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Traffic pollution has been recognized as directly worsening respiratory symptoms of allergic subjects, although whether urban air pollutants can also directly increase the allergenic potential of pollen has not yet been definitely proven. Therefore, the hypothesis that intra-urban air NO2 variation influences allergens expression in Cupressus sempervirens (Cs) L. pollen was tested.Mature microsporophylls were cut from Cs trees of similar age and height (14-17 m) present in three different sites of Florence (Italy) and processed in the laboratory. Cs pollen allergens amount was determined by a semi-quantitative analysis of electrophoretically separated pollen extracts fractions. NO2 air concentrations were recorded by air monitoring stations located at a distance not exceeding 50 m from each pollen collection site, and the relative annual mean values were acquired by a publicly available database (Tuscan Regional Agency for Environment Protection).Expression of three major Cs pollen allergens was non-linearly correlated with mean annual NO2 concentrations. Expression peak of all major allergens considered was reached at NO2 air concentration (67μg/m3), far below the value at risk for direct effect on the respiratory health (European Union Directive 2008/50/EC).The findings suggest that intra-urban NO2 variations do affect the expression of Cs pollen major allergens, and an apparent low risk NO2 concentration should be regarded as indirectly harmful for increasing the allergenic potential of pollen.

Objective To recapitulate the more recent epidemiologic studies on the association of air pollution with respiratory allergic diseases prevalence and to discuss the main limitations of current approaches used to establish a link between pollinosis and pollution. Data Sources Through the use of PubMed, we conducted a broad literature review in the following areas: epidemiology of respiratory allergic diseases, effect of pollution and climate changes on pollen grains, and immunomodulatory properties of pollen substances. Study Selections Studies on short- and long-term exposure to air pollutants, such as gaseous and particulate materials, on allergic sensitization, and on exacerbation of asthma symptoms were considered. Results Trend in respiratory allergic disease prevalence has increased worldwide during the last 3 decades. Although recent epidemiologic studies on a possible association of this phenomenon with increasing pollution are controversial, botanic studies suggest a clear effect of several pollutants combined to climatic changes on the increased expression of allergenic proteins in several pollen grains. The current literature suggests the need for considering both pollen allergen and pollutant contents for epidemiologic evaluation of environmental determinants in respiratory allergies. We propose that a measure of allergenic potential of pollens, indicative of the increase in allergenicity of a polluted pollen, may be considered as a new risk indicator for respiratory health in urban areas. Conclusion Because public greens are located in strict proximity to the anthropogenic sources of pollution, the identification of novel more reliable parameters for risk assessment in respiratory allergic diseases is an essential need for public health management and primary prevention area.

Eosinophils are major effectors of Th2-related pathologies, frequently found infiltrating several human cancers. We recently showed that eosinophils play an essential role in anti-tumor responses mediated by immunotherapy with the &lsquo, alarmin&rsquo, intereukin-33 (IL-33) in melanoma mouse models. Here, we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumor activities of eosinophils. We show that IL-33 recruits eosinophils indirectly, via stimulation of tumor cell-derived chemokines, while it activates eosinophils directly, up-regulating CD69, the adhesion molecules ICAM-1 and CD11b/CD18, and the degranulation marker CD63. In co-culture experiments with four different tumor cell lines, IL-33-activated eosinophils established large numbers of stable cell conjugates with target tumor cells, with the polarization of eosinophil effector proteins (ECP, EPX, and granzyme-B) and CD11b/CD18 to immune synapses, resulting in efficient contact-dependent degranulation and tumor cell killing. In tumor-bearing mice, IL-33 induced substantial accumulation of degranulating eosinophils within tumor necrotic areas, indicating cytotoxic activity in vivo. Blocking of CD11b/CD18 signaling significantly reduced IL-33-activated eosinophils&rsquo, binding and subsequent killing of tumor cells, indicating a crucial role for this integrin in triggering degranulation. Our findings provide novel mechanistic insights for eosinophil-mediated anti-tumoral function driven by IL-33. Treatments enabling tumor infiltration and proper activation of eosinophils may improve therapeutic response in cancer patients.

