Unrecognized volatile and semi-volatile organic compounds from brake wear.

Motor vehicles are among the major sources of pollutants and greenhouse gases in urban areas and a transition to "zero emission vehicles" is underway worldwide. However, emissions associated with brake and tire wear will remain. We show here that previously unrecognized volatile and semi-volatile organic compounds, which have a similarity to biomass burning emissions are emitted during braking. These include greenhouse gases or, these classified as Hazardous Air Pollutants, as well as nitrogen-containing organics, nitrogen oxides and ammonia. The distribution and reactivity of these gaseous emissions are such that they can react in air to form ozone and other secondary pollutants with adverse health and climate consequences. Some of the compounds may prove to be unique markers of brake emissions. At higher temperatures, nucleation and growth of nanoparticles is also observed. Regions with high traffic, which are often disadvantaged communities, as well as commuters can be impacted by these emissions even after combustion-powered vehicles are phased out.

Dimerisation increases the immunogenicity of recombinant Parj1/Parj2 allergens.

Purified recombinant Parj1 and Parj2 allergens bind an IgE repertoire common to the Parietaria species, allowing their use as marker molecules for diagnosis and therapy of allergic disease induced by the Urticaceae family. Preclinical studies on the in vivo immunogenicity of recombinant Parj1, Parj2 and their isoforms indicated differential capacity to induce IgG1 antibody responses, as indication of potential clinical use. A recombinant hetero-dimeric hybrid derivative (PjED), encompassing the shorter Parj1 isoform (Parj1.0201) and Parj2 allergen, was characterised. In vivo immunisation with PjED induces IgG1 antibodies capable of binding all the isoforms of Parietaria major allergens, overcoming the poor immunogenicity of single monomeric allergens. This feature makes PjED a promising candidate molecule to be further characterised for clinical applications in the treatment of Parietaria allergy.

Characterization of a Par j 1/Par j 2 mutant hybrid with reduced allergenicity for immunotherapy of Parietaria allergy.

BACKGROUND: Parietaria pollen is one of the major cause of pollinosis in the southern Europe. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease restoring a normal immunity against allergens. METHODS: We designed a recombinant molecule (PjEDloop1) comprised of genetic-engineered variants of the major allergens of the Parietaria pollen (Par j 2/Par j 1). Purity and chemical-physical properties of the derivative were analysed by RP-HPLC chromatography and Photon Correlation Spectroscopy. Immunological activity was evaluated by means of Western blotting, ELISA inhibition and PBMC proliferation assay in 10 Parietaria allergic patients. Basophil activation was studied in six subjects. The immunogenicity of the hybrid was studied looking at the immune responses induced in a mouse model of sensitization. RESULTS: The PjEDloop1 hybrid was produced as a purified recombinant protein with high stability in solution. Western blot, ELISA inhibition and basophil activation test showed that the PjEDloop1 displays a remarkable reduced IgE binding and anaphylactic activity. CD3 reactivity was conserved in all patients. Mice immunization with the rPjEDloop1 induced antibodies and T cell responses comparable to that obtained by the wild type allergens. Such antibodies shared the specificities to rPar j 1 and rPar j 2 with human IgE antibodies. CONCLUSION: Our results demonstrated that a mutant hybrid expressing genetically engineered forms of the major P. judaica allergens displayed reduced allergenicity and retained T cell reactivity for the induction of protective antibodies in vaccination approaches for the treatment of Parietaria pollinosis.

Oral therapeutic administration of a probiotic mixture suppresses established Th2 responses and systemic anaphylaxis in a murine model of food allergy.

