Clinical and Immunologic Changes due to Subcutaneous Immunotherapy With Cat and Dog Extracts Using an Ultrarush Up-Dosing Phase: A Real-Life Study.

OBJECTIVES: We aimed to evaluate the efficacy of and immunologic changes caused by subcutaneous immunotherapy (SCIT) in patients with allergy to cat and dog. METHODS: The study population comprised patients with rhinitis and/or asthma and allergy to cat or dog from a previous safety study. All patients had specific IgE to cat and/or dog. The SCIT maintenance dose was administered using an infusion pump over a single 4-hour session, followed by monthly administration over 6 months. Data were gathered on clinical outcomes, pulmonary function, FeNO, rhinitis and asthma symptoms, quality of life (QOL), and scores for the Asthma Control Test and symptom visual analog scale were recorded at baseline and then at 1, 3, and 6 months. Specific IgE and IgG antibody responses to cat and dog allergens were determined. RESULTS: The study population comprised 61 patients with a mean age of 35.6 (9.7) years, of whom 40 underwent SCIT for at allergy. A significant improvement was observed in rhinitis and asthma symptoms and in QOL, use of medication, visual analog scale score, and Asthma Control Test score at 1 month; these improvements persisted at month 6. The clinical improvement with cat extract was significantly more marked than with dog extract. Nearly half of the patients (49.09%) had an increase of >0.9 in the ESPRINT-15 QOL in allergic rhinitis questionnaire, and 58.18% had an increase of >0.5 in the Asthma Quality of Life Questionnaire score at month 6. Both differences represent the minimal clinical important difference. A significant increase was observed in specific IgG and IgE to different allergens at 3 and/or 6 months. CONCLUSION: Ultrarush SCIT with cat and dog extracts has substantial clinical value for many patients.

Allergy testing in children with persistent asthma: comparison of four diagnostic methods.

BACKGROUND: Multiple allergic sensitizations are common in persistent childhood asthma, and thorough assessment of allergy is crucial for optimal care of these children. Microarray testing offers opportunities for improved sIgE characterization, which has been projected to be useful in the management of multisensitized patients. OBJECTIVE: The aim of this study was to investigate the accuracy and information obtained by two microarray platforms applied on a well-characterized pediatric asthma cohort. METHODS: Seventy-one children were recruited from a nationwide Swedish study on severe childhood asthma. Severe (n = 40) and controlled (n = 31) asthmatics were assessed for allergic sensitization by two microarray systems (Microtest and ISAC) and by two standard diagnostic methods (ImmunoCAP and skin prick test). Data on clinical history, physical examination, spirometry, asthma control test, and doctor's diagnosis were collected. Results from the four diagnostic methods were analyzed and compared. RESULTS: A high prevalence of allergic sensitization was observed in this cohort. The pairwise concordance between two methods was 90-92% independently of methods compared. The sensitivity of the four methods against doctor's diagnosis was 0.77-0.88, and the specificity was 0.97-0.99. Microarray methods provided new information in 47% of the sensitized children in comparison with results obtained by standard diagnostic methods. CONCLUSION: The high prevalence of food and respiratory sensitization supports the clinical guideline recommendation that allergies should be evaluated in all children with suspected asthma. The microarray platforms studied here demonstrated acceptable accuracy and provided refined IgE characterization in 47% of the patients compared to standard extract-based methods.

IgG4 but no IgG1 antibody production after intralymphatic immunotherapy with recombinant MAT-Feld1 in human.