Introduction: natural air-born allergens can directly modulate the activity of dendritic cells (DCs) by enhancing ST2 expression and the IL-33-induced Th2 polarizing activity (PMID: 23608732), but what is their role in modify the immunological microenvironment of lung is largely unknown. The study evaluates the functional relevance of respiratory cells targeted modulatory activity by the major allergen Der f 1 and its mechanism of action. Authors: Afferni C, Lucarini V., Buccione C, Ziccheddu G., Schiavoni G., Mattei F. Methods: QRT-PCR, FACS analysis, ELISA. Results: natural Der f 1 stimulated the human type II pneumocytes-derived A549 cells to up-regulate the Th2-cells chemoattracting CCL20 mRNA, eosinophils chemoattracting CCL5 mRNA, IL13 mRNA and to release the Th2-polarizing IL33 cytokine. Of note IL33 chemoattracts mouse DCs as efficiently as CCL21, even if IL33 pretreated DCs migrated to higher extension toward IL33. We observed that final maturing bone-marrow derived mouse eosinophils by IL33 enable these cells to up-regulate IL13 and IL4 mRNA, to release the relative cytokines and to up-regulate the activation marker CD69. Conclusion: Der f 1 modulates the lung innate immunity by its effects on the alveolus lining cells, which are at the interface between the external environment and the mucosal immune system. In fact IL33 targeted DCs together with eosinophils-derived IL13 and IL4, and lung recruited Th2 cells could be sufficient for the sensitizing phase of the allergic response (PMID:23169007) and could be able to sustain the chronicity of an ongoing respiratory disease. The results are novel, important both for allergy immunotherapy and health impact of indoor environment.

The alarmin IL-33 plays pleiotropic roles in allergy, autoimmunity and inflammation through binding to its specific receptor ST2 expressed by most hematopoietic cells. Emerging evidences suggest an involvement of this cytokine also in cancer immunity, although its function remains ill-defined. Eosinophils (EOS) are a rare blood population playing critical roles in allergic inflammation and parasitic responses. We recently showed that EOS play an essential role in anti-tumor responses against melanoma growth and pulmonary metastasis mediated by IL-33 in vivo.In the present study we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumoral activities of EOS. We show that IL-33 indirectly stimulates the recruitment of EOS inducing tumor-derived chemokines CCL24 and CCL5. Furthermore, IL-33 directly activates EOS inducing the expression of adhesion molecules, such as the integrin CD11b, resulting in efficient contact-dependent tumor cell killing. In co-culture experiments, IL-33 activated EOS tightly bond to tumor cells, forming increased numbers of conjugates, with respect to resting eosinophils. Confocal laser-scanning microscopy (CLSM) of eosinophil-tumor cell conjugates revealed polarization of the pore-forming eosinophilic cationic protein (ECP) and of CD11b on the cell synapses exclusively in IL-33-activated, but not resting, EOS. Furthermore, we show that IL-33 activated EOS release larger amounts of extracellular vesicles (EV) with respect to resting EOS. Transmission electron microscopy (TEM) revealed increased degranulation and EV release of IL-33-activated EOS following cell contact with target tumor cells. Our results advocate for an eosinophil-mediated tumoricidal function promoted by IL-33, thus opening perspectives for novel cancer immunotherapy strategies. Citation Format: Fabrizio Mattei, Carla Buccione, Sara Andreone, Francesca Spadaro, Adele De Ninno, Jacopo Mancini, Cristiana Zanetti, Isabella Parolini, Francesca Iosi, Antonella Tinari, Valeria Lucarini, Annamaria Gerardino, Giovanna Ziccheddu, Luca Businaro, Claudia Afferni, Giovanna Schiavoni. IL-33 activates antitumoral toxicity in eosinophils through stimulation of contact-dependent degranulation [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A091.

