Domingo Barber

Lipid-transfer proteins are relevant allergens in fruit allergy

BACKGROUND Allergy to apple and Prunus fruits is frequently associated with birch pollinosis, with the principal cross-reacting allergens involved being members of the Bet v 1 family. However, a major 13-kd component, nonimmunologically related to Bet v 1, has been implicated as allergen in patients allergic to Prunoideae fruit but not to birch pollen. OBJECTIVE We sought to isolate and characterize the 13-kd allergen present in apple and peach. METHODS Sera from patients allergic to both fruits were selected on the basis of clinical symptoms, skin prick tests responses, and specific IgE levels. Allergens were purified by reverse-phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, specific IgE immunodetection, and immunoblot inhibition assays. RESULTS A 13-kd protein band was recognized in crude apple and peach extracts by 9 of 10 and 10 of 10 sera from patients allergic to fruit, respectively. The isolation and characterization of the corresponding allergens allowed their identification as lipid-transfer proteins, with a molecular mass of 9058 d for the apple protein and 9138 d for the peach protein. Both purified allergens were recognized by sera from patients allergic to fruit and fully inhibited the IgE binding by the 13-kd component present in the 2 crude fruit extracts. CONCLUSION Lipid-transfer proteins are relevant apple and peach allergens and, considering their ubiquitous distribution in tissues of many plant species, could be a novel type of panallergen of fruits and vegetables.

Lipid‐transfer proteins as potential plant panallergens: cross‐reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different IgE‐binding capacities

Lipid‐transfer proteins (LTPs), but not Bet v 1 homologues, have been identified as major allergens of apple and peach in the Rosaceae fruit‐allergic population in the Mediterranean area. Many of these patients show cosensitization to mugwort pollen. LTPs have an ubiquitous distribution in tissues of many plant species, and have been proposed as a novel type of plant panallergens.

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.

Randomized double-blind, placebo-controlled trial of sublingual immunotherapy with a Pru p 3 quantified peach extract.

Background:  Peach allergy is highly prevalent in the Mediterranean area; it is persistent and potentially severe, and therefore a prime target for immunotherapy. We aimed to study the efficacy and safety of sublingual immunotherapy (SLIT) with a peach extract quantified in mass units for Pru p 3, the peach lipid transfer protein.

Plant non‐specific lipid transfer proteins as food and pollen allergens

Several members of the plant non‐specific lipid transfer protein (LTP) family have been identified as relevant allergens in foods and pollens. These allergens are highly resistant to both heat treatment and proteolytic digestion. These characteristics have been related with the induction of severe systemic reactions in many patients, and with the possibility of being primary sensitizers by the oral route. A specific geographical distribution pattern of sensitization to LTP allergens has been uncovered. This allergen family is particularly important in the Mediterranean area, but shows a very limited incidence in Central and Northern Europe. The potential role in the plant, as well as the biochemical and allergenic properties of the LTP family, are reviewed here.

Understanding patient sensitization profiles in complex pollen areas: a molecular epidemiological study

Background:  Allergy diagnosis in patients exposed to multiple pollen species is complex and misdiagnosis is often a cause for unsuccessful specific immunotherapy.

Profilin sensitization detected in the office by skin prick test: a study of prevalence and clinical relevance of profilin as a plant food allergen.

Background Profilin, a pan‐allergen present in all eukaryotic cells, is one of the main causes of cross‐sensitization between pollen and plant‐derived foods, but its clinical relevance as a food allergen is still debated.

Members of the α-amylase inhibitors family from wheat endosperm are major allergens associated with baker's asthma

We have identified the major antigens or IgE binding components from wheat flour. Thirty‐five sera from patients with bakers asthma were used to analyze the reaction with wheat salt‐soluble proteins. We found a 15 kDa SDS‐PAGE band which reacted with all sera tested. Purified members of the α‐amylase inhibitor family, which are the main components of the 15 kDa band, were recognized by specific IgE when tested with a pool of reactive sera. Immunodetection after two‐dimensional electrophoretic fractionation of crude inhibitor preparations from wheat endosperms also detected several inhibitor subunits as major low‐molecular‐weight allergens.

Prevalence of sensitization to Artemisia allergens Art v 1, Art v 3 and Art v 60 kDa. Cross‐reactivity among Art v 3 and other relevant lipid‐transfer protein allergens

Background Artemisia vulgaris is a widespread weed in the Mediterranean area and several allergens have been detected in its pollen. One of them, Art v 3, belongs to the lipid‐transfer protein (LTP) family and its prevalence in Artemisia‐sensitized patients or its relationship with other LTP allergens is not clear.

Identification of IgE-binding epitopes of the major peach allergen Pru p 3

BACKGROUND Lipid transfer proteins (LTPs) are clinically relevant plant food panallergens and have been proposed as ideal tools to study true food allergy. Pru p 3, the major peach allergen in the Mediterranean area, is among the best-characterized allergenic members of the LTP family. Its diagnostic value for Rosaceae fruit allergy has been demonstrated both in vivo and in vitro. OBJECTIVE We sought to locate major IgE-binding epitopes of Pru p 3. METHODS A serum pool and individual sera from patients with peach allergy and positive skin prick test results to Pru p 3 were used. Three-dimensional modeling was achieved by using experimentally available structures of Pru p 3 homologues as templates. Theoretical prediction of potential IgE-binding regions was performed by selecting specific residues on the molecular surface displaying prominent electrostatic potential features. Point mutants of Pru p 3 were constructed by standard polymerase chain reaction procedures with the appropriate primers. Mutants were expressed in P pastoris by means of the pPIC 9 vector and purified from the corresponding supernatants by gel-filtration chromatography followed by RP-HPLC. IgE binding by Pru p 3 mutants was tested by immunodetection and quantified by ELISA and ELISA inhibition assays. Synthetic peptides (10 mer; 5 amino acids overlapping) covering the full Pru p 3 sequence were used to detect IgE epitopes by (125)I-anti-IgE immunodetection. RESULTS Pru p 3 showed a 3-dimensional structure comprising 4 alpha-helixes and a nonstructured C-terminal coil (residues 73 to 91). Regions around amino acids in positions 23 to 36, 39 to 44, and 80 to 91, particularly residues R39, T40, and R44, K80 and K91, were predicted as potential antibody recognition sites according to their relevant surface and electrostatic properties. Point mutants K80A and K91A were found to have an IgE-binding capacity similar to that of recombinant Pru p 3, but the triple mutant R39A/T40A/R44A showed a substantial decrease (approximately 5 times) of IgE binding. IgE immunodetection of synthetic peptides led to the identification of Pru p 3 sequence regions 11 to 25, 31 to 45, and 71 to 80 as major IgE epitopes. CONCLUSIONS Main IgE-binding regions of the Pru p 3 amino acid sequence were identified. The three major ones comprised the end of an alpha-helix and some residues of the following interhelix loop. These data can help to search for Pru p 3 hypoallergenic forms.