Allergy immunotherapy (AIT) mediates protection against allergen exposure in part due to allergen-specific antibodies. While immunization typically stimulated IgG1 and IgG2, AIT is often associated with production of IgG4. Here, twenty cat dander-sensitized patients were randomized to receive three injections of intralymphatic immunotherapy (ILIT) with MAT-Feld1 adsorbed to aluminum hydroxide or just aluminum hydroxide (placebo) in a double-blind setting (ClinicalTrials.gov NCT00718679). Whereas the clinical data, showing benefit of Mat-Feld1 ILIT was published in 2012 (Senti et al., J Allergy Clin Immunol, vol 129(5):1290-1296), the current study investigated the cat allergen-specific antibody responses. Blood was drawn prior to ILIT, as well as 1, 3, and 12 months after first ILIT. The sera were analyzed to characterize all IgG subclasses and IgE antibody responses. ILIT with MAT-Feld1 elicited high levels of total IgG that were maintained for at least 12 months. Interestingly, a strong increase in IgG4 and some increase in IgG2 were observed throughout the study, while production of cat-specific IgG1 and IgG3 was not stimulated by MAT-Feld1 ILIT. The IgE levels remained constant.

The cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1.

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat-sensitized Swedish patients and elucidated its allergenic activity and cross-reactivity with the dog lipocalin Can f 1. Sixty-five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross-reactive antisera. Fel d 7 is a common allergen in a Swedish cat-sensitized population that cross-reacts with Can f 1, and may contribute to symptoms in cat- but also in dog-allergic patients.

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.

Hypoallergenic derivatives of Fel d 1 obtained by rational reassembly for allergy vaccination and tolerance induction.

BACKGROUND AND OBJECTIVE: The major cat allergen Fel d 1 represents one of the most important respiratory allergens. Aim of this study was to engineer recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes for vaccination and tolerance induction. METHODS: Seven recombinant mosaic proteins were generated by reassembly of non-IgE-reactive peptides of Fel d 1 which contained the sequence elements for induction of allergen-specific blocking IgG antibodies and T cell epitopes. Mosaic proteins were expressed in Escherichia coli using codon-optimized synthetic genes and compared with Fel d 1 regarding structural fold by circular dichroism, IgE-binding capacity, activation of allergic patients' basophils and ability to induce allergen-specific blocking IgG antibodies upon immunization. RESULTS: Although each of the mosaic proteins had lost the alpha-helical fold typical for Fel d 1, a strong reduction in IgE reactivity as well as allergenic activity in basophil activation assays was only obtained for three constructs, two reassembled fragments (Fel d 1 MB, Fel d 1 MC) and a fusion of the latter two (Fel d 1 MF) in which the cysteines of Fel d 1 MC were replaced by serines. Immunization of rabbits with Fel d 1 MB, MC and MF induced high levels of IgG antibodies that inhibited IgE reactivity of cat-allergic patients to Fel d 1 in a comparable manner as IgG induced with the wild-type allergen. CONCLUSIONS: We report the development of hypoallergenic reassembled Fel d 1 proteins suitable for vaccination and tolerance induction in cat-allergic patients.

Recommendations for the allergy management in the primary care.

The majority of patients seeking medical advice for allergic diseases are first seen in a primary care setting. Correct diagnosis with identification of all offending allergens is an absolute prerequisite for appropriate management of allergic disease by the general practitioner. Allergy diagnostic tests recommended for use in primary care are critically reviewed in accordance with the significant workload in a primary care setting. Simplified pathways for recognition and diagnosis of allergic diseases are proposed, that should be further adapted to local (national) conditions.

Identification of galactose-α-1,3-galactose in the gastrointestinal tract of the tick Ixodes ricinus; possible relationship with red meat allergy.

Patients with IgE antibodies against the carbohydrate epitope galactose-α-1,3-galactose (α-Gal) have reported severe allergic reactions after consumption of red meat. Investigations have revealed associations between IgE to α-Gal and tick bites. We provide the first direct evidence that α-Gal is present within ticks thus potentially explaining the relationship between tick exposure and sensitization to α-Gal, with development of red meat allergy as a secondary phenomena. Serum from Swedish patients with delayed severe reactions to red meat was included in the study. A dose-dependent inhibition of IgE responses to α-Gal by the tick Ixodes ricinus is demonstrated. Furthermore, using cryostat-cut sections of I. ricinus, we show that both a monoclonal and a polyclonal antibody against α-Gal stains the gastrointestinal tract of the tick. The same pattern is seen when staining with patient sera IgE positive to α-Gal. These results confirm that the α-Gal epitope is present in I. ricinus and imply host exposure to α-Gal during a tick bite. This provides further evidence that tick bites are associated with IgE responses to α-Gal and red meat allergy.