Appropriate murine models of shrimp tropomyosin (ST) allergy would be useful in investigating the mechanisms underlying food allergy in human subjects, as well as for the pre-clinical evaluation of efficacy and safety of novel therapeutic approaches. These models should mimic immune and clinical features of human disease, including anaphylactic response. We sensitized C3H/HeJ mice by the oral route with purified ST using cholera toxin (CT) as adjuvant. ST-specific IgE, IgG1, IgG2a and IgA responses were evaluated by ELISA. Spleen cell proliferation and cytokine production by allergen-specific activation were assessed. Jejunum and colon fragments were collected to evaluate the local expression of cytokine genes by PCR. Local and systemic anaphylactic reactions induced by oral ST challenge were scored according to symptoms observed. Faecal samples were collected to assess local IgA production and histamine levels. Oral sensitization with ST plus CT induced in mice significant levels of serum IgE and IgG1 and faecal IgA. ST-specific cell proliferation and IL-4, IL-13 and IFN-gamma cytokine production were induced in the spleen. After oral challenge, 100% of mice had anaphylactic symptoms while no symptoms were observed in challenged naive mice. Faecal histamine content after ST challenge appeared significantly increased in sensitized mice when compared with that observed in pre-immune mice. Jejunum mRNA expression of T(h)2 cytokines was up-regulated by ST sensitization. These results support the importance of the oral way of sensitization and of the in-depth characterization of the anaphylactic response for the development of a suitable in vivo model of food allergy.

Summary 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.

Background: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. Methods: cDNA of Ole e 5 was amplified by nested 3′-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients’ IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). Results: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. Conclusions: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

Summary 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.

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.

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.

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. (J Allergy Clin Immunol 1998;101:755-61.)

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 cross-reactivity 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. (J ALLERGY CLIN IMMUNOL 1996;98:797-804.)

Background: There is increasing evidence for the relevance of Cupressaceae pollinosis among persons living in geographic areas where these species are native or imported. Objective: Previously reported problems in obtaining valid allergenic extracts to be used in the diagnosis of this winter pollinosis prompted us to assess the value of available Cupressaceae pollen extracts for in vivo and in vitro diagnosis. Methods: Commercial and in-house allergenic extracts from Cupressaceae and Taxodiaceae families were used for skin prick testing and specific IgE detection in six groups of subjects exposed to a high concentration of Cupressaceae pollen. Results: Four commercial and two in-house Cupressus sempervirens pollen extracts showed low cutaneous reactivity. Positive test results were recorded in 26% of the 713 subjects tested. C. arizonica in-house pollen extracts gave rise to larger cutaneous reactions. Furthermore, the skin prick test response was positive in a greater number of subjects (38%) of the same group. Six commercial immunoassays were able to detect specific IgE to C. sempervirens in rates ranging from 8.1% to 81.1%. Specific IgE to C. arizonica was detected by means of an in-house immunoenzymatic method in 70.3% of 54 patients with suspected "cypress" allergy, and specific IgE to C. sempervirens was detected in 75.9% of these patients by using a commercial system. High rates of cross-reactivity within the Cupressaceae family and with species of the Taxodiaceae family were recorded with both in vivo and in vitro tests. Conclusions: The use of C. sempervirens in vivo diagnostics should be carefully evaluated until better characterized extracts are developed. In-house–characterized extracts of C. arizonica seem to be more reliable in the diagnosis of Cupressaceae allergy by means of skin prick testing. The sensitivity of commercially available in vitro methods to detect specific IgE to C. sempervirens should be carefully evaluated; nevertheless, valid results can be obtained with some already available immunoassays. (J ALLERGY CLIN IMMUNOL 1996;98:21-31.)

