Carlo Pini

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment

Type I allergy is an immunoglobulin E (IgE)‐mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen‐containing sources but cannot identify the disease‐eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients’ IgE reactivity profiles to large numbers of disease‐causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

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.

The CREATE Project : development of certified reference materials for allergenic products and validation of methods for their quantification

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.

Characterization and comparison of commercially available mite extracts for in vivo diagnosis

To cite this article: Brunetto B, Tinghino R, Braschi MC, Antonicelli L, Pini C, Iacovacci P. Characterization and comparison of commercially available mite extracts for in vivo diagnosis. Allergy 2010; 65: 184–190.

Interleukin-12 Induces Expression of Interferon Regulatory Factor-1 via Signal Transducer and Activator of Transcription-4 in Human T Helper Type 1 Cells

IRF-1-deficient mice show a striking defect in the development of T helper 1 (Th1) cells. In the present report, we investigate the expression of IRF-1 during differentiation of human T helper cells. No significant differences of IRF-1 mRNA expression were found in established Th1 and Th2 cells; however, interleukin 12 (IL-12) induced a strong up-regulation of IRF-1 transcripts in Th1 but not in Th2 cells. We demonstrate that IL-12-induced up-regulation of IRF-1 is mediated by signal transducer and activator of transcription-4, which binds to the interferon (IFN)-γ-activated sequence present in the promoter of the IRF-1 gene. Strong IL-12-dependent activation of a reporter gene construct containing the IRF-1 IFN-γ-activated sequence element provides further evidence for the key role of signal transducer and activator of transcription-4 in the IL-12-induced up-regulation of IRF-1 transcripts in T cells. IRF-1 expression was strongly induced after stimulation of naive CD4+ T cells via the T cell receptor, irrespective of the cytokines present at priming, indicating that this transcription factor does not play a major role in initiating a Th1-specific transcriptional cascade in differentiating helper T cells. However, our finding that IRF-1 is a target gene of IL-12 suggests that some of the IL-12-induced effector functions of Th1 cells may be mediated by IRF-1.

Cupressaceae Pollinosis: Identification, Purification and Cloning of Relevant Allergens

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

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

Background: A rapid method for the purification of the major 43‐kDa all_ergen of Cupressus arizonica pollen, Cup a 1, was developed.

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).