Claudia Afferni

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.

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 all_ergen of Cupressus arizonica pollen, Cup a 1, was developed.

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.

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.

Purification and partial characterization of the major antigen of Echinococcus granulosus (antigen 5) with monoclonal antibodies

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.

Cypress allergy: an underestimated pollinosis

independent. In postmarketing surveillance studies, the overall incidence of ACEI-associated angioedema approximated 0.1-0.2% and was found to be more common in Afro-Americans. As in our patient, the time of onset is usually within hours or, at most, the first days of admitiistration (1); however, reports of delayed-onset angioedema occurring months to years after initiation of the drug have been published (1). Evidence has emerged that interruptions of the ACEI treatment may represent a possible risk factor for late-onset ACEI angioedema. Though the exact pathogenesis of ACEI-mediated angioedema remains unknown, the nonapeptide bradykiniti appears to play a key role. Bradykinin, cleaved from high-molecular-weight kininogens by kallikrein, is a potent activator of the L-arginine nitric oxide system that causes vasodilation and increases capillary leakage (4).

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 allergens 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.

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.

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.

Oral sensitization with shrimp tropomyosin induces in mice allergen-specific IgE, T cell response and systemic anaphylactic reactions

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.