Showing papers by "Domingo Barber published in 2004"

Plant non-specific lipid transfer proteins as food and pollen allergens.

Abstract: 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.

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

Abstract: Summary 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. Objective To assess the pattern of sensitization to an array of mugwort allergens in a Mediterranean population, and to study the cross-reactivity of Art v 3 with Pru p 3 and Par j 1, relevant LTP allergens in the area. Methods Skin prick test was performed with whole extracts (A. vulgaris, Parietaria judaica and peach) and pure natural allergens Art v 1, Art v 3, Art v 60 kDa and Par j 1 in 24 mugwort-allergic patients from a Mediterranean area. In vitro assays included measurement of specific IgE and ELISA inhibition among LTP allergens. Results The three Artemisia allergens elicited a positive skin response in 70–80% of the patients. Seven patients were clearly sensitized to Par j 1 and 11 to Pru p 3. There was no correlation between Par j 1 and Pru p 3 sensitization, but a highly significant correlation was found between peach extract and Art v 3 as regards the skin response. No IgE cross-reactivity was observed between Art v 3/Par j 1 or Pru p 3/Par j 1. In contrast, Art v 3 significantly inhibited the binding to Pru p 3 of IgE from three patients' sera out of six studied, but Pru p 3 was not able to inhibit the IgE binding to Art v 3. Conclusion Art v 3 is a major mugwort allergen and in some patients with IgE to both Art v 3 and Pru p 3, Art v 3 behaves as the primary sensitizing agent.

Respiratory allergy to peach leaves and lipid-transfer proteins.

Abstract: Summary Background Several lipid-transfer proteins (LTPs) have been identified as important food allergens, especially in fruits of the Rosaceae family The major peach (Prunus persica) allergen has been identified, sequenced and designated Pru p 3 Objective To present Pru p 3 as an aeroallergen able to induce occupational asthma Methods A thorough investigation was performed in a fruit grower with occupational asthma Skin prick–prick tests with peach leaves and prick tests with perennial respiratory allergens and pollens, fruits and peach leaf extracts were done Serum-specific IgE was tested for peach leaf, peach fruit, peach skin and respiratory allergens that were positive in skin prick tests Specific bronchial provocation tests (BPTs) with extracts of peach leaf were also done Before and 24 h after the BPT, BPTs with methacholine and sputum induction were done The IgE reactivity pattern to peach leaf and fruit extracts and to Pru p 3 was identified by using SDS-PAGE and immunoblotting Blotting inhibition of peach leaf extract by Pru p 3 was also performed The putative allergen was quantified in leaf and fruit skin extracts with ELISA based on an anti-Pru p 3 antibody Results Skin tests were positive for peach leaf and fruit The BPT was positive, with immediate and delayed response This test induced a decrease in PD20 (dose of agonist that induces a 20% fall in FEV1) methacholine and an increase in eosinophils and eosinophil cationic protein in sputum Peach leaf extract contained concentrations of Pru p 3 similar to those found in peach skin Specific IgE immunodetection showed that patient's sera reacted with Pru p 3, and with a single major band from the peach leaf extract fully inhibited by Pru p 3 Conclusion Pru p 3 from peach leaves can act as a respiratory allergen and cause occupational rhinoconjunctivitis and asthma

Occupational asthma due to grain pests Eurygaster and Ephestia.

Abstract: Background: Workers occupationally exposed to grain dust have a high prevalence of asthma. The pathogenesis of their respiratory symptoms remains obscure when sensitization to cereal allergens has not been proved. Given the ubiquity of arthropods in stored vegetable products, we have studied the allergenic potential of two very prevalent grain pests, Eurygaster and Ephestia, as a cause of occupational asthma. We have also studied the allergenic relationship between Anisakis simplex (AS)and these pests. Methods: We selected 15 asthmatic workers exposed to cereal dust, in whom sensitization to cereal allergens was not clear. As controls, we selected a patient who suffered from anaphylaxis after the ingestion of cereals, 6 patients sensitized to different arthropods, 1 patient who suffered from asthma after inhaling fish flour contaminated with AS, and a pool of 40 asthmatic patients with different ethiologies not due to arthropods or cereals. We performed prick tests with these pests, AS, and pure and paras...

Monoclonal antibody‐based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1

Abstract: Background: Pollen of Artemisia vulgaris (mugwort) is a relevant cause of pollinosis in temperate and humid regions. Recently, the major allergen of this pollen, Art v 1, has been characterized. Objective: To develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) to quantify Art v 1, and to assess the correlation of Art v 1 content with the biological activity of mugwort pollen extracts. Methods: Art v 1-specific mAbs were obtained from a BALB/c mouse immunized with high-performance liquid chromatography (HPLC)-purified Art v 1. One of these antibodies (Av 3.7), which recognizes the N-terminal defensin-like domain of Art v 1, was used as the capture antibody in an ELISA method for allergen quantitation. An anti-A. vulgaris rabbit serum was used as the second antibody. Art v 1 was purified by immunoaffinity chromatography and used as the standard in the assay. Results: The purity and identity of the affinity-purified Art v 1 was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), mass spectrometry, amino acid composition, and N-terminal amino acid sequencing. The prevalence of specific IgE against Art v 1, determined by radioallergosorbent test (RAST) in a population of 44 mugwort-allergic patients, was 79%. The Art v 1-ELISA developed displays a detection limit of 0.1 ng/ml, and a practical working range of 0.2–10 ng/ml. The concentration of Art v 1 was measured in 10 A. vulgaris pollen extracts, and a good correlation was observed between the Art v 1 content and the allergenic activity of the extracts. Conclusions: The results prove the usefulness of the Art v 1-ELISA for the standardization of A. vulgaris pollen extracts intended for clinical use.