Two monoclonal antibodies (MoAb) specific for Parietaria judaica allergenic components were selected on the basis of their capability to recognize either the Parietaria judaica major allergen (MoAb 1A6/1D1) or several other allergenic components (MoAb 1A4/2F8) except the major allergen. These two antibodies, either individually or combined, were used to develop an ELISA-inhibition system using a reference Parietaria judaica extract (in-house Reference preparation, IHR). The assays performed with these reagents were firstly standardized by testing the IHR several times. A good reproducibility, evaluated both at the level of 50% inhibition values, and in terms of analysis of the variance of the slopes of the regression curves, was obtained. Subsequently, the potency of several Parietaria judaica extracts, either obtained by manufacturing companies or produced in other laboratories, was evaluated by these tests. Data obtained by interpolation with the IHR values and expressed in terms of arbitrary units (AU) were compared with those obtained by classical human IgE inhibition, performed with sera from allergic patients. Results indicate that the monoclonal antibodies produced in our laboratory can be successfully employed, either individually or combined, in the standardization of allergenic preparations in addition to, and possibly replacing, the classical IgE-based standardization procedures which require human specimens often available in limited amounts only.

Background: The immunological mechanisms responsible for the immunomodulatory and anti-allergic effects of probiotic bacteria are still poorly defined. The combined effects of mixtures of different species of probiotic bacteria have been explored only in part. The present study describes the immunomodulatory activity of the VSL#3 probiotic preparation in in vitro and in vivo systems. Methods: The activation and cytokine production by in vitro probiotic-stimulated bone-marrow dendritic cells (BM-DCs) and spleen cells isolated from naïve or Par j 1-sensitized mice were investigated. Mice were intranasally administered a sonicate preparation of VSL#3 before immunization with rPar j 1. Serum antibody levels and cytokine expression in the lung were determined. Results: Both live and sonicated VSL#3 preparations induced maturation and cytokine production by BM-DCs. Cytokine production by spleen cells from naïve or Par j 1-sensitized mice was modulated by the probiotic preparations towards a Treg/Th0 profile, characterized by increased IL-10 and IFN-γ production. In vivo prophylactic treatment with VSL#3 induced a significant reduction of serum specific IgG1. At lung level, VSL#3 pre-treatment remarkably reduced IL-13 and IL-4 mRNA expression and increased IL-10 expression. Conclusions: The VSL#3 preparations have not only the capacity to bias primary immune responses towards a Treg/Th0-type profile, but also to modify in the same way the functional characteristics of established in vitro Th2 responses. In vivo studies on a mouse model of Par j 1 sensitization indicate that the prophylactic intranasal treatment with probiotic bacteria is able to modulate the development of Th2-biased responses.

Summary 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.

Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity.cDNA of Ole e 5 was amplified by nested 3'-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients' IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition).The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein.Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

Background: Calcium-binding plant allergens can be grouped in different families according to the number of calciumbinding domains (EF hands). Objective: We sought to identify pollens containing crossreactive calcium-binding allergens and to investigate structural and immunologic similarities of members belonging to different families of calcium-binding allergens. Methods: By means of multiple sequence alignment and molecular modeling, we searched for structural similarities among pollen allergens with 2 (Phl p 7, timothy grass; Aln g 4, alder), 3 (Bet v 3, birch) and 4 EF hands (Jun o 4, prickly juniper). Purified recombinant Aln g 4 and Jun o 4 were used to determine the prevalence of IgE recognition in 210 patients sensitized to different pollens and to search, by means of ELISA competition, for the presence of cross-reactive epitopes in pollens from 16 unrelated plant species. IgE cross-reactivity among the allergen families was studied with purified rPhl p 7, rAln g 4, rBet v 3, and rJun o 4 and 2 synthetic peptides comprising the N-terminal and C-terminal EF hands of Phl p 7 by means of ELISA competition. Results: Structural similarities were found by using molecular modeling among the allergens with 2, 3, and 4 EF hands. Pollens from 16 unrelated plants contained Aln g 4- and Jun o 4-related epitopes. Twenty-two percent of the patients with multiple pollen sensitization reacted to at least one of the calcium-binding allergens. A hierarchy of IgE cross-reactivity (rPhl p7 > rAln g 4 > rJun o 4 > rBet v 3) could be established that identified rPhl p 7 as the EF-hand allergen containing most IgE epitopes in the population studied. Conclusion: The demonstration that members of different families of calcium-binding plant allergens share similarities suggests that it may be possible to use representative molecules for the diagnosis and therapy of allergies to EF-hand allergens. (J Allergy Clin Immunol 2002;109:314-20.)

