Characterization of peach thaumatin-like proteins and their identification as major peach allergens.

BACKGROUND: Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross-reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin-like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen. OBJECTIVE: To identify members of the TLP family in peach fruit and to characterize putative allergens. METHODS: Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N-terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (beta-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). RESULTS: Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N-asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed beta-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. CONCLUSION: Three isoforms, belonging to the TLP family, were identified in peach as principal allergens. Their prevalence, observed in in vitro, ex vivo and in vivo analyses, suggests that they are important allergens and should therefore be included in the routine diagnosis of peach allergy, at least in the Mediterranean area.

Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms.

BACKGROUND: Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi-sensitized [skin prick test (SPT)-positive] and truly kiwi-allergic patients. OBJECTIVE: To isolate major kiwifruit allergens, and to explore their relevance by in vitro and in vivo methods in a large kiwi-sensitized and -allergic population. METHODS: A large group (n=92) of kiwi-sensitized patients with different clinical symptoms were selected, and double-blind, placebo-controlled, food challenges to kiwi were performed in 52 of them. The three major IgE-binding proteins from kiwifruit extracts were isolated and characterized by N-terminal amino acid sequencing and molecular size and glycosylation analysis. The allergenic potency of the three kiwi allergens, and of avocado Pers a 1 as a model allergen associated with the latex-fruit syndrome, was tested by specific IgE quantitation, immunodetection assays and SPTs. RESULTS: The isolated kiwifruit allergens were identified as actinidin Act d 1, glycosylated thaumatin-like Act d 2 and a novel 40 kDa glycoprotein designated as Act d 3.02. Specific IgE to each of the three allergens was found in over 60% of sera from kiwi-sensitized patients, and Act d 1 and Act d 2 induced positive SPT responses in over 50% of the tested patients. A significant link between IgE levels to Act d 1 and Act d 3 and anaphylaxis was uncovered. Avocado Pers a 1 showed an in vitro sensitization prevalence of around 45%, but a low in vivo reactivity. CONCLUSION: Act d 1, Act d 2 and Act d 3 are major allergens in the population studied. Severe symptoms after kiwi ingestion are associated with high IgE levels to Act d 1 and Act d 3.

Plant non-specific lipid transfer proteins: an interface between plant defence and human allergy.

Plant non-specific LTPs (lipid transfer proteins) form a protein family of basic polypeptides of 9 kDa ubiquitously distributed throughout the plant kingdom. The members of this family are located extracellularly, usually associated with plant cell walls, and possess a broad lipid-binding specificity closely related to their three-dimensional structure. The nsLTP fold is characterized by a compact domain composed of 4 alpha-helices, firmly held by a network of 4 conserved disulphide bridges. This fold presents a large internal tunnel-like cavity, which can accommodate different types of lipids. nsLTPs are involved in plant defence mechanisms against phytopathogenic bacteria and fungi, and, possibly, in the assembly of hydrophobic protective layers of surface polymers, such as cutin. In addition, several members of the nsLTP family have been identified as relevant allergens in plant foods and pollens. Their high resistance to both heat treatment and digestive proteolytic attack has been related with the induction by these allergens of severe symptoms in many patients. Therefore, they are probably primary sensitizers by the oral route. nsLTP sensitization shows an unexpected pattern throughout Europe, with a high prevalence in the Mediterranean area, but a low incidence in Northern and Central European countries.

Two different profiles of peach allergy in the north of Spain.

