Crystal structure of timothy grass allergen Phl p 12.0101 reveals an unusual profilin dimer.

Timothy grass pollen is a source of potent allergens. Among them, Phl p 1 and Phl p 5 are thought to be the most important, as a majority of timothy grass-allergic individuals have IgE antibodies directed against these two allergens. The profilin from timothy grass (Phl p 12) has been registered as a minor allergen, with up to 35% of individuals in populations of grass pollen allergic patients showing IgE binding to Phl p 12. Profilins are primarily minor allergens and are known for a high likelihood of co-sensitization as well as cross-reactivity situations caused by their sequence and structure similarity. The crystal structure of Phl p 12.0101 was determined and it revealed that this allergen may form an unusual dimer not previously observed among any profilins. For example, the Phl p 12 dimer has a completely different geometry and interface when compared with the latex profilin (Hev b 8) dimer that has its crystal structure determined. The structure of Phl p 12.0101 is described in the context of allergenic sensitization and allergy diagnostics. Moreover, the structure of the Phl p 12.0101 dimer is discussed, taking into account the production of recombinant allergens and their storage.

Soy ß-Conglycinin Peptide Attenuates Obesity and Lipid Abnormalities in Obese Model OLETF Rats.

We previously reported that soy ß-conglycinin (ßCG) improves obesity-induced metabolic abnormalities, but not obesity, in obese model Otsuka Long-Evans Tokushima fatty (OLETF) rats. In the present study, we aimed to investigate the effects of ßCG-derived peptide consumption on obesity and lipid abnormality in OLETF rats. To this end, wild-type Long-Evans Tokushima Otsuka and OLETF rats were provided a normal diet containing 20% casein for four weeks as a control. In addition, we prepared ßCG peptide by enzymatic hydrolysis, and OLETF rats were fed a diet in which half of the casein was replaced by ßCG peptide (ßCG peptide group). Consequently, rats in the ßCG peptide group showed decreased abdominal white adipose tissue weight and lipid content (serum and liver triglycerides, and serum and liver cholesterol) compared to control OLETF rats. Further analysis demonstrated that ßCG peptide consumption decreased lipogenic enzyme activity and increased lipolytic enzyme activity in the liver of OLETF rats. In addition, suppressive effects on both synthesis and absorption of cholesterol were observed in ßCG peptide-fed OLETF rats. These findings suggest that peptidization of ßCG enhanced the anti-obese and hypolipidemic effects of ßCG.

Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen.

INTRODUCTION: Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera. METHODS: Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract. RESULTS: Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too. CONCLUSION: The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.

Structural basis for cross-reactivity and conformation fluctuation of the major beech pollen allergen Fag s 1.

Fag s 1 is a member of the Pathogen Related protein family 10 (PR-10) and can elicit cross-reaction with IgE antibodies produced against the birch pollen allergen Bet v 1. The Nuclear Magnetic Resonance (NMR) structure of Fag s 1 is presented along with its dynamic properties. It shares 66% identity with Bet v 1 and exhibits the expected three α-helices and seven ß-sheets arranged as a semi-beta barrel and exposing the residues mapped as the Bet v 1 IgE epitope. The structural dynamics of Fag s 1 were monitored on the fast and intermediate timescales, using relaxation rates. The complex dynamics of Fag s 1 are closely related to the internal cavity, and they modulate IgE and ligand binding.

A subset of walnut allergic adults is sensitized to walnut 11S globulin Jug r 4.

BACKGROUND: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. OBJECTIVES: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. METHODS: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. RESULTS: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. CONCLUSIONS: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1.

Expression, purification and epitope analysis of Pla a 2 allergen from Platanus acerifolia pollen.