BACKGROUND: No effective treatment is available for food allergy and its primary management still consists of avoiding relevant allergens. Probiotics are claimed to beneficially affect the immune system. We sought to investigate the therapeutic potential of VSL#3 probiotic mixture on specific immune responses and anaphylactic reaction induced in mice by the major food allergen shrimp tropomyosin (ST). METHODS: The cytokine production by spleen cell from ST-sensitized mice upon allergen re-stimulation in the presence of VSL#3 was analysed. Next, the effects of oral administration of VSL#3 on allergen-induced anaphylaxis and Th2 response in the murine model of food allergy to ST was investigated by evaluating symptom score and histamine content in the faeces after allergen challenge, antibody response in serum and faeces, and cytokine and transcription factor expression in the jejunum. RESULTS: The in vitro studies on mouse spleen cells indicates that the VSL#3 preparation has the capacity to shift a polarized Th2 response to a Th1/T regulatory-type profile. Oral therapeutic administration of VSL#3 to ST-sensitized mice significantly reduces symptom score and histamine release in the faeces following allergen challenge, as well as specific IgE response. In the jejunum, IL-4, IL-5 and IL-13 tissue content was significantly reduced, whereas FOXP3 and IL-27 mRNA expression, IL-10, TGF-ß and IFN-γ tissue content were up-regulated. CONCLUSIONS: Oral therapeutic treatment with the probiotic mixture VSL#3 is effective in redirecting allergen-specific Th2-polarized immune responses towards Th1-T regulatory responses and in the protection against anaphylactic reactions induced by the allergen in a murine model of food allergy.

Evaluation of allergenicity of genetically modified soybean protein extract in a murine model of oral allergen-specific sensitization.

BACKGROUND: With the development of genetically modified crop plants there has been a growing interest in the approaches available to assess the potential allergenicity of novel gene products. For additional assessment of the potential allergenicity of expressed proteins, informative data can be generated using animal models. Soybean is one of the major source of protein in human and animal nutrition, and has also been well characterized as a major allergenic source. Advances in biotechnology have resulted in an increasing number of genetically engineered foods, and among these soybean is one of the most widespread. OBJECTIVE: To develop and characterize a murine model of IgE-mediated soybean sensitization induced by intragastric immunization, in the presence of Cholera Toxin, with wild-type soybean extract (wt-SE) or with genetically modified soybean extract (gm-SE). METHODS: Balb/c mice born in our animal facilities, from females fed on soy-free food, were fed with the same soy-free food and used in all the experiments. Mice were sensitized by gavages with soybean extracts, and allergen-specific IgE and IgG responses were studied by direct ELISA and ELISA inhibition. Antigen-specific cell proliferation and cytokine production were evaluated in spleen cell cultures. Results Sensitization with both soybean extracts induced high levels of antigen-specific IgE and IgG1 and low levels of specific IgG2a. Both wt-SE and gm-SE were able to inhibit the binding of specific IgE from mice immunized with gm-SE to the same antigen used for the ELISA coating. A comparable proliferative response was obtained with the homologous as well as with the heterologous extracts. CONCLUSION: In sensitized mice, we observed a predominantly T-helper type 2 (Th2)-type immune response, with increased soybean-specific IgE and IgG1 antibodies and a concomitant increase of IL-4 and IL-5 production. RESULTS: obtained by specific IgE ELISA inhibition and by antigen-specific T cell proliferation demonstrated that wt-SE and gm-SE shared B and T epitopes. The present murine model of soybean sensitization established by the oral route should provide valuable information about risk assessment for food allergy from new proteins of genetically modified foods.

Immunological characterization of a recombinant tropomyosin from a new indoor source, Lepisma saccharina.