Background: A rapid method for the purification of the major 43-kDa all_ergen 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 all_ergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and all_ergenic 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 all_ergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF3 (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF3/GnGXF3 (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF3/Gn(GF)XF3 (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. Conclusions: The rapid purification process of Cup a 1 all_owed 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.

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. (J Allergy Clin Immunol 1999;103:1005-11.)

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.

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 6×His-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni 2+ 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 Ca 2+ binding sites and showed a significant sequence similarity to calmodulins. Depletion of Ca 2+ 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. (J Allergy Clin Immunol 1998;101:772-7)

Background Cypress pollen causes respiratory syndromes with different grades of severity, including asthma. IL-33, its receptor ST2, and dendritic cells (DCs) have been implicated in human respiratory allergy. Objective We sought to define a new mouse model of allergy to cypress pollen that recapitulates clinical parameters in allergic patients and to evaluate the implications of DCs and the IL-33/ST2 pathway in this pathology. Methods BALB/c mice, either wild-type or ST2 deficient (ST2 −/− ), were sensitized and challenged with the Cupressus arizonica major allergen nCup a 1. Local and systemic allergic responses were evaluated. Pulmonary cells were characterized by means of flow cytometry. DCs were stimulated with nCup a 1 and tested for their biological response to IL-33 in coculture assays. Results nCup a 1 causes a respiratory syndrome closely resembling human pollinosis in BALB/c mice. nCup a 1–treated mice exhibit the hallmarks of allergic pathology associated with pulmonary infiltration of eosinophils, T cells, and DCs and a dominant T H 2-type immune response. IL-33 levels were increased in lungs and sera of nCup a 1–treated mice and in subjects with cypress allergy. The allergen-specific reaction was markedly reduced in ST2 −/− mice, which showed fewer infiltrating eosinophils, T cells, and DCs in the lungs. Finally, stimulation of DCs with nCup a 1 resulted in ST2 upregulation that endowed DCs with increased ability to respond to IL-33–mediated differentiation of IL-5– and IL-13–producing CD4 T cells. Conclusions Our findings define a novel preclinical model of allergy to cypress pollen and provide the first evidence of a functionally relevant linkage between pollen allergens and T H 2-polarizing activity by DCs through IL-33/ST2.

IgG antibody response to the inhalant allergen Parietaria judaica (Pj) and IgG subclass distribution were studied in 82 normal subjects, divided into three groups according to age (0-1, 1-20, and 20-60 years) and in 32 allergic subjects aged 20-60 years. Both normal and allergic subjects showed an IgG response, and all had IgG1 antibodies specific for PjE. Serum IgG2, IgG3, and IgG4 against PjE were detectable in 36%, 46%, and 22% of normal subjects, and in 58%, 31%, and 65% of allergic subjects, respectively. A significant difference in class distribution between allergic and age-matched normal subjects was found only for IgG4 antibodies against PjE (65% and 17%; P < 0.01). The ELISA results were also analyzed quantitatively, taking into account the relative proportion of specific antibodies. Thus, in normal subjects IgG1 antibodies showed a decreasing trend as the age rose, while no differences according to the age of the subject were found for IgG2 and IgG4. When data from allergic subjects (20-60 years) and the age-matched normal group were compared, they were different for the relative percentage of IgG2 only, showing for this a significantly lower value (P < 0.001). The present data indicate that normal and allergic subjects show differences in the IgG isotype distribution depending on their sensitivity and duration of allergen exposure.