Platanus acerifolia is one of the major sources of outdoor allergens to humans, and can induce allergic asthma, rhinitis, dermatitis and other allergic diseases. Pla a 2 is a polygalacturonase and represents the major allergen identified in P. acerifolia pollen. The aim of the present study was to express and purify Pla a 2, and to predict B and T cell epitopes of Pla a 2. The gene encoding Pla a 2 was cloned into the pET28a vector and subsequently transfected into ArcticExpress™ (DE3) Escherichia coli cells; purified Pla a 2 was analyzed by western blot analysis. The results of the present study revealed that the Pla a 2 allergen has the ability to bind immunoglobulin E within the sera of patients allergic to P. acerifolia pollen. In addition, the B cell epitopes of Pla a 2 were predicted using the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides and BepiPred 1.0 software; T cell epitopes were predicted using NetMHCIIpan ­3.0 and ­2.2. In total, eight B cell epitopes (15­24, 60­66, 78­86, 109­124, 232­240, 260­269, 298­306 and 315­322) and five T cell epitopes (62­67, 86­91, 125­132, 217­222 and 343­350) were predicted in the present study. These findings may be used to improve allergen immunotherapies and reduce the frequency of pollen­associated allergic reactions.

Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody.

Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET­28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody­containing ascites fluid, and the antibodies were purified using protein A­agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E­binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti­Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti­Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.

Expression, purification and epitope analysis of Pla a 3 allergen from Platanus acerifolia pollen.

Platanus acerifolia (P. acerifolia) is an important cause of pollinosis in cities. The use of allergen extracts on patients with allergic diseases is the most commonly applied method to attempt to treat pollinosis. Pla a 3, a non­specific lipid transfer protein, is a major allergen present in P. acerifolia pollen extracts. In the present study, the Pla a 3 gene was sub­cloned into a pSUMO­Mut vector using Stu I and Xho I sites and transformed into the Arctic Express™ (DE3) RP E. coli host strain. The purified Pla a 3 allergen was analyzed by western blotting and the results revealed that the Pla a 3 allergen has the ability to bind IgE in the P. acerifolia pollen of allergic patients' sera. Moreover, the authors predicted the potential B cell epitopes of the Pla a 3 allergen using the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server. In addition, the T cell epitopes were predicted by the SYFPEITHI database and the NetMHCII­2.2 server. As a result, two B cell epitopes (35­45 and 81­86) and four potential T cell epitopes including 2­15, 45­50, 55­61 and 67­73 were predicted in the present study. The current results can be used to contribute to allergen immunotherapies and useful in peptide­based vaccine designs of pollen allergy.

Purification and immunochemical characterization of Pla l 2, the profilin from Plantago lanceolata.

Profilins are small actin-binding proteins found in eukaryotes and involved in cell development, cytokinesis, membrane trafficking, and cell motility. From an allergenic point of view, profilins are panallergens usually involved in allergic polysensitization, although they are generally recognized as minor allergens. The objectives of this study were to identify and characterize the profilin from Plantago lanceolata pollen and to investigate the cross-reactivity between profilins from different pollen allergenic sources. Profilins from P. lancelolata (Pla l 2) and palm tree pollen (Pho d 2) were purified by affinity chromatography, deeply characterized and identified by mass spectrometry. Pla l 2 allergenicity was confirmed by immunoblot with serum samples from a patient population sensitized to profilin. Immunoblot inhibition was performed to study IgG reactivity between different pollen profilins. IgE cross-reactivity was demonstrated by ImmunoCAP inhibition. Pla l 2 is the second P. lanceolata allergen included in the IUIS Allergen Nomenclature database. Four peptides from purified Pla l 2 were identified with percentages of homology with other pollen profilins between 73 and 86%. Eighty-six percent (21/24) of the patient population recognized Pla l 2. The allergenic relatedness between Pla l 2, Pho d 2 and six pollen profilins was confirmed, and IgE cross-reactivity of Pla l 2 with rBet v 2 and rPhl p 12 was demonstrated. Pla l 2 is the profilin from P. lanceolata. The demonstrated allergenicity of this protein and its cross-reactivity with other pollen profilins support its use in profilin diagnostic assays.

T Cell Epitope-Containing Domains of Ragweed Amb a 1 and Mugwort Art v 6 Modulate Immunologic Responses in Humans and Mice.

