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The glycoprotein Pla l 1 is the major allergen from English plantain (Plantago lanceolata) pollen, which is a common cause of pollinosis in temperate areas. Three complete cDNAs for Pla l 1 isoforms were isolated by PCR using specific 3′ and 5′ primers. All three Pla l 1 cDNAs code for a 25-residue leader peptide and a 131-residue mature protein that contains two polymorphic positions, an N-glycosylation site at position 107 and six cysteine residues involved in three disulphide bridges. The allergen variant Pla l 1.0101 was produced in Pichia pastoris at a yield of 20 mg per litre of culture as a mixture of non-glycosylated (17 kDa), glycosylated (23 kDa) and dimeric (32–39 kDa) forms. Recombinant Pla l 1 (rPla l 1) was purified by affinity chromatography with an anti-natural Pla l 1 (anti-nPla l 1) monoclonal antibody, and its molecular and immunological properties were compared with the natural allergen by CD spectroscopic analysis, enzymic deglycosylation, lectin-binding assay, immunodetection and ELISA-inhibition assays using sera from plantain-allergic patients. The recombinant allergen is properly folded, as deduced from CD spectra, and the immunodominant allergenic epitopes of the natural allergen are preserved in rPla l 1. These results allow us to conclude that P. pastoris is a convenient system for the efficient production of biologically active rPla l 1, which could have a potential use for clinical purposes. Furthermore, a sequence similarity of Pla l 1 to the major allergen from the olive tree pollen, Ole e 1, is revealed in this work, and the allergenic cross-reactivity between both allergens has been studied.

Background: The peach lipid transfer protein Pru p 3 has been identified as a major allergen from this fruit. Homologous cross-reactive allergens have been found in several plant foods and pollens. Recombinant Pru p 3 has been recently produced in the yeast Pichia pastoris . Objective: We sought to evaluate the potential role of recombinant Pru p 3 as a novel tool for the diagnosis of fruit allergy. Methods: Circular dichroism analysis was used to compare the protein folding of natural Pru p 3 and recombinant Pru p 3. IgE binding by both molecular forms was quantified by means of ELISA and ELISA inhibition assays, and their biologic activity was estimated by using basophil activation, histamine release, and sulphidoleukotriene production tests. Individual sera or blood samples from patients with peach allergy (up to 17) were used in the assays. Results: A nearly identical circular dichroism spectra was shown by using natural Pru p 3 and recombinant Pru p 3, indicating that both protein forms are similarly folded. No difference was detected in the IgE-binding capacity of the 2 mo-lecular versions. Basophil activation and induction of sulphidoleukotriene production were positive in 9 of 10 patients, and histamine release was induced in at least half of the patients, with similar effects of the natural and recombinant forms in the 3 assays. Conclusion: Recombinant Pru p 3 shows a strong immunologic activity equivalent to that of its natural counterpart, and therefore it can be a useful tool for diagnosis (and future immunotherapy) of fruit allergy. (J Allergy Clin Immunol 2003;111:628-33.)

Background Lipid Transfer Protein (LTP) allergy constitutes the first cause of food allergy to vegetables in Southern Europe. LTPs are highly stable proteins, resistant to both gastric digestion and high temperatures. Allergic reactions to multiples vegetables, not phylogenetically related, are produced based on the cross-reactivity among these proteins, a phenomenon that has been named LTP syndrome. Tri a 14, the wheat LTP, is involved in baker s asthma and anaphylactic reaction. In the present work, we studied Weetabix ® foodstuff tolerance in four patients with wheat allergy, characterized by oral food challenge (OFC) and skin tests, trying to identify the source of this tolerance in wheat-based foodstuff.

Rationale In Southern Europe Pru p3 is the primary sensitizer of plants fruit and it is responsible of severe reactions. Specific immunotherapy (SIT) brings a new perspective to treat those patients. There is a lack of knowledge regarding cellular responses that include changes in the basophil activation during the IT. We aim to analyse early changes in the basophil response to Pru p 3 and other related allergen (Ara h 9) after the first month of sublingual immunotherapy (SLIT).