Dog saliva - an important source of dog allergens.

BACKGROUND: Allergy to dog (Canis familiaris) is a worldwide common cause of asthma and allergic rhinitis. However, dander extract in routine diagnostics is not an optimal predictor of IgE-mediated dog allergy. Our objective was to evaluate saliva as an allergen source for improved diagnostics of allergy to dog. METHODS: IgE-binding proteins in dog saliva and dander extract were analysed by immunoblot and mass spectrometry (LC-MS/MS) using pooled or individual sera from dog-allergic patients (n = 13). Sera from 59 patients IgE positive to dander and 55 patients IgE negative to dander but with symptoms to dog were analysed for IgE against saliva and dander by ELISA. Basophil stimulation with dog saliva and dander extract was measured by flow cytometry among three dog-allergic patients. Additionally, IgE-binding protein profiles of saliva from different breeds were investigated by immunoblot. RESULTS: Greater number and diversity of IgE-binding proteins was found in saliva compared to dander extract and varied among dog breeds. In saliva, Can f 1, 2, 3 and 6 were identified but also four new saliva allergen candidates. The majority of the 59 dog dander-positive sera (n = 44) were IgE positive to dog saliva. Among patients IgE negative to dander, but with symptoms to dog, 20% were IgE positive to saliva. The biological activity of saliva was confirmed by basophil degranulation. CONCLUSIONS: Dog saliva is an allergen source for improved diagnostics of dog allergy. The IgE-binding protein profile of saliva from different dogs varies.

IgE antibodies to animal-derived lipocalin, kallikrein and secretoglobin are markers of bronchial inflammation in severe childhood asthma.

BACKGROUND: Component-resolved allergy diagnostics enables the detection of crossreactive or species-specific allergen components. This study analysed Immunoglobulin E (IgE) profiles to single allergen components in relation to bronchial inflammation in severe childhood asthma. METHODS: Ninety-five schoolchildren were assessed, 39 with controlled mild-to-moderate asthma and 56 uncontrolled severe asthmatics. Allergen components (n = 111) of food allergens, pollen and perennial aeroallergens were analysed using an immunosolid-phase allergen chip. Blood eosinophils (10(9) × l(-1)), bronchial inflammation (FeNO, ppb), lung function (FEV(1)%) and bronchial hyper-responsiveness (BHR) (dose-response slope of methacholine challenge) were measured. RESULTS: A specific IgE response to more than three animal-derived components--lipocalin (nMus m 1, rEqu c 1, Fel d 4, rCan f 1, 2), kallikrein (rCan f 5) and secretoglobin (rFel d 1)--was more common among severe asthmatics compared to children with controlled asthma (n = 14 vs n = 3, P = 0.030). These subjects also displayed higher blood eosinophils (0.65 vs 0.39, P = 0.021), higher Fractional exhaled nitric oxide (38 ppb vs 25 ppb, P = 0.021) and increased BHR (112 vs 28, P = 0.002) compared to other severe asthmatics positive to fewer lipocalin/kallikrein/secretoglobin components. Among all sensitized subjects, there were correlations between specific IgE levels for rFel d 4 and nMus m 1 (r = 0.751, P ≤ 0.001) and for rFel d 4 and rEqu c 1 (r = 0.850, P ≤ 0.001). CONCLUSION: Multi-sensitization towards lipocalin, kallikrein and secretoglobin components is associated with increased bronchial inflammation in severe asthmatics. In addition, crossreactive patterns were observed between different lipocalin components.

