IgE to cyclophilins in pollen-allergic children: Epidemiologic, clinical, and diagnostic relevance of a neglected panallergen.

BACKGROUND: Cyclophilins are ubiquitous panallergens whose epidemiologic, diagnostic, and clinical relevance is largely unknown and whose sensitization is rarely examined in routine allergy practice. OBJECTIVE: We investigated the epidemiologic, diagnostic, and clinical relevance of cyclophilins in seasonal allergic rhinitis and its comorbidities. METHODS: We examined a random sample of 253 (25%) of 1263 Italian children with seasonal allergic rhinitis from the Panallergens in Pediatrics (PAN-PED) cohort with characterized disease phenotypes. Nested studies of sensitization prevalence, correlation, and allergen extract inhibition were performed in patients sensitized to birch pollen extract but lacking IgE to Bet v 1/2/4 (74/1263) or with highest serum level of IgE to Bet v 1 (26/1263); and in patients with sensitization to various extracts (ragweed, mugwort, pellitory, Plantago, and plane tree), but not to their respective major allergenic molecule, profilins, and polcalcins. IgE to cyclophilin was detected with recombinant Bet v 7, and extract inhibition tests were performed with the same rBet v 7. RESULTS: IgE to rBet v 7 was detected in 43 (17%) of 253 patients. It was associated with asthma (P < .028) and oral allergy syndrome (P < .017) in univariate but not multivariate analysis adjusted for IgE to profilins (Phl p 12), PR-10s (Bet v 1), and lipid transfer proteins (Pru p 3). IgE to rBet v 7 was also highly prevalent (47/74, 63%) among patients with unexplained sensitization to birch pollen extract. In patients with unexplained sensitization to ragweed, mugwort, pellitory, Plantago and plane tree pollen, the levels of IgE to those extracts correlated with the levels of IgE to rBet v 7, and they were also significantly inhibited by rBet v 7 (inhibition range 45%-74%). CONCLUSIONS: IgE sensitization to cyclophilin is frequent in pollen-allergic patients living in temperate areas and can produce "false" positive outcomes in skin prick and IgE tests to pollen extracts. Molecular diagnostic guidelines should include this panallergen family.

Targeting immunodominant Bet v 1 epitopes with monoclonal antibodies prevents the birch allergic response.

BACKGROUND: Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response. OBJECTIVES: This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein in birch pollen, can prevent the birch allergic response. METHODS: Bet v 1-specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy determined binding kinetics and structural data. Inhibition of IgE-binding, basophil activation, and mast cell degranulation were assessed via blocking ELISA, flow cytometry, and the passive cutaneous anaphylaxis mouse model. RESULTS: REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1- and birch pollen extract-induced basophil activation ex vivo and mast cell degranulation in vivo. Crystal structures of the complex of Bet v 1 with immunoglobulin antigen-binding fragments of REGN5713 or REGN5715 show distinct interaction sites on Bet v 1. Cryo-electron microscopy reveals a planar and roughly symmetrical complex formed by REGN5713/14/15 bound to Bet v 1. CONCLUSIONS: These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous binding of REGN5713, REGN5714, and REGN5715 with substantial areas of Bet v 1 exposed, suggesting that targeting specific epitopes is sufficient to block the allergic response.

Elevated Serum Level of CD48 in Patients with Intermittent Allergic Rhinitis.

BACKGROUND: In the pathogenesis of intermittent allergic rhinitis (IAR), the inflammatory reaction is of importance. CD48, belonging to the CD2 family, participates in mast cell-stimulating cross-talk, facilitates the formation of the mast cell/eosinophil effector unit, and is expressed by eosinophils. OBJECTIVES: To assess the serum level of soluble form of CD48 (sCD48) in patients with IAR during and out of the pollen season and correlate with the disease severity and with eosinophil-related parameters. MATERIALS AND METHODS: Sixty-three patients (female: 79%; mean age: 30.58) were included to the study. Forty-five patients were assessed during the pollen season and other 42 patients during out of the pollen season. Twenty-four patients (female: 37.50%; mean age: 27.90) were evaluated twice, during the pollen season and out of the pollen season. sCD48, ECP, eotaxin-1/CCL11 serum levels together with complete blood count, and fractional exhaled nitric oxide bronchial and nasal fraction (FeNO) were performed. The severity of symptoms was assessed using the Total Nasal Symptom Score (TNSS), and neutrophil-to-lymphocyte (NLR) and eosinophil-to-lymphocyte (ELR) ratios were calculated. RESULTS: sCD48 serum level, FeNO nasal and bronchial fractions, and TNSS were significantly higher in the IAR group in the pollen season compared with out of the pollen season. Differences in ECP, eotaxin-1/CCL11 serum levels, and NLR and ELR were not significant between season and out of the season. No correlations were found between sCD48 and eosinophil-related parameters. CONCLUSIONS: sCD48 may be a biomarker to the exacerbation phase in patients with IAR. One can assume that CD48 participates in the pathogenesis of IAR.

