and sequenced. The sequences were compared to a database, and distinct bacteria were identified. RESULTS: Seven of 76 independent bands were found to be inversely associated with asthma, atopic sensitization, and hay fever with odds ratios ranging from 0.17 to 0.73. The bands contained the sequences of Acinetobacter sp., Lactobacillus spp., Neisseria spp., Staphylococcus sciuri, Jeotgalicoccus sp., Corynebacterium spp., and others. CONCLUSIONS: In a diverse microbial environment, certain bacteria may account for the protective effect on the development of asthma and atopy.

Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma.

BACKGROUND: Leukotrienes (LTs) are potent pro-inflammatory mediators involved in asthma. Exosomes, nanosized vesicles released from various cells, can stimulate or down-regulate immune responses, depending on the state and nature of the originating cell. We have recently shown an altered exosome profile in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis, but their role in asthma is unknown. Our aims were to investigate whether exosomes from BALF have LT biosynthetic capacity and to explore phenotypic and functional characteristics of BALF exosomes in asthma. METHODS: Bronchoalveolar lavage fluid exosomes were collected from healthy individuals (n = 13) and patients with mild allergic asthma to birch pollen (n = 12) before and after birch allergen provocation. Exosomes were characterized by flow cytometry and Western blot. Their capacity to induce IL-8 and LT production in the human bronchial epithelial cell (BEC) line 16HB14o- was measured by ELISA and reverse-phase HPLC, respectively. RESULTS: Compared to BALF exosomes from healthy individuals, BALF exosomes from asthmatics displayed higher levels of exosome-associated markers, such as the tetraspanins CD63 and CD81 and the scavenger receptor CD36. No major differences were observed between BALF exosomes from before and after allergen provocation. Furthermore, we show that BALF exosomes contain enzymes for LT biosynthesis. The effect of exosomes to promote LTC(4) and IL-8 release in BEC was significantly increased for exosomes from asthmatics, and the CysLT(1) receptor antagonist Montelukast reduced exosome-induced IL-8 secretion. CONCLUSIONS: Bronchoalveolar lavage fluid exosomes from asthmatic and healthy individuals exhibit distinct phenotypes and functions. BALF exosomes from asthmatics might contribute to subclinical inflammation by increasing cytokine and LTC(4) generation in airway epithelium.

Cell-penetrating peptides as antifungals towards Malassezia sympodialis.

AIM: To determine whether different antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) are able to inhibit the growth of the commensal yeast Malassezia sympodialis, which can act as a trigger factor in different skin disorders, such as atopic eczema (AE), seborrhoeic eczema (SE) and dandruff. METHODS AND RESULTS: The antifungal activity of 21 different AMPs and CPPs was investigated by microdilution assay and plate counting to determine the number of colony forming units. Five CPPs and one AMP showed fungicidal activity at submicromolar concentrations. Importantly, no membrane damage on human keratinocytes was detected after peptide treatment. CONCLUSIONS: Several CPPs, while being nontoxic to mammalian cells, possess growth inhibitory activity on the very stringent yeast M. sympodialis. SIGNIFICANCE AND IMPACT OF STUDY: Our findings that five CPPs and one AMP that are harmless towards mammalian cells act as antifungal agents against M. sympodialis opens up the possibility to use these in the treatment for AE, SE and dandruff. To our knowledge, this is the first time peptides have been identified as antifungal agents against M. sympodialis. Further studies to elucidate the mechanism are warranted.

Lifestyle factors and sensitization in children - the ALADDIN birth cohort.