BACKGROUND: Peach allergy has two different patterns: central Europe with oral allergy syndrome (OAS) related to a primary sensitization to birch pollen Bet v 1 and profilins and southern Europe with mostly systemic symptoms, in many cases due to sensitization to lipid-transfer proteins. METHODS: Thirty peach-allergic patients with positive skin and food challenge tests and 29 control subjects were included. Skin prick tests (SPT) with inhalant allergens, commercial peach and apple extracts and native Pru p 3 were performed. In vitro specific immunoglobulin (Ig) E to grass pollen, birch pollen, peach, apple, rBet v 1, rBet v 2 and rPhl p 12 was determined by CAP, and rBet v 1, rMal d 1, rMal d 4, rMal d 3 and rPru p 3 using the ADVIA-Centaur platform. Basophil activation test (BAT) with commercial peach extract, commercial apple extract, nPru p 3, rMal d 3, rMal d 1 and rMal d 4 was also performed. RESULTS: Pru p 3 was the major allergen in the patient group from northern Spain. Sensitization to this allergen was found in 100% of the patients with systemic symptoms or contact urticaria. Only 60% of OAS patients were sensitized to Pru p 3, being all of them sensitized to profilins and 60% of them to allergens of the Bet v 1 family. Specific IgE determination and BAT using recombinant allergens (rPru p 3) show specificity and sensitivity values close to 100%. CONCLUSIONS: Most peach-allergic patients coming from the north of Spain present systemic symptoms after ingestion of peach, Pru p 3 being the main allergen. Patients with OAS present profilin-Bet v 1-related sensitization. Thus, in the north of Spain our patients show a mixed central-south Europe pattern with LTP-profilin-Bet v 1 sensitization depending on the symptoms presented. The use of natural and recombinant plant allergens, allows establishing the sensitization patterns to the different allergens studied.

Allergic hypersensitivity to the lentil pest Bruchus lentis.

BACKGROUND: Lentils are the most common legume involved in allergic reactions in the Mediterranean area and India. Lentil crops could be attacked by a wide range of insect species. Occupational asthma caused by the lentil pest Bruchus lentis has been described in the present study. OBJECTIVE: We studied the possibility of immunoglobulin (Ig)E-mediated hypersensitivity to lentil pests in 16 patients who suffered from allergic symptoms (asthma and anaphylaxis) related to inhalation of lentil particles or ingestion of lentils, in which sensitization to legume proteins was not clear. METHODS: Extracts prepared either from noninfested and infested lentils, and from the lentil parasite B. lentis were used for skin prick testing (SPT), bronchial and oral challenges and in vitro determinations. RESULTS: Skin prick test were positive to infested lentils and B. lentis in all patients and negative to noninfested lentil extracts. Five asthmatic patients reacted in the bronchial challenge test with Bruchus extract. Oral challenges performed with boiled infested lentils were positive in six of seven patients. Immunoglobulin E immunoblotting with Bruchus and infested lentils extracts revealed protein bands that were reactive to serum IgE from six and four, respectively, out of 16 patients. These patients had no IgE to lentil-specific proteins as determined by immunoblotting and Len c 1-specific IgE test. CONCLUSIONS: Lentil pest proteins can be a cause of IgE-mediated rhinoconjunctivitis and asthma in patients eating or inhaling infested lentil particles.

Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen.

BACKGROUND: Orange is among the most widely consumed fruits, and among the plant food sources causing allergic reactions according to popular perception. However, its relevant allergenic components are virtually unknown. Profilin is a well-defined minor plant panallergen, showing prevalences around 30% in fruits and vegetables. METHODS: Twenty-three orange-allergic patients were studied. Natural orange profilin, named Cit s 2, was purified by affinity chromatography and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry analysis and isolation of its coding cDNA. Reactivity to Cit s 2 was analyzed in vivo by skin prick tests (SPT) and in vitro by IgE immunodetection, specific IgE determination in individual sera and enzyme-linked immunosorbent assay-inhibition assays. RESULTS: The N-terminal amino acid sequence and molecular mass of natural Cit s 2, both fully in agreement with the complete amino acid sequence deduced from its coding cDNA, demonstrated its profilin nature. An unexpectedly high reactivity to Cit s 2 was found in vivo (78% of positive SPT responses) and in vitro (87% of sera from orange allergic patients had specific IgE to Cit s 2). The purified allergen inhibited around 50% of the IgE binding to an orange pulp extract. CONCLUSION: Orange profilin Cit s 2, unlike other plant food profilins, is a major and highly prevalent allergen.