Sublingual immunotherapy for 4 years increased the number of Foxp3+ Treg cells, which correlated with clinical effects.

OBJECTIVE: At least 3 years of sublingual immunotherapy (SLIT) is required to achieve long-term clinical tolerance for allergens. However, immunological changes with more than 3 years of SLIT have not yet been elucidated in detail. The present study investigated whether the numbers of regulatory T (Treg) cells and regulatory B (Breg) cells increased with 4 years of SLIT and if these increases correlated with clinical effects for pollinosis. METHODS: Seven Japanese cedar pollinosis patients received SLIT in 2014 or 2015 and continued treatment until May 2019. In May 2017 and May 2019, peripheral blood mononuclear cells (PBMCs) were collected from the patients, and analyzed by flow cytometer. RESULTS: (1) The visual analogue scale (VAS) was significantly higher in 2019 than in 2017. (2) The percentages of Foxp3+ Treg cells, type 1 regulatory T (Tr1) cells, and Breg cells in PBMCs were significantly higher in 2019 than in 2017. (3) The percentage of Foxp3+ Treg cells in PBMCs positively correlated with VAS, whereas those of Tr1 cells and Breg cells did not. CONCLUSIONS: These results suggest that 4 years of SLIT is needed to achieve sustained increases in Foxp3+ Treg cells, which are closely associated with the efficacy of SLIT.

Kidney-bean (Phaseolus Vulgaris) Dependent, Exercise-induced Anaphylaxis in Patients Comorbid with Mugwort (Artemisia Vulgaris) Pollinosis.

Background: The cross-reactive allergen between mugwort (Artemisia vulgaris) and kidney bean (Phaseolus vulgaris) has not yet been identified.Methods: A total of 24 patients were included in this study. The sera of patients were analyzed for the concentrations of specific IgE antibodies. The allergenicity and cross-reactivity were investigated by Western blotting and immunoblot inhibitory experiments.Results: The immunoblotting indicated the binding of patients' IgE to crude mugwort extract at ~26 kDa protein (15 cases), ~60 kDa (15 cases), and 10-15 kDa proteins (12 cases). The results of the immunoblot-inhibition assay showed that kidney bean seed extract inhibited specific IgE binding to mugwort at 10-15 kDa, ~26 kDa, and ~60 kDa in 4 (16.7%), 1 (4.2%) and 2 (8.3%) cases, respectively. On the other hand, mugwort extract was demonstrated to inhibit specific IgE binding to kidney bean seed at 10-15 kDa, 15-20 kDa, ~30 kDa, and 60 kDa in 1 (4.2%), 3 (12.5%), 4 (16.7%), and 3 (12.5%) cases, respectively.Conclusion: The 26-30 kDa, 10-15 kDa, and 60 kDa proteins are potential causative agents of the cross-reactivity between mugwort and kidney beans. The findings of this study improved the current understanding on the allergenicity of kidney beans and would provide insights into the refinement of treatment strategy for anaphylaxis.

Defining biomarkers to predict symptoms in subjects with and without allergy under natural pollen exposure.