Monoclonal antibody-based ELISA to quantify the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d 1.

Abstract: Background: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major

Mapping of Der p 2 antibody binding epitopes by site directed mutagenesis

Abstract: Methods rDer p 2 was subjected to site directed mutagenesis at six selected surface positions (K6, K15, H30, E62, H74, K82) distributed over the entire molecular surface. rDer p 2 mutants containing one (n=6), two (n=2), three (n=1), four (n=1) and six (n=1) mutated amino acids were expressed in Pichia pastoris and analyzed by IgE inhibition and monoclonal antibody (mAb) binding experiments. Results The IgE inhibition experiments demonstrated that mutations in position K6, H30 and E62 interfered marginally with IgE binding, whereas mutations at K15, H74 and K82 significantly reduced IgE binding. A cumulative effect was observed for mutations at H74 and K82, and the double mutant reduced IgE to a level comparable to that of the six position mutant. Three out of four mAbs raised against nDer p 2 were affected by the mutations. The single point mutations at K15 and H74 abolished the interaction between two of the mAbs and rDer p 2 mutants. Concomitant binding analysis of the mAbs indicated that at least 2 distinct epitopes could be identified on nDer p 2. Conclusions Site directed mutagenesis of rDer p 2 identified surface exposed amino acid residues, which participate in both IgE and mAb binding epitopes. Other surface exposed amino acid residues of Der p 2 seem to be located in areas not involved in the binding of the investigated antibodies.

Specific IgE-binding of rHev b 12 is restricted to fruit-allergic patients

Abstract: Rationale Lipid transfer proteins (LTP) have been identified as relevant allergens. Their role in latex allergy is unclear. This study aimed to produce a recombinant latex-LTP (rHev b 12) to examine its IgE-binding properties. Methods A recombinant generated Maltose-Binding-Protein (MBP)-rHev b 12 fusion protein was coupled to ImmunoCAP for the determination of specific IgE. 48 sera of atopic patients (14 from Spain, 34 from Germany) with fruit-allergy were examined. 25 of these sera displayed positive latex-specific IgE values. Results rHev b 12-specific IgE was measured in four Spanish peach-allergic patients (CAP-values: 0.88-2.27 kU/L), whereby one had an additional latex-sensitization. Two sera from cherry-allergic German patients with latex-sensitization displayed also rHev b 12-specific IgE antibodies (0.68 and 0.96 kU/L). All other sera and the MBP controls revealed negative results ( Conclusions Latex-LTP (rHev b 12)-specific IgE-binding in peach- and cherry-allergic patients seem to be a result of partially common IgE-binding epitopes with the LTPs of peach (homology to Pru p 3: 65%) and cherry (homology to Pru av 3: 61%). Although rHev b 12 seems to have minor relevance as a latex allergen in Central Europe, its significance as a cross-reactive allergen in Mediterranean countries like Spain has to be kept in mind.

Profile of sensitization to individual latex allergens among health care workers allergic to natural rubber latex

Abstract: Rationale We evaluated the profile of sensitization to different natural and recombinant natural rubber latex (NRL) allergens among health care workers (HCWs). Methods Thirty-nine HCWs allergic to NRL with respiratory and/or cutaneous symptoms as demonstrated by positive skin tests (ALK-Abello) and challenges tests were studied. Specific IgE to whole NRL extract (CAPk82) and different latex allergens was measured using CAP-FEIA (Pharmacia). Results Ten percent of the sera were negative to all determinations. The percentage of positive results was as follows: k82 (82%); mix rHev b1,5,6,8 (79%); in 30 sera both measurements were positive and in 6 both were negative, in 2 cases k82 was positive and mix negative, and in 4 cases k82 negative and mix positive. rHev b1 was positive 1/32 (3%), and 6/6 to nHev b2 (100%), 5 of these sera were negative to the recombinant mix, rHev b5 and rHev b6.01, but all them were positive to k82; 11/33 rHev b5 (33%); 23/33 rHev b6.01 (70%); 7 sera were positive to rHev b5 and rHev b6.01 and 6 were negative to both; 4/29 rHev b8 (14%); 0/9 rHev b9, 0/11 rHev b10, 1/10 rHev b11 (10%) and 1/6 rHev b13 (17%). Five sera were k82(-) and some recombinants(+). Conclusions Hev b6.01 and Hev b5 are major allergens recognized by our population of HCWs. Hev b2 also seems an important allergen in some patients. CAPk82 shows a good diagnostic sensitivity similar to the recombinant mix, but the use of a panel of allergens is necessary for in vitro diagnosis in some HCWs allergic to NRL.