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy. METHODS: Domains of Amb a 1 (Amb a 1α) and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT. RESULTS: The IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected. CONCLUSION: Accumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.

Numerical ragweed pollen forecasts using different source maps: a comparison for France.

One of the key input parameters for numerical pollen forecasts is the distribution of pollen sources. Generally, three different methodologies exist to assemble such distribution maps: (1) plant inventories, (2) land use data in combination with annual pollen counts, and (3) ecological modeling. We have used six exemplary maps for all of these methodologies to study their applicability and usefulness in numerical pollen forecasts. The ragweed pollen season of 2012 in France has been simulated with the numerical weather prediction model COSMO-ART using each of the distribution maps in turn. The simulated pollen concentrations were statistically compared to measured values to derive a ranking of the maps with respect to their performance. Overall, approach (2) resulted in the best correspondence between observed and simulated pollen concentrations for the year 2012. It is shown that maps resulting from ecological modeling that does not include a sophisticated estimation of the plant density have a very low predictive skill. For inventory maps and the maps based on land use data and pollen counts, the results depend very much on the observational site. The use of pollen counts to calibrate the map enhances the performance of the model considerably.

Pollen used to produce allergen extracts.

OBJECTIVE: To review the use of pollen for the production of allergen extracts to diagnose and treat allergic diseases, examine the associated regulations, and highlight candidate areas for improvement. DATA SOURCES: A PubMed search was performed using focused keywords combined with a review of regulatory documents and industry guidelines. STUDY SELECTIONS: The information obtained through literature, documents, and industry was scrutinized and used with personal experience and expertise to write this article. RESULTS: Both genetic and environmental factors affect the allergenic composition of pollen because it is a biologically active pharmaceutical ingredient obtained from nature. The potential effect of airborne contaminants in pollen requires major attention but can be properly addressed through careful collection practices, combined with a proper interpretation of the data on purity obtained for each pollen lot. The regulations associated with pollen used to manufacture allergen extracts in the United States and Europe and the numbers of pollen allergen extracts commercially available in both areas of the world differ. A critical parameter to select the appropriate extracts for diagnosis and allergen immunotherapy is to understand the phenomenon of cross-reactivity among pollen families, genera, and species. CONCLUSION: Physicians should be aware of the factors responsible for the qualitative and quantitative composition of pollen allergen extracts and the associated regulations to produce suitable extracts to diagnose and treat allergic diseases. Collaboration and cooperation among allergen manufacturing companies and regulatory agencies are necessary.

Molecular Cloning and Expression of Pro J 1: A New Allergen of Prosopis Juliflora Pollen.

Pollen from mesquite (Prosopis juliflora) is one of the important causes of immediate hypersensitivity reactions in the arid and semi-arid regions of the world. The aim of present study is to produce and purify the recombinant form of allergenic Ole e 1-like protein from the pollen of this allergenic tree. Immunological and cross-inhibition assays were performed for the evaluation of IgE-binding capacity of purified recombinant protein. For molecular cloning, the coding sequence of the mesquite Ole e 1-like protein was inserted into pTZ57R/T vector and expressed in Escherichia coli using the vector pET-21b(+). After purification of the recombinant protein, its immunoreactivity was analysed by in vitro assays using sera from twenty one patients with an allergy to mesquite pollen. The purified recombinant allergen was a member of Ole e 1-like protein family and consisted of 150 amino acid residues, with a predicted molecular mass of 16.5 kDa and a calculated isoelectric point (pI) of 4.75. Twelve patients (57.14%) had significant specific IgE levels for this recombinant allergen. Immunodetection and inhibition assays indicated that the purified recombinant allergen might be the same as that in the crude extract. Herein, we introduce an important new allergen from P. juliflora pollen (Pro j 1), which is a member of the Ole e 1-like protein family and exhibits significant identity and similarity to other allergenic members of this family.

Major allergen from Amaranthus palmeri pollen is a profilin: Isolation, partial characterisation and IgE recognition.