BACKGROUND: Pollen exposure induces local and systemic allergic immune responses in sensitized individuals, but nonsensitized individuals also are exposed to pollen. The kinetics of symptom expression under natural pollen exposure have never been systematically studied, especially in subjects without allergy. OBJECTIVE: We monitored the humoral immune response under natural pollen exposure to potentially uncover nasal biomarkers for in-season symptom severity and identify protective factors. METHODS: We compared humoral immune response kinetics in a panel study of subjects with seasonal allergic rhinitis (SAR) and subjects without allergy and tested for cross-sectional and interseasonal differences in levels of serum and nasal, total, and Betula verrucosa 1-specific immunoglobulin isotypes; immunoglobulin free light chains; cytokines; and chemokines. Nonsupervised principal component analysis was performed for all nasal immune variables, and single immune variables were correlated with in-season symptom severity by Spearman test. RESULTS: Symptoms followed airborne pollen concentrations in subjects with SAR, with a time lag between 0 and 13 days depending on the pollen type. Of the 7 subjects with nonallergy, 4 also exhibited in-season symptoms whereas 3 did not. Cumulative symptoms in those without allergy were lower than in those with SAR but followed the pollen exposure with similar kinetics. Nasal eotaxin-2, CCL22/MDC, and monocyte chemoattactant protein-1 (MCP-1) levels were higher in subjects with SAR, whereas IL-8 levels were higher in subjects without allergy. Principal component analysis and Spearman correlations identified nasal levels of IL-8, IL-33, and Betula verrucosa 1-specific IgG4 (sIgG4) and Betula verrucosa 1-specific IgE (sIgE) antibodies as predictive for seasonal symptom severity. CONCLUSIONS: Nasal pollen-specific IgA and IgG isotypes are potentially protective within the humoral compartment. Nasal levels of IL-8, IL-33, sIgG4 and sIgE could be predictive biomarkers for pollen-specific symptom expression, irrespective of atopy.

Characterization of a 7 kDa pollen allergen belonging to the gibberellin-regulated protein family from three Cupressaceae species.

BACKGROUND: Severe allergy to fruits mediated by a 7 kDa allergen belonging to the gibberellin-regulated protein (GRP) family is known to be associated with Cupressaceae pollinosis. OBJECTIVE: To identify and characterize Cupressaceae pollen allergens involved in GRP-related fruit allergy. METHODS: Pru p 7-related proteins from pollen of Cupressus sempervirens, Juniperus ashei and Cryptomeria japonica were identified using a rabbit anti-Pru p 7 antiserum, purified chromatographically and sequenced by mass spectrometry and bioinformatic comparisons. The C sempervirens protein was produced as a recombinant allergen in Pichia pastoris. IgE antibody binding to pollen GRP proteins was analysed in a peach allergic (n = 54) and a cypress pollen allergic (n = 88) patient population from southern France using ImmunoCAP. RESULTS: In each of the three Cupressaceae species studied, a 7 kDa pollen protein related to Pru p 7 was identified and found to comprise an amino acid sequence of 63 residues in length, 92%-98% identical to each other and 67%-68% identical to Pru p 7. The C sempervirens, J ashei and C japonica GRP allergens have been officially recognized by the WHO/IUIS Allergen Nomenclature Sub-Committee and named Cup s 7, Jun a 7 and Cry j 7, respectively. Recombinant Cup s 7 showed IgE antibody binding capacity comparable to that of the purified natural allergen. Among 51 peach allergic subjects sensitized to Pru p 7, substantially higher levels of IgE to Cup s 7 than to Pru p 7 were found. Further, the pollen protein was able to completely outcompete IgE binding to Pru p 7, while the reverse competition effect was modest, consistent with primary sensitization by the pollen allergen. CONCLUSION AND CLINICAL RELEVANCE: Pru p 7-related pollen allergens from three Cupressaceae species have been characterized and may become useful for the identification of pollinosis patients at risk of developing severe fruit allergy.

Peptide Glycodendrimers as Potential Vaccines for Olive Pollen Allergy.

