Challenges for Allergy Diagnosis in Regions with Complex Pollen Exposures

Over the past few decades, significant scientific progress has influenced clinical allergy practice. The biological standardization of extracts was followed by the massive identification and characterization of new allergens and their progressive use as diagnostic tools including allergen micro arrays that facilitate the simultaneous testing of more than 100 allergen components. Specific diagnosis is the basis of allergy practice and is always aiming to select the best therapeutic or avoidance intervention. As a consequence, redundant or irrelevant information might be adding unnecessary cost and complexity to daily clinical practice. A rational use of the different diagnostic alternatives would allow a significant improvement in the diagnosis and treatment of allergic patients, especially for those residing in complex pollen exposure areas

Pomegranate LTP isoforms identified by a new proteomic approach show different immunological properties

Background. Plant Lipid Transfer Proteins (LTPs) belong to a class of high conserved proteins extensively studied in the last decade. These proteins are recognized as food allergens capable of eliciting severe systemic symptoms, especially among the Mediterranean population. Several polypeptides belonging to this family have been characterized so far. For some LTPs different isoforms have been identified in a given plant. The sequence information was mainly derived from the cDNA, by contrast, the investigation of isoforms diversity is frequently not addressed. Allergen isoforms often exhibit different IgE binding capacities, hence contributing differently to the whole allergenic potencies (e. g. Bet v 1 and Mal d 1). Recently two different LTP isoforms have been isolated from pomegranate (PG) and characterized by mass spectrometry (MS). Here we applied a new proteomic approach based on acid-urea electrophoresis (AU-PAGE) followed by SDS-PAGE for the separation of LTP isoforms from PG and peach. Methods. Proteins from PG juice and peach peel were separated first by the AU-PAGE. The lanes were cut, equilibrated in SDS containing buffer, and subjected to 15% SDS-PAGE. The separated proteins were transferred to PVDF membrane and assayed with a polyclonal antibody raised against Pru p 3 (PAB). IgE immunoblotting assays were carried out using sera from two groups of patients: 1) allergic to pomegranate, 2) allergic to peach (sensitized to Pru p 3). For identification the proteins were subjected to MS. Results. By this approach up to 10 spots with an apparent MW of 9-12 kDa (depending on the redox conditions) were separated for PG. Five of these proteins were recognized by the PAB and by the patient\u2019s sera. IgE immunoblotting experiments clearly showed that PG LTP isoforms are differentially recognized. In contrast, 2D analysis of peach revealed only a single predominant 9kDa spot recognized by the PAB and by the patient's sera. Conclusions. Up to 5 LTP isoforms have been separated from PG by the proteomic approach. The power of this technique is demonstrated by the identification of a large number of IgE reactive LTP isoforms never described for other organisms. The immunological study of the identified isoforms indicates that they possess different IgE epitopes which might contribute differently to the whole allergenic potency of pomegranate

Identification and characterization of the major allergen of green bean (Phaseolus vulgaris) as a non-specific lipid transfer protein (Pha v 3)

BACKGROUND: Green bean (GB) has been reported to cause allergic reactions after ingestion, contact or inhalation of particles deriving from processing or cooking. Up-to-date no food allergens have been fully characterized in GB. OBJECTIVE: To characterize the GB major allergen(s) on a molecular level and to verify the involvement of non-specific lipid transfer proteins (nsLTPs) in GB allergy. METHODS: We recruited 10 Spanish patients reporting adverse reactions to GB. Skin prick tests, specific IgE detection and oral provocation were performed. Two nsLTP cDNAs were cloned from GB and over-expressed in Pichia pastoris. The recombinant LTPs (rLTPs) were characterized by circular dichroism spectroscopy and IgE-binding assays (immunoblotting and ELISA) with the patients' sera. Three natural LTPs (nLTPs) were further purified from GB fruit by chromatography. In vitro histamine release test was applied to compare the allergenic potency of rLTPs and nLTPs. RESULTS: Oral provocation test confirmed GB allergy. A 10kDa protein in GB extract was recognized by 80% of the sera and identified as nsLTP. The two rLTPs (named LTP1a and LTP1b), share 61.3% aa identity and present the typical nsLTP-like secondary structure. The IgE-binding and histamine release assays provided evidence that rLTPs and nLTPs possess different allergenic potency. CONCLUSIONS: nsLTP (Pha v 3) is the major allergen in GB and constitute a potential risk for patients affected by LTP-syndrome. GB encodes for several LTPs with different immune reactivity

Pomegranate LTP isoforms identified by a new proteomic approach show different immunological properties