Background Baker’s asthma is one of the most common types of occupational asthma and its prevalence is increasing in the last years. Diagnosis of occupational asthma is complex. The poor specificity of current diagnostic approaches may be associated with insufficient purity of wheat extracts or lack of inclusion of major allergens in them. In this work, we use microarray technology to characterize the allergenic profiles of baker’s asthma patients from three regions in Spain and to analyze the influence of other environmental allergens on the sensitization pattern.

Background: Lentils seem to be the most common legume implicated in pediatric allergic patients in the Mediterranean area. However, no lentil allergen has been isolated and characterized. Objective: We sought to purify and characterize relevant IgE-binding proteins from boiled lentil extracts. Methods: IgE-binding proteins from crude and boiled lentil extracts were detected with a pool of sera from patients with lentil allergy. Allergens were isolated by gel-filtration chromatography followed by cation- and anion-exchange chromatography or by reverse-phase HPLC. Their characterization included N-terminal amino acid sequencing, complex asparagine-linked glycan detection, specific IgE immunodetection with 22 individual sera from allergic patients, and immunoblot and CAP inhibition assays. Results: Heat treatment of lentils produced substantial changes in the SDS-PAGE patterns of whole extracts, mainly a strong increase of 12- to 16-kd bands and a decrease of 25- to 45-kd components. Major IgE-binding proteins from the boiled lentil extract were located in the 12- to 16-kd and 45- to 70-kd ranges. Two allergens of 16 kd, proteins L1 and L2, and another one of 12 kd, protein L3, were purified. N-terminal sequencing indicated that all 3 were related and allowed their identification as γ-vicilin subunits. Protein L1 was recognized by 68% of the individual sera tested and inhibited 64% of the IgE binding by commercial lentil CAPs. A second type of allergen of 66 kd, named protein H, was also isolated and identified as a seed-specific biotinylated protein. Protein H reacted with 41% of the individual sera and produced 45% inhibition in CAP inhibition assays. Conclusions: Two different types of allergens have been identified in boiled lentils. Those of 12 to 16 kd, called Len c 1, correspond to γ-vicilin subunits, and those of 66 kd, designated Len c 2, correspond to seed-specific biotinylated protein. Homology with proteins from other legume species can explain potential cross-reactions among these foods. (J Allergy Clin Immunol 2000;106:955-61.)

Diagnosis in patients sensitised to multiple pollens is difficult due to the relationship between pollen and food allergens. Misdiagnosis is often a cause for unsuccessful specific immunotherapy. Wheat is a potent allergen source and is one of the causes of baker's asthma, food and pollen allergy. Recently, we have performed a study on pollen sensitisation in our area, where cereal crops are very important. The clinical data from 19718 patients reviewed showed that grass pollen was the main source of clinical symptoms (6369 patients, 32.30% of asthmatics). However, wheat and cereal crop pollen showed very low prevalence. On the other hand, patients with wheat flour allergy after ingestion and/or with baker's asthma were not sensitised to wheat pollen, despite it containing some common allergens. In the same way, all our asthmatic bakers (135 patients) tolerated the ingestion of bread. Here we try to explain the reason for these surprising observations.

Background Banana allergy has been associated with the latex–fruit syndrome. Several IgE-binding components, the relevant ones being proteins of 30–37-kDa, have been detected in banana fruit, but none of them have been isolated and characterized yet. Objective To purify and characterize the 30–37 kDa banana allergens. Methods Fifteen patients allergic to banana were selected on the grounds of a latex-allergic population. Prick by prick tests to this fruit were performed. Total and specific IgE to banana were determined. Banana allergens were isolated by affinity chromatography, followed by cation-exchange chromatography. Their characterization includes N-terminal sequencing, enzymatic activity assays, immunodetection with sera from allergic patients and with antichitinase antibodies, and CAP and immunoblot inhibition tests. Skin prick tests with banana extracts and with the purified allergens were also carried out. Results Two major IgE-binding proteins of 34 and 32 kDa, also recognized by polyclonal antibodies against chestnut chitinases, were immunodetected in crude banana extracts. Purification and characterization of both proteins have allowed their identification as class I chitinases with an hevein-like domain. Each isolated allergen reached inhibition values higher than 90% in CAP inhibition assays, and fully inhibited the IgE-binding by the crude banana extract when tested by an immunoblot inhibition method. The two purified allergens provoked positive skin prick test responses in more than 50% of the banana-allergic patients. Conclusions Class I chitinases with an hevein-like domain are major allergens in banana fruit. Their presence in other fruits and nuts, such as avocado and chestnut, could explain the cross-sensitization among these foods.