Olive pollen is one of the most important causes of respiratory allergy, with Ole e 1 being the most clinically relevant sensitizing allergen. Peptide-based vaccines represent promising therapeutic approaches, but the use of adjuvants is required to strengthen the weak immunogenicity of small peptides. We propose the use of dendrimeric scaffolds conjugated to the T cell immunodominant epitope of Ole e 1 (OE109-130) for the development of novel vaccines against olive pollen allergy. Four dendrimeric scaffolds containing an ester/ether with nine mannoses, an ester succinimidyl linker with nine N-acetyl-glucosamine units or nine ethylene glycol units conjugated to OE109-130 peptide were designed, and their cytotoxicity, internalization pattern, and immunomodulatory properties were analyzed in vitro. None of the dendrimers exhibited cytotoxicity in humanized rat basophil (RBL-2H3), human bronchial epithelial Calu-3, and human mast LAD2 cell lines. Confocal images indicated that mannosylated glycodendropeptides exhibited lower colocalization with a lysosomal marker. Moreover, mannosylated glycodendropeptides showed higher transport tendency through the epithelial barrier formed by Calu-3 cells cultured at the air-liquid interface. Finally, mannosylated glycodendropeptides promoted Treg and IL10+Treg proliferation and IL-10 secretion by peripheral blood mononuclear cells from allergic patients. Mannosylated dendrimers conjugated with OE109-130 peptide from Ole e 1 have been identified as suitable candidates for the development of novel vaccines of olive pollen allergy.

IgE antibody repertoire in nasal secretions of children and adults with seasonal allergic rhinitis: A molecular analysis.

BACKGROUND: There is growing interest both in testing IgE in nasal secretions (NS) and in molecular diagnosis of seasonal allergic rhinitis (SAR). Yet, the reliability of nasal IgE detection with the newest molecular assays has never been assessed in a large cohort of pollen allergic patients. OBJECTIVE: To investigate with microarray technology and compare the repertoires of specific IgE (sIgE) antibodies in NS and sera of a large population of children and adults with SAR. METHODS: Nasal secretions were collected with an absorbent device (Merocel 2000® , Medtronic) and a minimal dilution procedure from 90 children and 71 adults with SAR. Total IgE (tIgE) (ImmunoCAP, Thermo Fisher Scientific (TFS)) and sIgE antibodies against 112 allergen molecules (ISAC-112, TFS) were measured in NS and serum. RESULTS: Nasal sIgE was detectable in 68.3% of the patients. The detected nasal sIgE antibodies recognized airborne (88%), vegetable (10%), and animal food or other (<1%) allergen molecules. The prevalence and average levels of sIgE in NS and serum were highly interrelated at population level. A positive nasal sIgE antibody to a given molecule predicted the detection of the same antibody in the patient's serum with a specificity of 99.7% and a sensitivity of 40%. CONCLUSIONS: The concentration of sIgE is much lower in nasal secretions than in the serum. sIgE assays with very high analytical sensitivity and sampling methods with minimal dilution will be therefore needed to validate nasal secretions as alternative to serum in testing the sIgE repertoire.

Clinical symptoms and biomarkers of Bermuda grass-induced allergic rhinitis using the nasal allergen challenge model.

BACKGROUND: Bermuda grass is a prevalent allergen that flourishes in tropical climates. Its exposure is traditionally believed to be low in Ontario due to the colder environment. However, high sensitization rates have been observed in Kingston, Ontario. OBJECTIVE: We sought to investigate whether its allergens can provoke allergic rhinitis (AR) symptoms in sensitized participants from south-eastern Ontario and determine if nasal allergen challenge (NAC) model is appropriate to study Bermuda grass-induced AR. METHODS: Twenty-one participants sensitized to Bermuda grass and 12 nonallergic participants completed a titrated NAC with increasing allergen concentrations at a screening visit. Total nasal symptom score (TNSS) and peak nasal inspiratory flow were collected before allergen exposure and 10 minutes after delivery of each concentration. Twelve participants with a Bermuda grass allergy who met the qualifying criteria (TNSS ≥ 8 and peak nasal inspiratory flow fall ≥ 50%) and 11 nonallergic controls returned for single-dose NAC visit. RESULTS: At titrated NAC, 19 of 21 sensitized participants met the criteria of positive allergic response when challenged. During single-dose NAC, participants with allergy had significantly greater TNSS between 15 minutes and 3 hours after NAC than controls. Likewise, allergic participants had a significantly increased number of nasal lavage eosinophils at both 1 and 6 hours after NAC. Bermuda grass-specific immunoglobulin E was significantly increased in Bermuda grass allergic participants at NAC than screening visit. CONCLUSION: Although Bermuda grass is a non-native allergen in Ontario, it can induce AR symptoms in sensitized participants, and the NAC model is appropriate to study Bermuda grass-induced AR.