Background: Plant Lipid Transfer Proteins (LTPs) belong to a class of high conserved proteins extensively studied in the last decade. These proteins are recognized as food allergens capable of eliciting severe systemic symptoms, especially among the Mediterranean population. Several polypeptides belonging to this family have been characterized so far. For some LTPs different isoforms have been identified in a given plant. The sequence information was mainly derived from the cDNA, by contrast, the investigation of isoforms diversity is frequently not addressed. Allergen isoforms often exhibit different IgE binding capacities, hence contributing differently to the whole allergenic potencies (e.g. Bet v 1 and Mal d 1). Recently two different LTP isoforms have been isolated from pomegranate (PG) and characterized by mass spectrometry (MS). Here we applied a new proteomic approach based on acid-urea electrophoresis (AU-PAGE) followed by SDS-PAGE for the separation of LTP isoforms from PG and peach. Methods: Proteins from PG juice and peach peel were separated first by the AU-PAGE. The lanes were cut, equilibrated in SDS containing buffer, and subjected to 15% SDS-PAGE. The separated proteins were transferred to PVDF membrane and assayed with a polyclonal antibody raised against Pru p 3 (PAB). IgE immunoblotting assays were carried out using sera from two groups of patients: 1) allergic to pomegranate, 2) allergic to peach (sensitised to Pru p 3). For identification the proteins were subjected to MS. Results: By this approach up to 10 spots with an apparent MW of 9\u201312 kDa (depend- ing on the redox conditions) were separated for PG. Five of these proteins were recognized by the PAB and by the patient\u2019s sera. IgE immunoblotting experiments clearly showed that PG LTP isoforms are differentially recognized. In contrast, 2D analysis of peach revealed only a single predominant9kDa spot recognized by the PAB and by the patient\u2019s sera. Conclusions: Up to 5 LTP isoforms have been separated from PG by the proteomic approach. The power of this technique is demonstrated by the identification of a large number of IgE reactive LTP isoforms never described for other organisms. The immunological study of the identified isoforms indicates that they possess different IgE epitopes which might contribute differently to the whole allergenic potency of pomegranate

Pomegranate ( Punica granatum L.) expresses several nsLTP isoforms characterized by different immunoglobulin E-binding properties.

BACKGROUND: Pomegranate allergy is associated with sensitization to non-specific lipid transfer proteins (nsLTPs). Our aim was to identify and characterize the non-specific nsLTPs expressed in pomegranate at the molecular level and to study their allergenic properties in terms of immunoglobulin E (IgE)-binding and cross-reactivity with peach nsLTP (Pru p 3). METHODS: A non-equilibrium two-dimensional (2-D) electrophoretic approach based on acid-urea PAGE and sodium dodecyl sulfate PAGE was set up to separate pomegranate nsLTPs. Their immunoreactivity was tested by immunoblotting carried out with anti-Pru p 3 polyclonal antibodies and sera from pomegranate-allergic patients. For final identification, pomegranate nsLTPs were purified by chromatography and subjected to trypsin digestion and mass spectrometry (MS) analysis. For this purpose, the sequences obtained by cDNA cloning of three pomegranate nsLTPs were integrated in the database that was subsequently searched for MS data interpretation. RESULTS: Four nsLTPs were identified by 2-D immunoblotting. The detected proteins showed different IgE-binding capacity and partial cross-reactivity with Pru p 3. cDNA cloning and MS analyses led to the identification of three nsLTP isoforms with 66-68% amino acid sequence identity named Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301. CONCLUSIONS: By 2-D electrophoresis, we could separate different nsLTP isoforms possessing different IgE-binding properties, which might reflect peculiar allergenic potencies. The contribution of Pru p 3 to prime sensitization is not central as in other plant nsLTPs

Erratum: Comparison of IgE-Binding Capacity, Cross-Reactivity and Biological Potency of Allergenic Non-Specific Lipid Transfer Proteins from Peach, Cherry and Hazelnut

<i>Background:</i> Whether the observed clinical pattern of non-specific lipid transfer protein (nsLTP)-mediated food allergies is attributable to a primary sensitization by Pru p 3 from peach and subsequent cross-reactivity with Rosaceae- and non-Rosaceae-derived foods expressing homologous allergens is still unclear. <i>Objective:</i> To investigate the allergenic properties of nsLTPs from Rosaceae and non-Rosaceae foods. <i>Methods:</i> In peach-, cherry- or hazelnut-allergic patients, prevalence of sensitization, IgE-binding capacity, cross-reactivity and allergenic potency of Pru p 3 was compared with Pru av 3 (cherry) and Cor a 8 (hazelnut). <i>Results:</i> Frequency of sensitization to corresponding nsLTPs was 88, 85, and 77% in peach-, hazelnut- and cherry-allergic patients, respectively. Concomitant allergic reactions to cherry and hazelnut were reported in 51 and 44% of peach-allergic patients, respectively. In contrast to cherry allergy, hazelnut allergy was not strictly associated to peach allergy. Sensitization to Cor a 8 or Pru av 3 was strongly correlated with IgE reactivity to Pru p 3, even when subjects tolerated peach. Specific IgE was highest for Rosaceae LTPs, and cross-inhibition experiments confirmed a stronger IgE-binding capacity of Pru p 3 than Cor a 8. The biological potency of Pru p 3 and Pru av 3 was similar but stronger for both nsLTPs than that of Cor a 8. <i>Conclusion:</i> Clinical cross-reactivity of food-allergic patients in the Mediterranean area is likely attributed to a primary sensitization to Pru p 3 and serological cross-reactivity with homologous food nsLTPs. In comparison to Cor a 8, Rosaceae nsLTPs showed a stronger IgE-binding capacity and allergenic potency indicating a different epitope pattern

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 projec