S U N D A Y 313 Baked Milk Oral Food Challenges: A Single-Center Pediatric Hospital Experience Rushani W. Saltzman, MD, Mary E. Kelleher, RN, CPN, AE-C, Jonathan M. Spergel, MD, PhD, FAAAAI; The Children’s Hospital of Philadelphia, Voorhees, NJ, Children’s Hospital of Philadelphia, Philadelphia, PA, Perelman School of Medicine at Univ of Pennsylvania. RATIONALE: Cow’s milk (CM) allergy is the most common food allergen in children. Recent research has shown that a majority of children with unheated or native cow’s milk (N-CM) allergy can tolerate the extensively heated form (H-CM) due to epitope structure changes from thermal processing. Furthermore, those H-CM tolerant appear to have accelerated development of tolerance to N-CMwhen compared with those strictly avoiding N-CM. METHODS: Graded, open baked milk oral food challenges (OFC) were performed for CM allergic patients [clinical history and/or positive CM skin prick testing (SPT)]. A standard protocol (1 cup CM baked in standard cake recipe, 3508Fahrenheit, minimum 30 minutes) was followed. Baked milk OFCs conducted at our institution (July 2011-early September 2012) were evaluated by retrospective chart review. Baseline demographics and characteristics were analyzed. RESULTS: Eighteen baked milk OFCs were performed and 61% were baked milk tolerant (ingested 2 gm baked CMwithout reaction). Male sex, younger age, history of other food allergies, history of atopic dermatitis, and larger median CM SPTwheal size were trends noted in the baked CM reactive cohort. Median cumulative dose eliciting clinical reaction was 0.34 gm baked CM. CONCLUSIONS: Our study confirms that a majority of CM allergic patients can tolerate baked milk. Dietary introduction of baked milk after successful completion of a supervised OFC improves one’s quality of life through a broadened diet. Additionally, there is likely accelerated development of tolerance to N-CM. Further studies are needed to elucidate such food allergy phenotypes and establish baked milk OFC patient selection criteria.

Journal Article; Research Support, Non-U.S. Gov't; BACKGROUND Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. OBJECTIVE To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma MATERIALS AND METHODS An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. RESULTS Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA-), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p

Background Sunflower seeds (Helianthus annuus) can trigger anaphylactic reactions, generalized urticaria, angioedema, oral allergy syndrome and other symptoms after ingestion. These reactions have been attributed to 2S albumins (SFA-8) and LTP (Hel a 3). We aimed to characterize the basophil response to storage proteins and oleosins from sunflower seed in patients allergic to sunflower. Methods The proteins 2S, 11S and oleosins were purified from a raw sunflower seed extract by FPLC/HPLC and identified by specific antibodies and peptide mass fingerprinting. We tested the immunological recognition of these proteins by basophil activation test (BAT). Four concentrations (1, 0.2, 0.1, 0.02 ig/ml) of each protein and the sunflower roasted extract were used. Ten patients were selected by clinical history and skin prick test positive to commercial extract. Twelve subjects with skin prick test negative to commercial extract and not food allergy were included as controls. Results All patients showed a positive basophil response to roasted extract. BAT was positive in 87.5% of cases for 2S albumin, 60% for oleosins, and 57.14% for 11S albumin. 50% of patients were positive to the 3 proteins, 37.5% only for 2S albumin and 12.5% for storage proteins (both 2S and 11S albumin). In 40% of controls the concentration 1 ig/ml of 2S albumin induced low basophil activation. Conclusions

Background Lipid transfer proteins (LTP) are considered true food allergens because sensitization takes place in the gastrointestinal tract, in contrast with other food allergens like profilins or Bet v 1-homologues, where sensitization is a consequence of an allergic sensitization to inhaled allergens. Although allergy to peach LTP (pru p 3) is frequently associated to sensitization to plane tree pollen LTP (Pla a 3) and/or mugwort LTP (Art v 3), it has never been elucidated if this cross-reactivity could lead to a respiratory allergy. The aim of this study is to demonstrate that a LTP-dependent food allergy with the adequate exposure can induce a LTP-dependent respiratory allergy.