A new molecular multiplex IgE assay for the diagnosis of pollen allergy in Mediterranean countries: A validation study.

BACKGROUND: The identification of the primary sensitizing pollen is difficult in Southern European patients with Seasonal Allergic Rhinitis (SAR) if sensitized to various pollen sources with overlapping seasonality. A more precise diagnosis is obtained by IgE assays to allergen molecules, currently available as singleplex or microarrays. OBJECTIVES: To test the analytical performance of a multi-parameter immunoblot molecular "Pollen Test" specifically designed to test IgE antibodies to pollen extracts and molecules clinically relevant in Southern Europe. METHODS: Sera were obtained from 101 children and 98 adults with SAR and tested with a customized multiplex immunoblot assay (EUROLINE Southern European Pollen Profile [ESEP]; EUROIMMUN AG, Luebeck, Germany) containing a comprehensive panel of allergen extracts and molecules. ESEP's outcomes were then compared in selected sera (ESEP positive to negative = 2:1) with those of singleplex IgE assays (ImmunoCAP; ThermoFisher Scientific, Uppsala, Sweden). For each of the examined reagents, qualitative (sensitivity, specificity, accuracy), semi-quantitative (classes) and quantitative (Spearman's rank correlation, Bland-Altmann plots) comparisons were performed. RESULTS: Compared to ImmunoCAP, cumulative ESEP's sensitivity and specificity were 87% (95% CI 84%-90%) and 88% (83%-93%) for extracts and 99% (98%-100%) and 87% (83%-91%) for molecules. Cohen's kappa coefficients (κC ) ranged for extracts from 0.18 (Pellitory) to 0.50 (Cypress) and for molecules from 0.21 (Ole e 1) to 0.68 (Phl p 7). The quantitative outcomes of the two diagnostic tests were highly correlated, with Spearman's rank correlation coefficients always exceeding 0.80. Bland-Altmann plots showed a tendency of ESEP to overestimate serum specific IgE levels, when compared to ImmunoCAP. CONCLUSIONS AND CLINICAL RELEVANCE: Sensitivity and specificity of ESEP in testing serum IgE antibodies against pollen allergen extracts and molecules, in Italian patients with SAR, both exceeded 85%. The advantages and limitations of a multiplex customized immunoblot assay, in the routine clinical use of molecular diagnostics in Southern European pollen allergic patients, deserve to be tested.

Diverse age-incidence patterns of atopic sensitization in an unselected Finnish population up to 12 years.

BACKGROUND: The temporal sequence in which allergic sensitization to different allergens emerges is not well characterized at the level of general population. OBJECTIVE: We describe the incidence patterns of atopic sensitization to different allergens from birth up to 12 years of age in an unselected Finnish population. METHODS: The study population comprised all children born between 2001 and 2006 identified from the nationwide population register as residents of the province of South Karelia, Finland (n = 5564). The results of allergy tests (22,380 results from skin prick tests, immunoglobulin E [IgE] antibodies, and open food challenges [OFCs], performed in 1827 children) were collected from patient records of all the health care units in the area. RESULTS: The incidence rates of positive results for food and animal allergens as well as positive OFCs for cow's milk showed prominent peaks at 5 months of age. Positive results for pollen allergens started to emerge after 1.5 years of age. The 12-year cumulative incidence of sensitization to food, animal, pollen, and any allergens was 12%, 8%, 10%, and 18%, respectively. The cumulative incidence of sensitization to house dust mites was 1% and to molds or latex less than 1%. Firstborn boys had the highest, and those who were not firstborn girls and children born in rural municipalities had the lowest early incidence of sensitization to inhalation allergens. CONCLUSION: In the unselected population, the atopic sensitization against food and animal allergens began before 6 months of age and was followed by sensitization to pollen allergens before 2 years of age. Primary prevention of sensitization to food and inhalation allergens should therefore occur in early infancy.

Proteomic Identification of Allergenic Proteins of Morus alba L. Pollenexacerbation.