BACKGROUND: Pollens represent a rich source of proteins that are also potential elicitors of IgE-mediated pollen allergy. Sensitisation to panallergens could play an important role in diagnosis and specific immunotherapy, because these molecules are present in different plant pollens and plant foods and have marked structural similarity in different species. Profilins are one of the most common panallergens to be studied because they are responsible for a large number of sensitisations and are clearly related to cross-reactivity and co-sensitisation. This study aimed to isolate and characterise a new allergen of Amaranthus palmeri pollen and to determine its allergenicity. METHODS: A. palmeri pollen profilin was purified using poly-l-proline-Sepharose affinity chromatography followed by anion exchanger chromatography. Identification of purified protein was carried out by mass spectrometry. Specific IgE was estimated in sera of patients with positive skin prick test to A. palmeri pollen extract, by enzyme-linked immunosorbent assay (ELISA). PRINCIPAL FINDINGS: Purified protein appeared as a single band at 14 kDa in SDS-PAGE gel. Mass spectrometric analysis of the gel band identified two highly conserved peptides corresponding to allergenic profilins from pollen of other plants. Sera from about 60% of allergic patients have IgE that recognises the purified A. palmeri protein. CONCLUSION: A 14 kDa protein of A. palmeri pollen was purified and identified as allergenic profilin, which was recognised by sera from pollen allergic patients.

A recombinant Sal k 1 isoform as an alternative to the polymorphic allergen from Salsola kali pollen for allergy diagnosis.

BACKGROUND: The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, Salsola kali is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of S. kali pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. METHODS: Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) Escherichia coli cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from S. kali pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. RESULTS: rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as Platanus acerifolia and Oleaceae members. CONCLUSIONS: rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.

Pollensomes as Natural Vehicles for Pollen Allergens.

Olive (Olea europaea) pollen constitutes one of the most important allergen sources in the Mediterranean countries and some areas of the United States, South Africa, and Australia. Recently, we provided evidence that olive pollen releases nanovesicles of respirable size, named generically pollensomes, during in vitro germination. Olive pollensomes contain allergens, such as Ole e 1, Ole e 11, and Ole e 12, suggesting a possible role in allergy. The aim of this study was to assess the contribution of pollensomes to the allergic reaction. We show that pollensomes exhibit allergenic activity in terms of patients' IgE-binding capacity, human basophil activation, and positive skin reaction in sensitized patients. Furthermore, allergen-containing pollensomes have been isolated from three clinically relevant nonphylogenetically related species: birch (Betula verrucosa), pine (Pinus sylvestris), and ryegrass (Lolium perenne). Most interesting, pollensomes were isolated from aerobiological samples collected with an eight-stage cascade impactor collector, indicating that pollensomes secretion is a naturally occurring phenomenon. Our findings indicate that pollensomes may represent widespread vehicles for pollen allergens, with potential implications in the allergic reaction.

Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients.

BACKGROUND: Sublingual immunotherapy (SLIT) applied to type I respiratory allergies is commonly performed with natural allergen extracts. Herein, we developed a sublingual tablet made of pharmaceutical-grade recombinant Bet v 1.0101 (rBet v 1) and investigated its clinical safety and efficacy in birch pollen (BP)-allergic patients. METHODS: Following expression in Escherichia coli and purification, rBet v 1 was characterized using chromatography, capillary electrophoresis, circular dichroism, mass spectrometry and crystallography. Safety and efficacy of rBet v 1 formulated as a sublingual tablet were assessed in a multicentre, double-blind, placebo-controlled study conducted in 483 patients with BP-induced rhinoconjunctivitis. RESULTS: In-depth characterization confirmed the intact product structure and high purity of GMP-grade rBet v 1. The crystal structure resolved at 1.2 Å documented the natural conformation of the molecule. Native or oxidized forms of rBet v 1 did not induce the production of any proinflammatory cytokine by blood dendritic cells or mononuclear cells. Bet v 1 tablets were well tolerated by patients, consistent with the known safety profile of SLIT. The average adjusted symptom scores were significantly decreased relative to placebo in patients receiving once daily for 5 months rBet v 1 tablets, with a mean difference of 17.0-17.7% relative to the group treated with placebo (P < 0.025), without any influence of the dose in the range (12.5-50 µg) tested. CONCLUSION: Recombinant Bet v 1 has been produced as a well-characterized pharmaceutical-grade biological drug. Sublingual administration of rBet v 1 tablets is safe and efficacious in patients with BP allergic rhinoconjunctivitis.

