G. Di Felice

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.

Oral therapeutic administration of a probiotic mixture suppresses established Th2 responses and systemic anaphylaxis in a murine model of food allergy.

To cite this article: Schiavi E, Barletta B, Butteroni C, Corinti S, Boirivant M, Di Felice G. Oral therapeutic administration of a probiotic mixture suppresses established Th2 responses and systemic anaphylaxis in a murine model of food allergy. Allergy 2011; 66: 499–508.

In vitro production of cytokines by peripheral blood mononuclear cells from hydatid patients

The role of cytokines in human hydatidosJs (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL‐4, IL‐10 and interferon‐gamma (IFN‐γ) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In ceil cultures from hydatid patients parasite and non‐parasite antigen stimulation significantly increased IL‐4 production (P·0·005). Spontaneous and milogen‐driven IL‐4 production was similar in patients and controls. IL‐10 and IFN‐γ production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen‐driven IFN‐γ concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite‐driven cytokine production. High IgE and IgG4 responders produced high IL‐4 and IL‐10 concentrations. High IgE responders showed decreased IFN‐γ production, but high IgG4 responders had IFN‐γ levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL‐4 and the high IL‐10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen‐driven IFN‐γ production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.

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.

The recombinant major allergen of Parietaria judaica and its hypoallergenic variant: in vivo evaluation in a murine model of allergic sensitization.

Background  Par j 1 represents the major allergenic component of Parietaria judaica pollen. Its three‐dimensional structure is stabilized by four disulphide bridges. A family of three‐dimensional mutants of the recombinant Par j 1 (rPar j 1) allergen, showing reduced allergenicity and retained T cell recognition has been recently developed by site‐directed mutagenesis.

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.

A Hybrid Expressing Genetically Engineered Major Allergens of the Parietaria Pollen as a Tool for Specific Allergy Vaccination

Background: Allergy is an immunological disorder affecting about 25% of the population living in the industrialized countries. Specific immunotherapy is the only treatment with a long-lasting relief of allergic symptoms and able to reduce the risk of developing new allergic sensitizations and inhibiting the development of clinical asthma in children treated for allergic rhinitis. Methods: By means of DNA recombinant technology, we were able to design a head to tail dimer expressing disulphide bond variants of the major allergen of the Parietaria pollen. IgE binding activity was studied by Western blot, ELISA inhibition assays and the skin prick test. T cell recognition was studied by peripheral blood mononuclear cell proliferation. The immunogenicity of the hybrid was studied in a mouse model of sensitization. Results: In vitro and in vivo analysis showed that the disruption of specific cysteine residues in both allergens caused a strong reduction in IgE binding activity of the PjEDcys hybrid. In addition,we were able to show that a reduction in the IgE epitope content profoundly reduced the anaphylactic activity of the hybrid (from 100 to 1,000 times less than wild-type allergens) without interfering with the T cell recognition. Sera from BALB/c mice immunized with the hybrid were able to bind the natural Parietaria allergens and to inhibit the binding of human IgE to wild-type Par j 1 and Par j 2 allergens up to 90%. Conclusion: Our results demonstrate that hybrid-expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and maintained T cell reactivity for induction of protective antibodies.

Immunological characterization of a recombinant tropomyosin from a new indoor source, Lepisma saccharina

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.

Characterization of a Par j 1/Par j 2 mutant hybrid with reduced allergenicity for immunotherapy of Parietaria allergy.

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.