Allergenic reactivity of the melon profilin Cuc m 2 and its identification as major allergen.

BACKGROUND: Melon allergy is commonly associated with oral allergy syndrome (OAS) and with hypersensitivity to pollens and other plant foods. No melon allergen responsible for these clinical characteristics has yet been isolated, although profilin has been proposed as a potential target. OBJECTIVE: To isolate natural and recombinant melon profilin, to evaluate its in vivo and in vitro reactivity, and to analyse its behaviour in simulated gastric fluid (SGF) and heat treatments. METHODS: A pool or individual sera from 23 patients, and an additional group of 10 patients, all of them with melon allergy, were analysed by in vitro and in vivo tests, respectively. Natural melon profilin (nCuc m 2) and its recombinant counterpart (rCuc m 2) were isolated by poly-l-proline affinity chromatography, and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization analysis, DNA sequencing of cDNAs encoding rCuc m 2, and immunodetection with anti-profilin antibodies. In vitro analysis included IgE immunodetection, specific IgE determination, ELISA-inhibition assays, and histamine release (HR) tests. In vivo activity of nCuc m 2 was established by skin prick testing (SPT). The effect of SGF and heat treatment on rCuc m 2 was followed by immunodetection, ELISA inhibition, and HR assays. RESULTS: Both purified forms of melon profilin were recognized by rabbit anti-profilin antibodies and IgE of sera from allergic patients, and showed molecular sizes typical of the profilin family. nCuc m 2 had a blocked N-terminus, whereas rCuc m 2 rendered the expected N-terminal amino acid sequence, its full protein sequence being highly similar (98--71% identity) to those of profilins from plant foods and pollens. The natural allergen displayed a slightly higher IgE-binding capacity than its recombinant counterpart. Specific IgE to nCuc m 2 and rCuc m 2 was found in 100% and 78% of the 23 individual sera analysed, respectively. nCuc m 2 evoked positive SPT responses in all (10/10) patients tested, and rCuc m 2 induced HR in two out of three sera assayed. SGF treatment readily inactivated rCuc m 2, as shown by its loss of recognition by anti-profilin antibodies, lack of IgE binding, and inability to induce HR. In contrast, heat treatment did not affect the IgE-binding capacity of rCuc m 2. CONCLUSIONS: Profilin is highly prevalent in melon-allergic patients, and promptly inactivated by SGF, as expected for an allergen mainly linked to OAS.

Latex-vegetable syndrome due to custard apple and aubergine: new variations of the hevein symphony.

An increasing number of vegetables with crossreactions to latex are being described in patients with latex-vegetable syndrome. We present two of these vegetables, custard apple linked in two previous cases with latex sensitisation, and aubergine, that had not been described up to now in patients with latex sensitisation. The diagnosis of both cases was based on the clinical history, positive skin prick test (SPT) and specific IgE to the offending vegetables, as well as to positive SPT and specific IgE levels to latex and the major fruits involved in the latex-fruit syndrome (avocado, banana, and chestnut). Further, crude extracts from latex, custard apple and aubergine, as well as the purified allergens Hev b 6.02 and Prs a 1 were used in in vitro and in vivo assays: IgE immunodetection, histamine release (HRT) and basophil activation (BAT) tests and skin prick tests. In case 1, both purified Hev b 6.02 and Prs a 1 induced positive responses in skin prick tests, high levels of basophil activation and histamine release. Specific IgE immunodetection uncovered a reactive band of 45 kd in the crude custard apple extract, which was also recognized by anti-chitinase monospecific antibodies. The serum from patient 1 also detected Prs a 1 in immunodetection. Hev b 6.02 produced positive skin responses and showed high biological activity in HRT and BAT in the case of patient 2. However, Prs a 1 was reactive neither in SPT nor in IgE immunodetection. In fact, no band was detected using the serum of patient 2 in avocado or aubergine extracts. By contrast, Prs a 1 reached high values of basophil activation and over 10% of histamine release in case 2.