Characterization of two pollen allergens of the London plane tree in Shanghai.

Platanus acerifolia, London plane tree, a significant source of airborne allergens, is widely grown in Shanghai and other cities in China. In recent decades, little has been known regarding the influence of the allergens on sensitizing the population in the Shanghai area. The aims of this study were to purify and characterize the two major allergens and to confirm the immunological activities of these pollen allergens in Shanghai. Crude extract was purified with a HiTrap SP column and a Sephedex G75 column. Immunodetection was performed with ELISA and immunoblotting. Following gel proteolytic digestion and mass spectrometry, the tandem MS (MS/MS) peptide mass fingerprint was obtained and the MASCOT search engine was used to identify the peptide. The accession number of the interesting homologous data and all the sequence information was acquired by an internet database and the evolutionary trees were drawn with Mega 4.0 software. Two proteins with molecular weights of 43 kDa and 18 kDa were purified from P. acerifolia pollen extract. The purified proteins were identified as pollen allergen Pla a 1 and Pla a 2 via mass spectrometry. The proteins have immunological activities with human IgE antibodies. According to the ELISA results, 12% (5/41) of the subjects were sensitive to Pla a 1 and 9% (4/41) were sensitive to Pla a 2. Pla a 1 and Pla a 2 are thus important allergens for patients with an allergic reaction to P. acerifolia pollen in Shanghai.

Component-resolved diagnosis of baker's allergy based on specific IgE to recombinant wheat flour proteins.

BACKGROUND: Sensitization to wheat flour plays an important role in the development and diagnosis of baker's asthma. OBJECTIVES: We evaluated wheat allergen components as sensitizers for bakers with work-related complaints, with consideration of cross-reactivity to grass pollen. METHODS: Nineteen recombinant wheat flour proteins and 2 cross-reactive carbohydrate determinants were tested by using CAP-FEIA in sera of 101 bakers with wheat flour allergy (40 German, 37 Dutch, and 24 Spanish) and 29 pollen-sensitized control subjects with wheat-specific IgE but without occupational exposure. IgE binding to the single components was inhibited with wheat flour, rye flour, and grass pollen. The diagnostic efficiencies of IgE tests with single allergens and combinations were evaluated by assessing their ability to discriminate between patients with baker's allergy and control subjects based on receiver operating characteristic analyses. RESULTS: Eighty percent of bakers had specific IgE levels of 0.35 kUA/L or greater and 91% had specific IgE levels of 0.1 kUA/L or greater to at least one of the 21 allergens. The highest frequencies of IgE binding were found for thiol reductase (Tri a 27) and the wheat dimeric α-amylase inhibitor 0.19 (Tri a 28). Cross-reactivity to grass pollen was proved for 9 components, and cross-reactivity to rye flour was proved for 18 components. A combination of IgE tests to 5 components, Tri a 27, Tri a 28, tetrameric α-amylase inhibitor CM2 (Tri a 29.02), serine protease inhibitor-like allergen (Tri a 39), and 1-cys-peroxiredoxin (Tri a 32), produced the maximal area under the curve (AUC = 0.84) in receiver operating characteristic analyses, but this was still lower than the AUC for wheat- or rye flour-specific IgE (AUC = 0.89 or 0.88, respectively). CONCLUSIONS: Component-resolved diagnostics help to distinguish between sensitization caused by occupational flour exposure and wheat seropositivity based on cross-reactivity to grass pollen. For routine diagnosis of baker's allergy, however, allergen-specific IgE tests with whole wheat and rye flour extracts remain mandatory because of superior diagnostic sensitivity.