Characterization of the dog lipocalin allergen Can f 6: the role in cross-reactivity with cat and horse.

BACKGROUND: Allergy to the domestic dog (Canis familiaris) affects 5-10% of the population in affluent countries. Three of four patients are allergic to more than one pet, which can only partially be explained by cross-reactivity between serum albumins. The lipocalin protein family harbours allergens in mammalian species. METHODS: We set out to clone and characterize a novel dog allergen, and investigate its potential role in cross-sensitization between dog, cat and horse. The gene encoding Can f 6 was amplified from dog skin and bladder cDNA libraries. The corresponding allergen was produced and purified by recombinant techniques and evaluated by SDS-PAGE, size exclusion chromatography, circular dichroism spectra, ELISA and basophil activation test. RESULTS: IgE antibodies to Can f 6 were found in serum from 38% of dog-sensitized subjects. Sequence similarities between the lipocalin allergens Can f 6, Fel d 4 (cat) and Equ c 1 (horse) suggested a probability for cross-reactivity, which was demonstrated by competitive ELISA. The biological relevance of Can f 6 was confirmed by basophil activation test in dog-allergic patients. CONCLUSION: Can f 6 is a new lipocalin dog allergen that cross-reacts with lipocalins from horse and cat. Can f 6 and homologous allergens may contribute to multisensitization and symptoms in individuals allergic to mammals.

Treatment with a Fel d 1 hypoallergen reduces allergic responses in a mouse model for cat allergy.

BACKGROUND: A hypoallergen of the major cat allergen Fel d 1, recombinant (r) Fel d 1 (DTE III), was previously shown to have retained T-cell reactivity and strongly reduced IgE-binding capacity compared to unmodified rFel d 1. Here, we evaluated the therapeutic capacity of rFel d 1 (DTE III) in a mouse model for cat allergy. METHODS: Mice were subcutaneously (s.c.) sensitized with rFel d 1 and subsequently treated (s.c.) with 50 or 200 µg rFel d 1 (DTE III), or 50 µg rFel d 1, prior to intranasal challenge with cat dander extract. Airway hyperreactivity (AHR), cells and cytokines in bronchoalveolar lavage fluid, splenocyte in vitro response, and serum immunoglobulins were analyzed. Seven cat-allergic patients and ten healthy controls were tested for skin prick test (SPT) reactivity to rFel d 1 (DTE III) and rFel d 1. RESULTS: Mice treated with 50 and 200 µg rFel d 1 (DTE III), and 50 µg rFel d 1, produced increased serum levels of rFel d 1-specific IgG1 and IgG2a compared to sham-treated mice. IgG from all treatment groups could block binding of patients' IgE to rFel d 1. The 200 µg rFel d 1 (DTE III) treatment tended to reduce AHR. All mice tolerated treatment with rFel d 1 (DTE III), in contrast to only four of ten treated with rFel d 1. Compared to rFel d 1, the hypoallergen showed a tendency of reduced SPT reactivity. CONCLUSION: The rFel d 1 (DTE III) hypoallergen might be a promising candidate for application in immunotherapy of cat allergy with improved safety and efficacy.

The non-proteolytic house dust mite allergen Der p 2 induce NF-kappaB and MAPK dependent activation of bronchial epithelial cells.