BACKGROUND: The presence of specific IgE antibodies to invertebrates is common among patients with rhinitis and asthma. Tropomyosin has been described as an invertebrate cross-reactive allergen. We have recently characterized an allergenic extract from silverfish (Lepisma saccharina). Since this insect could be a new source of tropomyosin in the indoor environment, we have thought important to clone and characterize the tropomyosin from it. METHODS: Recombinant tropomyosin was cloned and characterized by means of immunoblotting with tropomyosin-specific monoclonal antibodies, rabbit polyclonal antibodies and IgE from allergic patients. Its allergenic activity was investigated in histamine release assays. Immunoblotting and ELISA inhibition were carried out to identify the natural tropomyosin in the silverfish extract and to study the cross-reactivity among other arthropod tropomyosins. RESULTS: Tropomyosin-specific antibodies recognized in immunoblotting the natural tropomyosin in the insoluble fraction of silverfish extract. The silverfish tropomyosin (Lep s 1) was cloned and fully expressed. It shared high homology with other arthropod tropomyosins. rLep s 1 was recognized by tropomyosin-specific monoclonal and polyclonal antibodies and by IgE of allergic patients. It was able to inhibit the IgE binding to the insoluble fraction of silverfish extract, and to induce histamine release by an arthropod-allergic serum. Inhibition experiments revealed IgE cross-reactivity between rLep s 1 and other arthropod tropomyosins. CONCLUSION: rLep s 1 is the first allergen cloned and characterized from silverfish extract. It enabled us to identify the natural counterpart in the insoluble fraction of silverfish extract, suggesting that the tropomyosin is not readily extractable with a classic aqueous extraction procedure. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.

Allergen skin weal/radioallergosorbent test relationship in childhood populations that differ in histamine skin reactivity: a multi-national survey.

BACKGROUND: Histamine skin reactivity (HSR, the dimension of the skin weal elicited by histamine 10 mg/mL) is a variable that differs in children from different European countries and increases over time in the same place (Italy). OBJECTIVE: In this epidemiologic study, we investigated to what extent differences in HSR influence the relationship between positive allergen skin prick tests (ASPTs) and serum-specific IgE concentrations. METHODS: Between October 2001 and February 2002, 591 unselected 9-10-year-old schoolchildren drawn from five small towns in central Poland (Starachowice), central Italy (Ronciglione, Guardea) and Libya (Al-Azyzia, near the Mediterranean sea and Samno, 900 km south of the coast) were analysed for histamine, common ASPT and for serum total and specific IgE. RESULTS: HSR differed markedly in children from the three countries (Libya>Italy>Poland) whereas serum total IgE concentrations remained the same. The prevalence of children with measurable serum specific IgE (> or = 0.35 kU) or with a positive ASPT for five common allergens was high in Italy, lower in Poland and far lower in Libya. A 3-mm ASPT weal corresponded to a serum-specific IgE concentration that was two to threefold higher in children with low HSR compared with children with high HSR (P = 0.008). CONCLUSION: These findings suggest that HSR--a variable that differs in schoolchildren populations from the three countries studied--independently influences the results of ASPT and its influence should be considered when ASPT are assessed in international studies. The HSR differences found in the populations reported here probably reflect a complex, dynamic, environmental interaction that should be monitored in the different parts of the world.

Preparation and characterization of silverfish (Lepisma saccharina) extract and identification of allergenic components.

BACKGROUND: Airborne insect antigens represent important aeroallergens which have been widely investigated. Although it has been demonstrated that house dust contains significant silverfish (Lepisma saccharina) levels, none of the extracts obtained so far has been extensively characterized. Thus, we have prepared and characterized a silverfish extract and investigated its IgE-reactive components by testing the reactivity of sera from patients allergic to inhalant insect allergens. METHODS: The extract from silverfish insect bodies was prepared by homogenizing frozen silverfish in Tris-HCl buffer. The soluble material (Sup) was filtered and the insoluble material (Ppt) was resuspended in 100 mM Tris pH 10.6. The two fractions were characterized by biochemical and immunochemical methods. IgE reactivity was investigated on both fractions before and after periodate treatment. RESULTS: Protein content and total carbohydrates was 2 and 3% w/w for Sup and 1 and 0.3% w/w for Ppt. The SDS-PAGE profile of the two fractions showed a different pattern in the MW range of 5-175 kD. Sup and Ppt, probed with allergic sera, showed a complex pattern of IgE reactivity. When periodate-treated fractions were tested, IgE reactivity was either completely abrogated, reduced or not affected, depending on the allergic serum employed. CONCLUSIONS: The results obtained indicate that the classic aqueous-extraction procedures that have been used up to now for other insects might not be completely satisfactory, since several allergenic components are not soluble at the normally used pH. We developed a dedicated extraction procedure allowing the detection of a certain degree of reactivity in sera negative to allergens extracted following classic procedures.