BACKGROUND: Several cross-sectional studies indicate that an anthroposophic lifestyle reduces the risk of allergy in children. We initiated the Assessment of Lifestyle and Allergic Disease During Infancy (ALADDIN) birth cohort to elucidate the role of specific factors supposed to mediate this effect. The aims of this study are to describe the ALADDIN cohort and to report patterns of exposure and allergic sensitization during the first years of life. METHODS: The ALADDIN study is a prospective birth cohort study of 330 children from families with an anthroposophic, partly anthroposophic, or nonanthroposophic lifestyle. The children and their parents were following an extensive data collection scheme, including repeated questionnaires and biological samples. Blood samples were collected from the parents and from the child at birth as well as at 6, 12, and 24 months of age. RESULTS: Several lifestyle factors differed between the groups, such as diet, medication, and place of delivery. Children of families with an anthroposophic lifestyle had a markedly decreased risk of sensitization during the first 2 years of life compared with children of nonanthroposophic families with adjusted OR 0.25 (95% CI 0.10-0.64) and P-value 0.004. A similar situation held true for children from families with a partly anthroposophic lifestyle, adjusted OR 0.31 (95% CI 0.15-0.54), and P-value 0.002. CONCLUSIONS: The anthroposophic lifestyle comprises several factors of interest for allergy development and is here shown to be associated with reduced risk of IgE sensitization already in infancy. Identifying the factors responsible for this association would be of significant clinical importance.

Mast cells generated from patients with atopic eczema have enhanced levels of granule mediators and an impaired Dectin-1 expression.

BACKGROUND: The disrupted skin barrier of patients with atopic eczema (AE) might facilitate contact between mast cells (MCs) in the skin and environmental triggers of the disease. One such trigger is the skin-colonizing yeast Malassezia sympodialis (M. sympodialis). In this study, we investigated the interaction of MC with M. sympodialis. METHODS: Mast cells were generated from peripheral blood CD34(+) progenitor cells of healthy controls (HC) and M. sympodialis-sensitized AE patients. Biopsy specimens were taken from HC and lesional AE skin for immunohistological stainings. RESULTS: The progenitor-derived MCs expressed the macrophage-inducible C-type lectin receptor Mincle, and exposure of these cells to M. sympodialis induced up-regulation of the mRNA expression of Mincle. Furthermore, we demonstrate that, when compared to HC, the progenitor-derived MCs from AE patients (i) contain more intrinsic granule mediators such as histamine, (ii) exhibit enhanced IL-6 release in response to M. sympodialis exposure, and (iii) have an impaired up-regulation of the fungal recognition receptor Dectin-1. In addition, analysis of skin sections from HC and AE patients revealed MCs as the predominant Dectin-1-expressing cell type in the skin. CONCLUSION: Our data indicate that progenitor-derived MCs from AE patients differ from those from HC. Further investigations with skin-derived MCs are necessary to confirm the observed differences which could provide new insights into the pathogenic mechanisms underlying AE.

IgE in the human placenta: why there?

Immunoglobulin E (IgE) antibodies are key effector molecules in the allergic inflammatory response and are also involved in the protection against extracellular parasites. Allergic symptoms often develop early in life, and the intrauterine environment has been proposed to play an important role in affecting the risk of later allergy development. The placenta constitutes a selective barrier between the maternal and foetal circulation. Recently, we reported that maternal IgE antibodies are present on foetal macrophages in the villous tissue of the human placenta irrespective of maternal allergy status. This review discusses the presence of IgE antibodies in the human placenta and its possible roles in normal and pathologic pregnancy. It also deals with the relationship between placental IgE and development of allergy during childhood. A better understanding of the role of IgE in placenta could give us clues on how to prevent allergy development in the future generations.

Lifestyle and parental allergen sensitization are reflected in the intrauterine environment at gene expression level.

BACKGROUND: Environmental factors, including the intrauterine environment, can influence the risk of allergy development. In the present study, we investigated whether lifestyle and parental allergen sensitization status are reflected at gene expression level in the intrauterine environment. METHODS: mRNA expression of 17 genes was determined by means of quantitative real-time PCR in term placenta of 36 families participating in the ALADDIN study (Assessment of Lifestyle and Allergic Disease During Infancy). Data were analysed using a linear regression model to estimate the influence of lifestyle and parental allergen sensitization on the relative mRNA expression levels. Immunohistochemistry on placenta biopsies was used to verify protein expression. RESULTS: Significant differences in mRNA expression levels were detected at the foetal side of the placenta, where CD14 was expressed at higher levels in placentas from families living on a farm compared to not living on a farm, and IL-12(p40) was expressed at lower levels when the father was sensitized compared to nonsensitized. At the maternal side of the placenta, higher expression of STAT4 and lower expression of GATA3 were detected in families with sensitized compared to nonsensitized mothers, and IL-12(p40) was lower expressed when the families were living on a farm compared to not living on a farm. Immunohistochemistry performed for STAT4 and GATA3 showed that protein and mRNA levels correlated well. CONCLUSION: Living on a farm and parental allergen sensitization are reflected in the intrauterine environment at the gene expression level.