BACKGROUND: House dust mites (HDM) are well-known as a source of indoor aeroallergens and for causing allergic airway diseases. Some proteolytic HDM allergens are known to activate respiratory epithelial cells to produce pro-inflammatory mediators, while there is limited knowledge regarding such activity among non-proteolytic HDM allergens. OBJECTIVE: To investigate whether Der p 2, a major non-proteolytic allergen of Dermatophagoides pteronyssinus, activates respiratory epithelial cells to produce mediators involved in asthma pathogenesis and to elucidate the mechanism of such activation. METHODS: The human bronchial epithelial cell line BEAS-2B, normal human bronchial epithelial (NHBE) cells and the alveolar epithelial cell line A549 were exposed to recombinant Der p 2. Following exposure, we analysed a panel of soluble mediators and cell adhesion receptors involved in asthma pathogenesis by promoting recruitment, survival and binding of inflammatory cells. The involvement of nuclear factor (NF)-kappaB and mitogen-activated protein kinases (MAPKs) was studied using specific inhibitors. RESULTS: Der p 2 activated bronchial BEAS-2B and NHBE cells, but not alveolar A549 cells. In BEAS-2B cells Der p 2 induced dose-dependent up-regulation in both mRNA level and protein secretion of granulocyte-macrophage colony-stimulating factor, IL-6, IL-8, monocyte-chemotactic protein-1 and macrophage inflammatory protein-3alpha. Secretion as well as surface expression of intercellular adhesion molecule (ICAM)-1 was also up-regulated, which was associated with increased adhesion of monocytes to the epithelial cells. The release of cytokines and chemokines was regulated by NF-kappaB and MAPK activation in different ways, while expression of ICAM-1 was solely dependent on NF-kappaB activation. CONCLUSION: These results show that Der p 2 activates respiratory epithelial cells, indicating that this non-proteolytic allergen, in addition to its immunogenic properties, can aggravate respiratory airway disease by adjuvant-like activation of the lung epithelium.

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.

Higher immunoglobulin E antibody levels to recombinant Fel d 1 in cat-allergic children with asthma compared with rhinoconjunctivitis.

BACKGROUND: Current diagnosis of allergy and asthma to cat is confirmed using cat dander extract (CDE). We have previously engineered a recombinant major cat allergen, rFel d 1, with properties identical to the natural molecule. OBJECTIVE: The aim of the study was to evaluate IgE and IgG4 antibodies to rFel d 1 among sera from cat-allergic children and adults suffering from asthma and/or rhinoconjunctivitis (RC) in populations from Sweden and Austria. METHODS: Cat-allergic children and adults from Sweden (n=27 and 31, respectively) and Austria (n=41 and 41) with RC and/or asthma were selected. Sera were tested for IgE and IgG4 antibodies to CDE and rFel d 1 by CAP, and IgE to rFel d 1 by ELISA. Healthy subjects and non-cat-allergic patients (n=75) were included as controls. RESULTS: There was a high correlation between IgE responses to rFel d 1 and CDE among the 140 patients (r(s)=0.85, P<0.001); however, measured levels to rFel d 1 were on average 30% higher (P<0.0001). Ninety-eight percent of patients and none of the controls showed IgE to rFel d 1 and there was a threefold increased risk of asthma for half of the children with the highest IgE levels [odds ratio 3.23; 95% confidence interval (CI), 1.19-8.79] by ELISA. IgE responses to rFel d 1 among children with asthma were higher (median 19.4 kU/L) compared with children with RC (median 6.6 kU/L, P<0.05) and adults with asthma (median 3.0 kU/L, P<0.01). Furthermore, children with asthma displayed higher IgG4 levels than the asthmatic adults. CONCLUSION: A single recombinant molecule, rFel d 1, is at least as sensitive for in vitro diagnostics of cat allergy as the current extract-based test. Elevated IgE antibody levels to Fel d 1 are suggested to be a risk factor for asthma in cat-allergic children.

Variability of IgE reactivity profiles among European mite allergic patients.