Comparison between the native glycosylated and the recombinant Cup a1 allergen: role of carbohydrates in the histamine release from basophils.

BACKGROUND: Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract. OBJECTIVE: To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one. METHODS: Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils. RESULTS: Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity-after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation. CONCLUSION: A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy.

Cupressaceae pollinosis: identification, purification and cloning of relevant allergens.

Allergy to Cupressaceae pollen is a worldwide pollinosis caused by several species. Pollen extracts prepared from allergenic species belonging to this family are characterised by low protein and high carbohydrate content. The allergenic components represented in the pollen extracts from different species of the Cupressaceae family show high levels of cross-reactivity when probed with human IgE from allergic subjects and share a number of common epitopes also identified by polyclonal rabbit antisera and monoclonal antibodies. A close relationship has also been described with the Taxodiaceae and Podocarpaceae families. Although both proteic and carbohydrate epitopes appear to be involved in IgE recognition and allergenic cross-reactivity, a large portion of the IgE reactivity of Cupressaceae-allergic patients seems to be associated with sugar moieties present on the relevant allergenic molecules. From this point of view, Cupressaceae/Taxodiaceae allergens constitute a particularly useful model to study IgE cross-reactivity, as they have been shown to display different levels of homology. Moreover, the availability of the purified allergens, together with their recombinant counterparts, may shed light on the actual role played by carbohydrate in allergic sensitisation, IgE recognition and allergenic cross-reactivity.

Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen.

BACKGROUND: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. METHODS: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. RESULTS: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF(3)/Gn(GF)XF(3) (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. CONCLUSION: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.

A monoclonal antibody specific for a carbohydrate epitope recognizes an IgE-binding determinant shared by taxonomically unrelated allergenic pollens.

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.

Role of carbohydrate moieties in IgE binding to allergenic components of Cupressus arizonica pollen extract.

BACKGROUND: A reduction of IgE immunoreactivity after periodate-treatment has been previously reported for various glycoprotein allergens. OBJECTIVE: The aim of this study was to investigate the role of glycan moiety of a C. arizonica extract in the binding of patients' IgE and to identify the carbohydrates possibly involved. METHODS: The reactivity of IgE with C. arizonica extract, before and after periodate-treatment, was evaluated by immunoblotting and ELISA inhibition. The specificity of carbohydrate-reactive IgE was evaluated by ELISA using unrelated glycoproteins with known sugar composition and structure, such as pineapple bromelain, honeybee venom phospholipase A2, and ovalbumin, before and after periodate treatment. RESULTS: When periodate-treated C. arizonica extract was probed after SDS-PAGE and immunoblotting with patients' IgE, no reactivity could be detected. Furthermore, a very poor inhibitory activity of the periodate-treated C. arizonica extract as compared with the untreated sample could be observed in the ELISA inhibition experiments performed using C. arizonica extract as antigen. When phospholipase A2 and bromelain were used as antigens in ELISA, they were recognized by patients' IgE, whereas ovalbumin was negative. Treatment of phospholipase A2 and bromelain with periodate completely abolishes the IgE reactivity. CONCLUSION: A large portion of the IgE reactivity of Cupressaceae-allergic subjects appears to be associated with sugar moieties of C. arizonica extract which appear to be shared by bromelain and phospholipase A2, thus suggesting that the IgE of patients reacting with such epitopes probably react with beta 1 --> 2 xylose, alpha 1 --> 3 fucose and/or alpha 1 --> 6 fucose.

Specific IgE to cross-reactive carbohydrate determinants strongly affect the in vitro diagnosis of allergic diseases.