Vicilin and convicilin are potential major allergens from pea.

BACKGROUND: Allergic reactions to pea (Pisum sativum) ingestion are frequently associated with lentil allergy in the Spanish population. Vicilin have been described as a major lentil allergen. OBJECTIVE: To identify the main IgE binding components from pea seeds and to study their potential cross-reactivity with lentil vicilin. METHODS: A serum pool or individual sera from 18 patients with pea allergy were used to detect IgE binding proteins from pea seeds by immunodetection and immunoblot inhibition assays. Protein preparations enriched in pea vicilin were obtained by gel filtration chromatography followed by reverse-phase high-performance liquid chromatography (HPLC). IgE binding components were identified by means of N-terminal amino acid sequencing. Complete cDNAs encoding pea vicilin were isolated by PCR, using primers based on the amino acid sequence of the reactive proteins. RESULTS: IgE immunodetection of crude pea extracts revealed that convicilin (63 kDa), as well as vicilin (44 kDa) and one of its proteolytic fragments (32 kDa), reacted with more than 50% of the individual sera tested. Additional proteolytic subunits of vicilin (36, 16 and 13 kDa) bound IgE from approximately 20% of the sera. The lentil vicilin allergen Len c 1 strongly inhibited the IgE binding to all components mentioned above. The characterization of cDNA clones encoding pea vicilin has allowed the deduction of its complete amino acid sequence (90% of sequence identity to Len c 1), as well as those of its reactive proteolytic processed subunits. CONCLUSIONS: Vicilin and convicilin are potential major allergens from pea seeds. Furthermore, proteolytic fragments from vicilin are also relevant IgE binding pea components. All these proteins cross-react with the major lentil allergen Len c 1.

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.

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. 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.

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.

Diversity of asparagus allergy: clinical and immunological features.

BACKGROUND: Asparagus (Asparagus officinalis) is an extensively grown and consumed vegetable. To a lesser extent than other Liliaceae vegetables, allergic contact dermatitis (ACD) due to asparagus has been reported. However, only a few case reports of asparagus IgE-mediated allergy have been published. In a previous study, we demonstrated that two lipid transfer proteins (LTPs) (Aspa o 1.01 and Aspa o 1.02) were relevant allergens of asparagus. OBJECTIVE: We retrospectively analysed the 27 patients diagnosed with asparagus allergy during the last 5 years. All of them reported adverse symptoms after either asparagus ingestion or handling. We describe their clinical features and evaluate whether they were associated to immunological findings (immunoblot pattern and skin reactivity to LTPs). METHODS: Patients underwent skin prick and patch tests with standard panels of vegetables and aeroallergens. Besides crude asparagus extract, two purified LTPs were prick and patch tested. Total and specific IgE measurements and asparagus extract IgE immunoblotting were performed. Patients reporting asthma symptoms underwent specific inhalation challenge to asparagus. RESULTS: Of the 27 subjects, eight had ACD, 17 had IgE-mediated allergy and two had both ACD- and IgE-mediated allergy. Positive patch tests with the crude asparagus extract but not with LTPs were observed in subjects with ACD (n=10). Of 19 patients with IgE-mediated disease, 10 had contact urticaria after asparagus handling. Of them, five subjects and five others without skin allergy showed respiratory symptoms; of them, eight were diagnosed with occupational asthma confirmed by positive asparagus inhalation challenge, whereas the remaining two had isolated rhinitis. Four patients suffered from immediate allergic reactions related to asparagus ingestion (food allergy); three of them reported anaphylaxis whereas the other had oral allergic syndrome. Positive IgE immunoblotting (bands of 15 and 45-70 kDa) was observed in 10 subjects. Of 10 subjects with positive prick test to LTPs, six showed bands at 15 kDa. Either IgE-binding bands or positive prick tests to LTPs were observed in asthma (62%) and anaphylaxis (67%). CONCLUSION: Asparagus is a relevant source of occupational allergy inducing ACD and also IgE-mediated reactions. Severe disease (anaphylaxis or asthma) is common and LTPs seem to play a major role. The clinical relevance of LTP sensitization among patients with mild disease or symptom-free subjects should be addressed in prospective studies.