Cross-reactivity between the different components in Parietaria judaica pollen extract has been investigated by polyclonal as well as monoclonal antibodies before and after chemical deglycosylation obtained by trifluoromethanesulphonic acid (TFMS) treatment of the extract. In western blotting a polyclonal rabbit antiserum, obtained by injecting purified Par j I, was able to recognise many components of the native extract. However, its reactivity was restricted, after chemical deglycosylation of the extract, to the major allergen alone, indicating that its cross-reactivity was due to sugar moieties. Moreover, out of several monoclonal antibodies raised by injecting the whole Parietaria judaica extract, one (1A4/2F8) was also able in western blotting to recognise an epitope shared by many components of the extract except the major allergen Par j I. However, in this case the broad reactivity of the antibody was not affected by the deglycosylating procedure. When the reactivity of Parietaria judaica extract was tested before and after sugar removal, against specific IgE from a pool of patient sera, no differences could be demonstrated, thus indicating that carbohydrates are not strongly involved in the binding of Parietaria judaica-specific IgE. The results indicate that both proteic and carbohydratic cross-reactive epitopes are shared by many components of Parietaria judaica pollen extract.

In a previously work we have reported that T8+ lymphocytes decrease can be obtained by incubation of lymphocytes from normal blood donors with serum taken from patients in the acute phase of the disease. In this paper is described a partial characterization of the masking serum factor present in the acute phase of the disease. It displays a m.w. of approximately 640,000 d and its effect disappears either after heating 60 min. at 37 degrees C or 30 min. at 56 degrees C, after dialysis against 1M NaCl or lyophilization. Another serum factor, insoluble at 50% ammonium sulphate final saturation, resistant to heating and lyophilization, able to remove (in vitro) the former masking factor from the T8+ cells, is present in the remission phase.

A monoclonal antibody specific for antigen 5 of Echinococcus granulosus was isolated and partially characterized. Purification of antigen 5 was accomplished by affinity chromatography using an immunoabsorbent prepared with this monoclonal antibody. Pure antigen 5 was identified by immunoelectrophoresis, double diffusion in agar gel, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. The pure antigen displayed the electrophoretic mobility typical of antigen 5 and gave a single precipitin band in double diffusion with both monoclonal antibody and rabbit anti-pH5PPT hydatid fraction serum. Two bands of 66 and 56 kDa could be detected in the pure antigen 5 after sodium dodecyl sulphate-polyacrylamide gel electrophoresis when performed in non-reducing conditions: both bands were reactive with the monoclonal antibody in immunoblotting. After reduction with 2-mercaptoethanol, antigen 5 displayed one band only, of 39 kDa. Antigen 5 purified by this procedure was found to retain reactivity with human sera from hydatid patients in a DD5 test.

A simple and sensitive immunoenzymatic assay for detection of specific IgE in hydatid disease is presented. Sera from 43 patients with hydatidosis, eight with schistosomiasis, two with taeniasis, two with cancer and 16 healthy blood donors were tested by both the immunoenzymatic technique and the classical radioallergosorbent test. Data were analysed in terms of different sensitivity and specificity of the two methods. The results indicate that the enzymatic method performed in microplates is as sensitive as the radioimmunological one (77% of positivity for both the tests), but the latter suffers from an higher rate of false positive reactions with sera from patients with other helminthic diseases (80% vs 20%). When the specific IgE evaluation is compared with other classical serological techniques, such as indirect haemagglutination and double diffusion in terms of different sensitivity in revealing an anti-Echinococcus immunological reactivity, the results suggest that specific IgE evaluation represents a useful addition to the classical serodiagnostic tests.