BACKGROUND: Cross-reacting carbohydrate determinants (CCDs) are antigenic structures shared by allergenic components from taxonomically distant sources. The case history of a patient with a great discrepancy between skin test and specific IgE results led us to investigate the role of these determinants in his specific case and in an allergic population. OBJECTIVE: We sought to determine the role of CCDs in causing false-positive and clinically irrelevant results in in vitro tests. METHODS: The involvement of CCDs was studied by specific IgE inhibition by using glycoproteins with a known carbohydrate structure. Direct and inhibition assays were performed by commercially available systems, in-house ELISA, and the immunoblotting technique. The binding to the periodate-oxidated carbohydrate structure of glycoproteins and allergenic extracts was also evaluated. A comparative study between skin test and specific IgE responses to the antigens studied was carried out in 428 consecutive allergic subjects. RESULTS: All the tests performed suggested that cross-reacting carbohydrate epitopes were the cause of false-positive specific IgE results in one of the commercial systems and the high reactivity in all the solid-phase in vitro tests. None of the cross-reacting carbohydrate allergens yielded a positive skin test response. Periodate treatment caused variable degrees of reduction of IgE binding to the different antigens studied, indicating that CCDs played a different role in each of them. About 41% of patients allergic to pollen had specific IgE for a glycoprotein, without a positive skin test response to the same molecule. CONCLUSIONS: CCDs must be taken into account when evaluating the clinical relevance of positive results in in vitro specific IgE assays, at least in the diagnosis of patients with pollen allergy. Commercial systems should be carefully assessed for the ability to detect specific IgE for carbohydrate determinants to avoid false-positive or clinically irrelevant results.

Arizona cypress (Cupressus arizonica) pollen allergens. Identification of cross-reactive periodate-resistant and -sensitive epitopes with monoclonal antibodies.

Species of the Cupressaceae family are a worldwide cause of respiratory allergies. We used monoclonal antibodies (mAbs) to investigate the presence and the nature of cross-reacting epitopes shared by various components within Cupressus arizonica pollen extract (CaE) or by CaE and pollen extract from C. sempervirens (CsE). mAbs were produced in mice immunized with whole CaE (4A6 and 5E6) or with the major allergen components (2D5). Their reactivity was investigated by ELISA and immunoblotting before and after CaE periodate treatment. Cross-reactivity was evaluated by ELISA inhibition and immunoblotting. mAbs 2D5 and 4A6 recognized periodate-resistant epitopes, whereas the mAb 5E6 reacted with a periodate-sensitive determinant. The former mAbs recognized epitopes present on CaE major allergen and also shared by other components. mAb 5E6 showed a spread reactivity on CaE, with exclusion of the major allergen. When the three mAbs were tested with CsE, a restricted pattern of reactivity to mAbs 2D5 and 4A6 was obtained, whereas mAb 5E6 maintained a spread reactivity. The CaE major allergen is represented by two components recognized by human IgE and sharing common epitopes, as proven by mAbs reactivity. The use of these mAbs demonstrates that cross-reactivity within CaE components and between CaE and CsE is due to the presence of periodate-sensitive as well as -resistant epitopes.

Juniperus oxycedrus: a new allergenic pollen from the Cupressaceae family.