Peach profilin: cloning, heterologous expression and cross-reactivity with Bet v 2.

BACKGROUND: Peach is among the main foods causing allergic reactions in the Mediterranean adult population. Only a single peach allergen, named Pru p 3, has been characterized. However, a potential role of profilin has also been suggested in grass pollen-associated allergy to peach. METHODS: Complementary DNA clones for two different peach profilin isoforms were obtained by reverse transcriptase polymerase chain reaction using non-degenerated primers. Expression of recombinant peach profilin was performed in Escherichia coli, and confirmed using rabbit polyclonal antibodies to sunflower pollen profilin. Twenty-nine individual sera from patients with peach allergy proved by double-blind, placebo-controlled food challenges (DBPCFC), either with (n = 15) or without (n = 14) specific IgE to Bet v 2, were used in immunodetection assays to test recombinant peach profilin reactivity. RESULTS: Each peach profilin cDNA included an open reading frame coding for a 131 amino acid protein. The peach profilin isoforms, designated Pru p 4.01 and Pru p 4.02, showed 80% of amino acid sequence identity, and were very similar (>70% identity) to allergenic profilins from plant foods and pollens. Recombinant Pru p 4.01 was expressed in E. coli as a nonfusion protein, displaying the expected molecular size and reacting with anti-profilin antibodies. rPru p 4.01 was recognized by all sera (15 of 15) with specific IgE to Bet v 2, whereas no sera (zero of 14) without IgE to this birch allergen reacted with rPru p 4.01. CONCLUSIONS: Peach profilin Pru p 4 is very closed to other allergenic profilins from plant foods and pollens. A complete correlation between reactivity to rPru p 4 and rBet v 2 has been found in sera from peach allergic patients.

Allergy after ingestion or inhalation of cereals involves similar allergens in different ages.

BACKGROUND: Cereals are among the major foods that account for food hypersensitivity reactions. Salt-soluble proteins appear to be the most important allergens contributing to the asthmatic response. In contrast, very limited information is available regarding cereal allergens responsible for allergic reactions after ingestion of cereal proteins. OBJECTIVE: The aim of this study was to evaluate the allergenic reactivity of ingested and inhaled cereal allergens in different ages, in order to investigate if the response to different allergens would depend on the sensitization route. METHODS: We included 66 patients in three groups. Group 1: 40 children aged 3 to 6 months who suffered from diarrhoea, vomiting, eczema or weight loss after the introduction of cereal formula in their diet and in which a possibility of coeliac disease was discarded. Group 2: 18 adults with food allergy due to cereals tested by prick tests, specific IgE and food challenge. Group 3: eight patients previously diagnosed as having baker's asthma. Sera pool samples were collected from each group of patients and IgE immunoblotting was performed. RESULTS: We found an important sensitization to cereal in the 40 children. The most important allergens were wheat followed by barley and rye. Among the adults with cereal allergy, sensitization to other allergens was common, especially to Lolium perenne (rye grass) pollen. Immunoblotting showed similar allergenic detection in the three groups. CONCLUSION: Clinically significant reactivity to cereal may be observed in early life. Inhalation and ingestion routes causing cereal allergy seem to involve similar allergens. The diet control was more effective in children. The possibility of cereal allergy after the introduction of cereal formula during the lactation period should not be underestimated.

cDNA cloning and heterologous expression of the major allergens from peach and apple belonging to the lipid-transfer protein family.