Maternal allergic disease does not affect the phenotype of T and B cells or the immune response to allergens in neonates.

BACKGROUND: It is hypothesized that the in utero environment in allergic mothers can affect the neonatal immune responses. The aim of this study was to analyse the effect of maternal allergic disease on cord blood mononuclear cell (CBMC) phenotype and proliferative responses upon allergen stimulation. METHODS: Peripheral blood mononuclear cells (PBMC) from 12 allergic and 14 nonallergic mothers and CBMC from their children were analysed. In the mothers, we determined cell proliferation, production of IL-4 and expression of FOXP3 in response to allergen stimulation. In the children, we evaluated cell proliferation and FOXP3 expression following allergen stimulation. Furthermore, expression of different homing markers on T cells and regulatory T cells and maturity of the T cells and B cell subsets were evaluated directly ex vivo. RESULTS: The timothy- and birch-allergic mothers responded with increased proliferation and/or IL-4 production towards timothy and birch extract, respectively, when compared to nonallergic mothers. This could not be explained by impairment of FOXP3(+) regulatory T cells in the allergic mothers. CBMC proliferation and FOXP3 expression in response to allergens were not affected by the allergic status of the mother. Also, phenotype of T cells, FOXP3(+) regulatory T cells and B cells was not affected by the allergic status of the mother. CONCLUSION: Our results suggest that maternal allergic disease has no effect on the neonatal response to allergens or the phenotype of neonatal lymphocytes. The factors studied here could, however, still affect later development of allergy.

Enhanced expression of the antimicrobial peptide LL-37 in lesional skin of adults with atopic eczema.

BACKGROUND: Atopic eczema (AE) is a common multifactorial chronic skin disease associated with a defective skin barrier and increased susceptibility to skin infections. The human cathelicidin LL-37 plays a role in the host defence of skin. Studies have demonstrated deficient expression of LL-37 in skin of AE patients. OBJECTIVES: The aim of this study was to investigate the expression of LL-37 in lesional skin compared with nonlesional skin in patients with different severity of AE, patients with other eczema and healthy subjects. METHODS: Twenty patients with AE, four patients with other eczema and 10 healthy subjects were included. Severity of AE was graded using SCORing of atopic dermatitis (SCORAD). Skin biopsies were taken from lesional and nonlesional skin from all patients and from skin of healthy controls. The levels of LL-37 mRNA were analysed by quantitative reverse transcriptase-polymerase chain reaction. Evaluation of dermal and epidermal protein expression of LL-37 and the degree of inflammation was performed by immunohistochemical stainings. RESULTS: Patients with AE and patients with other eczema had significantly (P < 0.05) higher levels of LL-37 in lesional skin than in nonlesional skin. The expression of LL-37 was not statistically associated to severity of AE valued by SCORAD. Nonlesional skin from patients did not differ from skin of healthy subjects in terms of LL-37 expression. In the presence of epidermal injury or vesicles the LL-37 peptide was always detected. CONCLUSIONS: Patients with AE exhibit enhanced expression of LL-37 in lesional skin compared with nonlesional, suggesting a role of LL-37 in AE that might be associated with the process of re-epithelialization.

Evidence for allergen-specific IgE of maternal origin in human placenta.

BACKGROUND: Immunoglobulin E (IgE) has been identified on macrophage-like cells in the villi of human placenta, irrespective of the serum IgE levels or allergy status of the mother. The origin of placental IgE is debated and it is not known if it is spontaneously produced, so-called 'natural IgE', or if it has any specificity for certain allergens. The aim of this study was to investigate if placental IgE originates from mother or child and to analyse its specificity. METHODS: Immunoglobulin E was eluted from placenta by lowering the pH. Total and allergen-specific IgEs were measured in placenta eluate, maternal and cord blood plasma by means of ImmunoCAP (Phadia AB). The levels of natural antibodies were determined with an anti-phosphorylcholine (PC) enzyme-linked immunosorbent assay, as natural IgE has been shown in one previous publication with this assay. RESULTS: Detectable amounts of IgE were eluted from 11/12 full-term placentas. Natural (anti-PC) IgE antibodies were detected in low amounts in maternal plasma but not in the placental eluate or in cord blood plasma. There was a significant correlation between the amount of total IgE eluted from placenta and the levels of total IgE in maternal plasma; however, not between maternal and cord blood plasma. Allergen-specific IgE was only found in placental eluates from mothers with specific IgE towards these allergens. Furthermore, there was a significant correlation between the amount of allergen-specific IgE eluted from placenta and the levels of allergen-specific IgE in maternal plasma. Allergen-specific IgE could not be detected in cord blood. CONCLUSION: These results suggest a maternal origin of placental IgE, which can be allergen-specific.