BACKGROUND: Cupressaceae allergy is a worldwide pollinosis caused by several species. Some species in limited geographic areas pollinate in fall and winter. Juniperus oxycedrus matches these features. OBJECTIVE: We sought to define the immunochemical, allergologic, and environmental aspects of J. oxycedrus pollen. METHODS: Pollen extract from J. oxycedrus was prepared and characterized by biochemical analysis and human specific IgE binding by means of ELISA and immunoblotting. A 3-year phenological study was conducted to define the pollinating period of J. oxycedrus. Forty consecutive patients allergic to cypress were recruited in two areas and divided into two groups according to their exposure to J. oxycedrus pollen. Clinical evaluation, skin prick tests, and specific IgE determination with J. oxycedrus, J. ashei, and Cupressus arizonica extracts were carried out on both groups. RESULTS: J. oxycedrus pollen extract was obtained, and it showed specific IgE binding and wide cross-reactivity with other Cupressaceae species. The extract caused a positive skin test response in all the patients tested, with about 80% of them having detectable specific IgE. Symptoms related to J. oxycedrus pollen exposure were recorded in 72% of the directly exposed patients and occasionally in 9% of the nonexposed patients. In the Mediterranean coastal area considered, J. oxycedrus was the first Cupressaceae species that started to pollinate at the beginning of November and ended in the first part of December. CONCLUSIONS: J. oxycedrus represents a newly characterized pollen species of the Cupressaceae family that cross-reacts with other members of the same family. Subjects with cypress allergy have in vivo and in vitro positive test responses for J. oxycedrus and can show symptoms when exposed to its pollen. Finally, the most important feature of J. oxycedrus is its early pollinating period in southern Europe (Italy), causing a further extension of the cypress pollen season in areas where other Cupressaceae species are present.

Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: a novel calcium-binding allergen.

BACKGROUND: Species belonging to the Cupressaceae family are a relevant source of allergens that are present in a wide number of countries. OBJECTIVE: We sought to identify, purify, and characterize recombinant allergens from Juniperus oxycedrus, a species belonging to the Cupressaceae family. METHODS: Double-stranded cDNA was synthesized from mRNA and cloned into the lambda-ZAP expression vector. IgE screening of the library was performed with a pool of sera from subjects allergic to Cupressaceae. A recombinant 6xHis-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni2+ affinity chromatography. It was studied further by immunoblotting inhibition with pollen extracts from other Cupressaceae, Oleaceae, Urticaceae, and Graminaceae. The role of protein-bound calcium on the allergen's IgE-binding capacity was tested in a plaque assay in the presence or absence of EGTA. RESULTS: A cDNA coding for a newly identified Juniperus oxycedrus pollen allergen, rJun o 2, was isolated. The deduced amino acid sequence contained four typical Ca2+ binding sites and showed a significant sequence similarity to calmodulins. Depletion of Ca2+ in the plaque assay led to a loss of IgE-binding capacity of rJun o 2. Immunoblotting inhibition revealed that J. oxycedrus, J. ashei, Cupressus arizonica, C. sempervirens, Parietaria judaica, Olea europaea, and Lolium perenne pollen extracts were able to inhibit IgE binding to blotted rJun o 2 at different concentrations. CONCLUSION: rJun o 2 contains IgE-binding epitopes shared by taxonomically unrelated species, and therefore it can be regarded as a new panallergen. These findings could contribute to an explanation for the phenomenon of multiple positive test results in polysensitized patients and the potential symptom-eliciting role of allergenic sources previously not encountered.

Cross-reactivity between Cupressus arizonica and Cupressus sempervirens pollen extracts.

BACKGROUND: Cupressus arizonica and C. sempervirens, two species belonging to the Cupressaceae family, are recognized as an important cause of respiratory allergies in countries with a Mediterranean climate. OBJECTIVE: The relationship between pollen extracts from these two species was studied by evaluating the reactivity with polyclonal rabbit antisera and human IgE. METHODS: The two extracts were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Cross-reactivity was evaluated by ELISA and immunoblotting inhibition experiments. RESULTS: The electrophoretic patterns of the two extracts are quite different, although some components display identical molecular weights. The immunoblotting developed with human IgE from subjects allergic to members of the Cupressaceae family indicated that two major IgE-reactive components, displaying molecular weights of about 43,000 and 36,000 d, were similarly detected in both extracts. Inhibition experiments showed a high degree of crossreactivity between the two extracts when tested with rabbit polyclonal antibodies against C. arizonica and C. sempervirens. When tested with human IgE inhibition methods, both extracts were able to reciprocally inhibit all of the IgE-reactive bands, although C. arizonica extract was always a better inhibitor. CONCLUSIONS: C. arizonica and C. sempervirens extracts are highly cross-reactive at the IgE level and share a number of common epitopes also identified by polyclonal rabbit antisera.