BACKGROUND: Tree pollens are well-known aeroallergens all over the world. Little is known about the allergenicity of Morus alba (white mulberry) pollen. OBJECIVE: We aimed to explore the potential allergens of this pollen and its clinical relevance in tree pollen allergic patients living in Istanbul, Turkey. METHODS: Twenty three seasonal allergic rhinitis patients with a confirmed tree pollen allergy and 5 healthy control subjects underwent skin prick and nasal provocation tests with M.alba pollen extract. The pollen extract was then resolved by gel electrophoresis, and immunoblotted with sera from patients/control individuals to detect the potential allergenic proteins. The prevalent IgE binding proteins from 1D-gel were analyzed by MALDI-TOF/TOF. RESULTS: Eleven out of 23 patients were reactive to the extract with skin prick tests. Seven of those patients also reacted positively to the nasal provocation tests. The most common IgE-binding pollen proteins were detected between 55-100 kDa, and also at molecular weights lower than 30 kDa for some patients. Mass spectrometry analyses revealed that the principal IgE-binding protein was methionine synthase (5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase), which is then proposed as a novel allergen in M.alba pollen. CONCLUSION: This study provides the first detailed information for the potential allergens of Morus alba pollen of Istanbul. Methionine synthase with an apparent molecular weight of 80 to 85 kDa has been recognized as one of the allergens in Morus alba pollen for the first time.

Analysis of factors associated with cedar pollen sensitization and development of pollinosis in a young Japanese adult population.

BACKGROUND: Genetic and environmental factors are proposed to be involved in cedar pollen allergy sensitization and onset. The impact of these factors will provide key information for the prevention of cedar pollen sensitization and allergy onset, which we investigated in this cross-sectional study. METHODS: Subjects were 382 young adult volunteers who completed a self-administered questionnaire on self-reported subjective symptoms of pollinosis, physician-diagnosed pollinosis, and background factors. We also measured their serum IgE antibody titers specific for cedar, cypress, and mites. Factors associated with subjective symptoms, physician diagnosis, and the three specific antigens were determined using both univariate and multivariate analyses. RESULTS: Sensitization to cedar, cypress, and mites, defined as specific IgE levels of class 1 or above, was found in 78.8%, 64.4%, and 56.0% of subjects, respectively. The prevalence of cedar pollinosis was 41.2% based on subjective symptoms and 22.2% based on physician diagnosis. Factors associated with increased cedar pollen sensitization were mite sensitization, comorbid allergic rhinitis, and family history of cedar pollinosis. Risk-reducing factors for cedar pollen sensitization were keeping a cat, number of common colds, and hours of sleep. Risk-increasing factors for both subjective pollinosis symptoms and physician-diagnosed pollinosis were comorbid allergic rhinitis and family history of cedar pollinosis. CONCLUSIONS: Sensitization to cedar pollen in this population was extremely high. Both common and distinct factors were associated with sensitization to pollen and with the development of pollinosis. The distinct factors were associated with sensitization to cedar and cypress antigens.

Analysis of the Serum Cytokine Profile in Allergic Patients Opens a Way to Personalized Treatment of Allergy.

Plant lipid transfer proteins and homologues of the main birch pollen allergen Bet v 1 are involved in the development of allergic reactions of varying severity to plant foods and pollen. In this study, the sera from patients with tree and weed pollen allergies in the Moscow region were examined. The levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, IFNγ, TNFα, and TNFß cytokines were determined in the sera of patients with specific IgE antibodies to Bet v 1 and Pru p 3 allergens. It was confirmed that patients with pollen allergy are often characterized by Th2 response of the immune system, though other mechanisms of allergy development occurred in some cases. The data obtained demonstrate the necessity of detailed analysis of the individual mechanism of allergic reactions and patient-centered approach to the personalized allergy treatment.

Diagnostic relevance of IgE sensitization profiles to eight recombinant Phleum pratense molecules.

