Search

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.

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.

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., (Copyright 2002 S. Karger AG, Basel)

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: 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.

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 fo 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.

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.

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 less than 0.001); the specific IgE, measured by RAST, were surprisingly decreased (from 7.76 to 4.74 PRU/ml, P less than 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 less than 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.

Summary 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 ( Pj EDloop1) 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 Pj EDloop1 hybrid was produced as a purified recombinant protein with high stability in solution. Western blot, ELISA inhibition and basophil activation test showed that the Pj EDloop1 displays a remarkable reduced IgE binding and anaphylactic activity. CD3 reactivity was conserved in all patients. Mice immunization with the r Pj EDloop1 induced antibodies and T cell responses comparable to that obtained by the wild type allergens. Such antibodies shared the specificities to r Par j 1 and r Par 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. [ABSTRACT FROM AUTHOR]

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]