Cypress pollen: an unexpected major sensitizing agent in different regions of Italy

Objectives: In this multicenter survey, we assessed the impact of sensitization to cypress in atopic patients in Italy and determined whether cypress pollen concentration changed over time. Methods: Allergists were required to collect the results of 100-200 consecutive skin prick tests (SPTs) performed during 2012. Seasonal symptoms were also recorded, as were airborne cypress pollen concentrations (data from the Italian Aerobiology Association) in 1998- 2000 and 2010-2012. Results: We examined 2258 atopic outpatients (56% females; age, 2-84 years) sensitized to at least 1 of the aeroallergens tested (Dermatophagoides species, grass, pellitory, olive, cypress, birch, Alternaria tenuis, and dog and cat dander). We found that 62.9%, 16.1%, and 32.7% of patients living in central, northern, and southern Italy, respectively, were sensitized to cypress (P<.0001). The cypress pollen concentration peak was delayed from February to March in 1998-2000 and 2010-2012 in all 3 regions, with a shift in pollination towards spring. Patients who were monosensitized to cypress reported mainly rhinitis (90.7%-97.6%) and conjunctivitis (38.1%-100%). In polysensitized patients, the prevalence of rhinitis, conjunctivitis, and asthma increased progressively (P<.0001) from southern to northern Italy. The same trend was observed for the prevalence of reported winter symptoms typical of cypress allergy (28%-65%). Conclusions: Today, cypress pollen is the most frequent sensitizing aeroallergen (assessed by SPT) in several areas of central Italy. Variations in the timing of the cypress pollination period may have favored this increased sensitization. Rhinitis and conjunctivitis are the predominant symptoms. The clinical impact of this allergy was poor in southern Italy and increased in central areas before reaching its peak in northern regions (AU)

Antecedentes: Se trata de una encuesta multicéntrica realizada en Italia para evaluar el impacto de la sensibilización a polen de ciprés en sujetos atópicos y establecer si la concentración de este polen en el aire ha cambiado a lo largo del tiempo. Métodos: El estudio fue realizado por alergólogos que recopilaron 100-200 sujetos consecutivos con pruebas cutáneas positivas (Prick) realizadas en 2012. Se recogieron los síntomas estacionales, junto con la concentración de polen de ciprés (obtenida por la asociación italiana de aerobiología) en 1998-2000 y 2010-2012. Resultados: En cuanto a los resultados obtenidos fueron examinados 2258 pacientes atópicos (56% mujeres; edad 2-84), sensibilizados frente al menos uno de los aeroalérgenos testados (Dermatophagoides, gramíneas, parietaria, olivo, cipres, abedul, Alternaria tenuis y epitelio de gato). El 62.9%, 16.1% y 32.7% de los pacientes que vivían en el centro, norte y sur de Italia, respectivamente, mostraron sensibilización a polen de ciprés (p<0.0001). Observamos un pico de concentración de polen de ciprés de febrero a marzo en los años 1998-2000 y 2010-2012, en todas las áreas. Los pacientes monosensibilizados a ciprés mostraron de forma prevalente rinitis (90.7-97.6%) y conjuntivitis (38.1-100%). La prevalencia de rinitis, conjuntivitis y asma se incrementa progresivamente (p<0.0001) del sur hacia el norte de Italia en los sujetos polisensibilizados. La misma tendencia se observó en los síntomas invernales típicos de la alergia al ciprés. Conclusiones: En conclusión, actualmente el polen de ciprés es el aeroalérgeno sensibilizante más frecuente (según resultados de prueba cutánea) en varias áreas de Italia central. Las variaciones del periodo de polinización pueden favorecer el incremento observado en la sensibilización a este polen. Los síntomas predominantes son rinitis y conjuntivitis. El impacto clínico de esta alergia es pobre en áreas del sur de Italia, siendo alto en las áreas del norte (AU)