BACKGROUND: Grass pollen-related seasonal allergic rhinoconjunctivitis (SARg) is clinically heterogeneous in severity, comorbidities, and response to treatment. The component-resolved diagnostics disclosed also a high heterogeneity at molecular level. Our study aimed at analyzing the characteristics of the IgE sensitization to Phleum pratense molecules and investigating the diagnostic relevance of such molecules in childhood. METHODS: We examined 1120 children (age 4-18 years) with SARg. Standardized questionnaires on atopy were acquired through informatics platform (AllergyCARD™). Skin prick tests were performed with pollen extracts. Serum IgE to airborne allergens and eight P. pratense molecules (rPhl p 1, rPhl p 2, rPhl p 4, rPhl p 5b, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12) were tested by ImmunoCAP FEIA. RESULTS: The analysis of IgE responses against eight P. pratense molecules showed 87 profiles. According to the number of molecules recognized by IgE, the more complex profiles were characterized by higher serum total IgE, higher grass-specific serum IgE, and higher number and degree of sensitization to pollens. The most frequent IgE sensitization profile was the monomolecular Phl p 1. Sensitization to Phl p 7 was a reliable biomarker of asthma, whereas Phl p 12 of oral allergy syndrome. Sensitization to Phl p 7 was associated with a higher severity of SARg, and complex profiles were associated with longer disease duration. CONCLUSIONS: In a large pediatric population, the complexity of IgE sensitization profiles against P. pratense molecules is related to high atopic features although useless for predicting the clinical severity. The detection of serum IgE to Phl p 1, Phl p 7, and Phl p 12 can be used as clinical biomarkers of SARg and comorbidities. Further studies in different areas are required to test the impact of different IgE molecular profiles on AIT response.

Do Plantago lanceolata Skin Prick Test-Positive Patients Display IgE to Genuine Plantain Pollen Allergens? Investigation of Pollen Allergic Patients from the North-East of France.

BACKGROUND: English plantain (Plantago lanceolata) is an important weed pollen allergen source triggering allergic symptoms during summer. To elucidate genuine versus cross-reactive sensitization, we investigated IgE reactivity patterns and inhibition capacities of plantain-sensitized patients. METHODS: Sera of 35 rhinoconjunctivitis patients from the north-east of France with positive skin prick tests (SPT) to Plantago lanceolata pollen were tested with clinically relevant allergen sources using ELISA, ImmunoCAP, and immunoblot inhibition. RESULTS: The patients were multisensitized with additional reactivity to grass (94.3%), ash (74.3%), birch (71.4%), and mugwort (55.2%) pollen in SPT. Sensitization prevalence to allergen molecules was 34.3% (Pla l 1), 94.3% (Phl p 1/5), 60.0% (Ole e 1), 65.7% (Bet v 1), 37.1% (profilin), and 40.0% (CCD). In immunoblot, IgE reactivity to plantain pollen was inhibited with relevant pollen extracts and purified rPla l 1. Two sera did not reveal any IgE cross-reactivity, while reactivity to plantain was efficiently inhibited by grass pollen in the sera of 10 patients. The sera from 17 different patients could be inhibited by grass, birch, or ash pollen to varying degrees. Thus, only 37.1% of our patients demonstrated true plantain pollen sensitization, while 62.9% were solely positive due to IgE cross-reactive molecules from other clinically relevant pollen. CONCLUSIONS: Plantain pollen-sensitized patients are multi-reactors demonstrating varying and complex IgE-reactivity profiles. In vivo and in vitro tests using extracts are typically blurred due to the presence of homologous allergens or CCD in grass, birch, or ash pollen. So far, Pla l 1 represents the only indicative marker allergen for the diagnosis of genuine plantain pollen sensitization.

Misleading Allergens in the Diagnosis of Latex Allergy: Profilin and Cross-Reactive Carbohydrate Determinants.

