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Notes: The in vitro proliferative response to separated immunologically relevant components of Parietaria judaica pollen extract (PjE) was investigated by proliferation assay and limiting dilution analysis, in peripheral blood mononuclear cells from Parietaria-allergic subjects and nonallergic controls. In the same subjects, the profile of the antibody response to the PjE fractions was also studied by immunoblotting to evaluate the functional significance of allergen-induced T-cell activation in the two groups. The estimated frequency of PjE-reactive T cells in peripheral-blood mononuclear cells was low in both groups. No difference was found between the Parietaria-allergic subjects and nonallergic controls. To assess the overall contribution to the cellular response of PjE components of different molecular weights, we separated the extract by the SDS-PAGE technique, and the fractions were blotted onto nitrocellulose and solubilized. Almost all the 14 fractions tested induced T-cell proliferation, at different degrees of magnitude. Responses were similar in the allergic subjects and nonallergic controls. Immunoblotting demonstrated specific IgG antibodies to the 14 PjE fractions not only in the allergic subjects, but also in the healthy controls, whereas IgE antibodies were found, as expected, only in the sera from atopic subjects. These findings indicate that PjE fractions elicit similar T-cell activation and IgG production in allergic and normal subjects.

Notes: 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.

Notes: 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.

Notes: 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.

Notes: 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 antiseruni, 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.

Notes: A major allergen of Parietaria officinalis, a species responsible for a large number of respiratory allergies in Mediterranean areas, has been identified and characterized. This allergen (Pol) was found in the fraction which precipitates between 70 and 100% ammonium sulphate saturation. Pol showed a molecular weight of 15,000 daltons as determined by SDS-PAGE and HPLC. The pI of Pol was in the pH region 4-6, IEF showing four major bands. Two major bands were shown by CIE, CRIE and immuno-blotting; major contaminants or aggregates were also revealed by the latter technique and by HPLC. Pol showed an allergic specific activity 2 times higher than the crude extract; moreover it was shown to be a major allergen since it inhibited 29 out of 30 sera from allergic patients sensitive to P. officinalis.

Notes: Changes in specific skin reactivity, specific IgE and specific IgG after immunotherapy (IT) were investigated in olive pollinosis. Thirty patients, receiving IT with commercial extracts, were studied in comparison with a control group of seven patients, receiving only drug therapy. Skin reactivity, IgE and IgG were assessed before starting IT and 1 year later. Definite changes in the three considered parameters occurred in patients given IT with Olea europaea extracts; no variation was observed in the control group. The specific skin reactivity, evaluated by means of quantitative skin prick tests, significantly decreased (Skin Index geometrical mean from 2.73 to 0.88, P〈0.001); the specific IgE, measured by RAST, were surprisingly decreased (from 7.76 to 4.74 PRU/ml, P 〈 0.001); the specific IgG, measured by ELISA, in basic conditions were detectable only in nine patients of 30, while, after IT, they were found in almost all patients with a remarkable increase (from 5.48 to 266.89 AU/ml, P 〈 0.001). No correlation was found among the changes in the considered parameters, suggesting that, at least in olive pollinosis, specific skin reactivity, specific IgE and specific IgG are three variables depending on IT but reciprocally independent.

Notes: 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.

Notes: 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 arizoniea 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.