Prolonged antigen-exposure with carbohydrate particle based vaccination prevents allergic immune responses in sensitized mice.

BACKGROUND: Defined particles carrying tightly bound allergens at high density have been suggested as alternatives in allergy vaccination. Carbohydrate based particles (CBP), sized 2 microm, provide a platform for covalent coupling of allergens. OBJECTIVE: To investigate the mechanisms of antigen presentation by CBP, as well as cellular and humoral responses after vaccination with the major cat allergen Fel d 1, covalently coupled to CBP. METHODS: Mice (n = 10/group) were subcutaneously vaccinated with CBP-rFel d 1, CBP or phosphate buffer saline (PBS) before sensitization with rFel d 1 and challenged with cat dander extract. Fluorescent and (75)Se-radiolabeled tracking of allergens and particles were performed with flow cytometry and whole-body autoradiography. Humoral, cellular and regulatory immune responses were analyzed by ELISA and flow cytometry. Cytokines were measured in bronchoalveolar lavage fluid and splenocyte cultures. RESULTS: CBP-rFel d 1 prevented induction of airway inflammation and induced allergen-specific T-cell anergy. CBP-rFel d 1 also induced rapid IgM and IgG1-responses compared with soluble rFel d 1. Particles were phagocytosed by antigen-presenting cells and transported to draining lymph nodes and spleen. Moreover, antigen coupled to CBP remained longer at the injection site compared with alum. CONCLUSIONS: Covalent coupling of rFel d 1 to CBP induces rapid antibody production, prevents induction of allergic immune responses and systemic allergen spreading. Thus, CBP comprise several attractive adjuvant features for use in allergy vaccination. CLINICAL IMPLICATIONS: Prolonged allergen exposure through covalent coupling to particles suitable for phagocytosis, provides an adjuvant for safer and efficient allergy vaccination.

The protective effect of farm animal exposure on childhood allergy is modified by NPSR1 polymorphisms.

BACKGROUND: Little is known about the asthma candidate gene neuropeptide S receptor 1 (NPSR1) in relation to environmental exposures, but recent evidences suggest its role as an effect modifier. OBJECTIVES: To explore the interaction between NPSR1 polymorphisms and environmental exposures related to farming lifestyle and to study the in vitro effects of lipopolysaccharide (LPS) stimulation on NPSR1 expression levels. METHODS: We studied 3113 children from PARSIFAL, a European cross-sectional study on environmental/lifestyle factors and childhood allergy, partly focused on children brought up on a farm. Information on exposures and outcomes was primarily obtained from parental questionnaires. Seven tagging polymorphisms were analysed in a conserved haplotype block of NPSR1. Multivariate logistic regression was used to evaluate a multiplicative model of interaction. NPSR1 protein and messenger RNA (mRNA) levels in monocytes were measured after LPS stimulation by fluorescence activated cell sorting (FACS) and quantitative real-time polymerase chain reaction (PCR). RESULTS: A strong interaction was seen between current regular contact to farm animals and several NPSR1 polymorphisms, particularly rs323922 and rs324377 (p<0.005), with respect to allergic symptoms. Considering the timing of initiation of such current regular farm animal contact, significant interactions with these and two additional polymorphisms (SNP546333, rs740347) were revealed. In response to LPS, NPSR1-A protein levels in monocytes were upregulated (p = 0.002), as were NPSR1-A mRNA levels (p = 0.02). CONCLUSIONS: The effect of farm animal contact on the development of allergic symptoms in children is modified by NPSR1 genetic background.