BACKGROUND: It has been suggested that latex-specific IgE analysis may lead to false-positive results, especially in patients with pollen allergy. In the present study, the reasons underlying clinically irrelevant latex-specific IgE positivity were investigated. METHODS: Thirty patients with latex allergy (group 1), 89 patients sensitised to aeroallergens (group 2a), and 98 healthy individuals without allergy (group 2b) were enrolled. Participants from all 3 groups were subjected to skin prick tests with aeroallergens including latex, latex-specific IgE analysis (ImmunoCAP), and nasal provocation test with latex. All cases demonstrating positive latex-specific IgE also underwent specific IgE tests (ImmunoCAP) with latex profilin, birch pollen profilin, peach lipid transfer protein, and pineapple bromelain as cross-reactive carbohydrate determinants. RESULTS: Comparison of the atopic and healthy control groups showed that the rate of positive latex-specific IgE was significantly higher in group 2a. Latex profilin-, birch pollen profilin-, and bromelain-specific IgE were remarkably higher in group 2a. CONCLUSION: False positivity to latex-specific IgE in ImmunoCAP analysis may be observed in approximately 19% of patients with pollen allergy. Profilins and bromelain are the main contributors to clinically irrelevant positive latex-specific IgE.

Molecular sensitization patterns and influence of molecular diagnosis in immunotherapy prescription in children sensitized to both grass and olive pollen.

BACKGROUND: The overlapping grass and olive pollen seasons in Spain and the phenomenon of cross-reactivity can make it difficult to determine the true causative agent of seasonal allergic rhinitis when only skin prick tests with whole extracts are used. The aim of the GRAMOLE study was to determine sensitization patterns to the major grass and olive pollen allergens detected using specific recombinant IgE and to explore how this knowledge affected physicians' choice of allergen-specific immunotherapy. METHODS: Epidemiological, observational, multicenter, cross-sectional study. Results from children under 18 years of age diagnosed with seasonal allergic rhinitis by positive skin prick tests to olive and grass pollen were analyzed. Specific IgE to Phl p 1+5, Ole e 1, and Phl p 7+12 was determined. Investigators specified the optimal composition of allergen immunotherapy before and after knowing the results of the molecular diagnosis. RESULTS: A total of 281 patients with a mean age of 13.4 years were included. Double sensitization to both major allergens was found in vitro in 76% of children for an IgE cutoff point of 0.35 kU/L. When the molecular diagnosis results were known, specialists changed the composition of the prescribed immunotherapy in 52.87% of cases. CONCLUSIONS: Double sensitization to grass and olive pollen is common in Spain and also occurs in the pediatric population. Molecular diagnosis using specific IgE may help improve immunotherapy selection in polysensitized patients.

Intradermal grass pollen immunotherapy increases TH2 and IgE responses and worsens respiratory allergic symptoms.

BACKGROUND: Repeated low-dose grass pollen intradermal allergen injection suppresses allergen-induced cutaneous late-phase responses comparably with conventional subcutaneous and sublingual immunotherapy. OBJECTIVE: We sought to evaluate the efficacy and safety of grass pollen intradermal immunotherapy in the treatment of allergic rhinitis. METHODS: We randomly assigned 93 adults with grass pollen-induced allergic rhinitis to receive 7 preseasonal intradermal allergen injections (containing 7 ng of Phl p 5 major allergen) or a histamine control. The primary end point was daily combined symptom-medication scores during the 2013 pollen season (area under the curve). Analysis was by intention to treat. Skin biopsy specimens were collected after intradermal allergen challenges, and late-phase responses were measured 4 and 7, 10, or 13 months after treatment. RESULTS: There was no significant difference in the primary end point between treatment arms (active, n = 46; control, n = 47; median difference, 14; 95% CI, -172.5 to 215.1; P = .80). Among secondary end points, nasal symptoms were worse in the intradermal treatment group, as measured based on daily (median difference, 35; 95% CI, 4.0-67.5; P = .03) and visual analog scale (median difference, 53; 95% CI, -11.6 to 125.2; P = .05) scores. In a per-protocol analysis intradermal immunotherapy was further associated with worse asthma symptoms and fewer symptom-free days. Intradermal immunotherapy increased serum Phleum pratense-specific IgE levels (P = .001) compared with those in the control arm. T cells cultured from biopsy specimens of subjects undergoing intradermal immunotherapy had higher expression of the TH2 surface marker CRTH2 (P = .04) and lower expression of the TH1 marker CXCR3 (P = .01), respectively. Late-phase responses remained inhibited 7 months after treatment (P = .03). CONCLUSION: Intradermal allergen immunotherapy suppressed skin late-phase responses but was not clinically effective and resulted in worsening of respiratory allergic symptoms.