BACKGROUND: House dust mites (HDM) Dermatophagoides pteronyssinus are a frequent indoor allergen source. Our aim was to determine the frequencies of IgE reactivity to purified HDM allergen molecules in mite allergic patients from different parts of Europe in order to establish an allergen panel for diagnosis of HDM allergy. MATERIALS AND METHODS: Populations of D. pteronyssinus-allergic patients from Austria (n = 56), France (n = 55), Italy (n = 67) and Sweden (n = 65) and storage mite allergic patients from Sweden (n = 31) were analysed for IgE reactivity to eight purified natural (n) and recombinant (r) D. pteronyssinus allergens (nDer p 1, rDer p 2, nDer p 4, rDer p 5, rDer p 7, rDer p 8, rDer p 10 and rDer p 14) in RAST-based dot blot assays. RESULTS: Using a combination of Der p 1 and Der p 2, at least 97% of the D. pteronyssinus-allergic patients could be diagnosed in each of the HDM allergic populations. However, more than 50% of the patients also reacted with other allergens and significant variabilities regarding the frequencies of IgE reactivity to individual allergen molecules were found. Patients with a predominant storage mite allergy showed none or only very weak IgE reactivity to purified D. pteronyssinus allergens. CONCLUSIONS: Purified Der p 1 and Der p 2 are sufficient for the diagnosis of > or = 97% of D. pteronyssinus allergic patients in Europe, but other allergens may also play an important role for the diagnosis and treatment of HDM allergy.

Carbohydrate-based particles reduce allergic inflammation in a mouse model for cat allergy.

BACKGROUND: Allergen-specific immunotherapy (ASIT) is the only treatment of allergic disease that gives long-lasting relief of symptoms. However, concerns for safety and efficiency have highlighted the need for improvement of the therapy. We have previously suggested carbohydrate-based particles (CBPs) as a novel adjuvant and allergen carrier for ASIT. Our aim of this study was to evaluate the therapeutic potential of CBPs in ASIT, employing a mouse model for cat allergy. METHODS: BALB/c mice were subcutaneously immunized with the recombinant (r) cat allergen Fel d 1 followed by intranasal challenge with cat dander extract (CDE). The sensitized mice were therapeutically treated with rFel d 1 covalently coupled to CBPs (CBP-rFel d 1). Airway hyper-reactivity (AHR), infiltration of leucocytes in bronchoalveolar lavage (BAL) fluid, allergen-specific serum immunoglobulin levels and in vitro splenocyte responses were evaluated. RESULTS: Mice treated with CBP-rFel d 1 showed reduced features of allergic inflammation. They responded with (i) significantly decreased AHR and infiltration of eosinophils in BAL fluid after CDE challenge, (ii) the serum level of rFel d 1-specific IgE was reduced and the level of IgG(2)a was more pronounced after CBP-rFel d 1 treatment, and (iii) there was also a tendency of decreased allergen-specific cellular response. CONCLUSIONS: Carbohydrate-based particles are effective tools as adjuvant and allergen carriers for use in ASIT and constitutes a promising strategy to improve allergy treatment.

The immunoglobulin-like modules Cepsilon3 and alpha2 are the minimal units necessary for human IgE-FcepsilonRI interaction.

Atopic allergy is a genetically determined immunodisorder that affects almost 20% of the population worldwide. Immediate symptoms of type I allergy are caused by the release of biologic mediators from effector cells induced by IgE-allergen complexes that cross-link the high-affinity receptor for IgE (FcepsilonRI). Chronic disease manifestations result from allergen-specific T-cell activation, a process that is enhanced when allergens are presented via FcepsilonRI-bound IgE. We report the baculovirus expression, as soluble recombinant proteins, of the minimal units required for human IgE and FcepsilonRI interaction: Cepsilon3 represents the third constant domain of the IgE heavy chain, and alpha2 is the membrane-proximal Ig-like module from FcepsilonRIalpha. Native overlay experiments showed binding of human FcepsilonRIalpha to recombinant Cepsilon3 and of natural or recombinant human IgE to recombinant alpha2. Moreover, recombinant Cepsilon3 inhibited binding of natural IgE antibodies to alpha2, and preincubation of human IgE with alpha2 inhibited anti-IgE-triggered histamine release from human basophils. Isolated Cepsilon3 and alpha2 can now be used for the molecular and structural analysis of the IgE-FcepsilonRI interaction, as well as for diagnostic and therapeutic applications.