BACKGROUND: Lipid-transfer proteins (LTPs) have been identified as major allergens of Rosaceae fruits in populations living in areas virtually free of Fagales trees, such as several Mediterranean communities. Pru p 3 and Mal d 3, the allergens from peach and apple, respectively, have a main clinical relevance in these areas. OBJECTIVE: To isolate and characterize cDNAs for Pru p 3 and Mal d 3, and to produce recombinant Pru p 3 in the yeast Pichiapastoris. METHODS: cDNAs for both allergens were isolated by polymerase chain reaction using nondegenerated primers. Expression of Pru p 3 was performed in P. pastoris using the pPIC 9 vector. The recombinant product was purified by gel-filtration chromatography followed by RP-HPLC. Immunodetection and immunoblot inhibition assays were carried out with sera from peach-allergic patients. RESULTS: The cDNAs for both Pru p 3 and Mal d 3 showed a 273 open reading frame coding for the 91 amino acid mature polypeptides. The deduced amino acid sequences exhibited N-terminal regions fully identical to those previously determined for the natural peach and apple allergens. Pru p 3 was expressed in P. pastoris at 20 mg/L of culture medium. The recombinant allergen showed the same N-terminal sequence (plus a glutamic acid added for proper extracellular expression) and apparent molecular size as natural Pru p 3. Both the recombinant and natural forms of Pru p 3 displayed similar immunoglobulin (Ig)E-binding capacity in immunodetection and immunoblot inhibition assays. CONCLUSIONS: Comparison of the complete primary structures of mature Pru p 3 and Mal d 3 deduced from their corresponding cDNA clones supports the close relationship between both allergens. Recombinant Pru p 3 binds IgE in vitro like its natural counterpart. Therefore, it can be a useful tool for specific diagnosis and structural studies.

What is the role of the hevein-like domain of fruit class I chitinases in their allergenic capacity?

BACKGROUND: Class I chitinases are the major panallergens in fruits associated with the latex-fruit syndrome. These enzymes contain an N-terminal hevein-like domain homologous to latex hevein, and a larger catalytic domain. The role of these domains in their allergenic capacity is still controversial. OBJECTIVE: We sought to evaluate the role of both domains of class I chitinases in their IgE-binding properties, using Cas s 5, the major allergen from chestnut, as a model. METHODS: Recombinant Cas s 5 and its deleted form, lacking the hevein-like domain, designated rCat, were expressed in Pichia pastoris using the pPIC 9 vector. Both recombinant products were purified from the supernatants of transformed yeast cultures by gel-filtration and cation-exchange chromatography. The isolated proteins were characterized by N-terminal sequencing, enzymatic activity and N-glycosylation tests, anti-chitinase and specific IgE immunodetection. Immunoblot, RAST and CAP inhibition assays were also performed. RESULTS: Both purified rCas s 5 and rCat showed the expected N-terminal amino acid sequences and an enzymatic activity similar to that of their natural counterparts isolated from chestnut seeds, and were strongly recognized by anti-chitinase antibodies. In contrast, only rCas s 5, but not rCat, bound specific IgE from sera of patients suffering from the latex-fruit syndrome, and fully inhibited IgE-binding to natural Cas s 5 in immunoblot inhibition assays. Latex hevein also exerted a strong immunoblot inhibition of IgE-binding to chestnut Cas s 5. RAST and CAP inhibition using whole chestnut extract on the solid phase, rendered inhibition levels around 70-90% for rCas s 5 and 60% for rCat, in contrast to the immunoblotting results. CONCLUSIONS: Recombinant Cas s 5 behaves like natural Cas s 5 in IgE-binding assays in vitro. The hevein-like domain of allergenic class I chitinases seems to include all their main IgE-binding epitopes when tested by immunodetection and immunoblot inhibition experiments. RAST and CAP inhibition assays, on the contrary, suggest that relevant epitopes are also harboured in the catalytic domain of these allergens.

Anaphylaxis associated with antiphospholipid syndrome.

BACKGROUND: To our knowledge, no previously published reports have described food-induced anaphylaxis associated with the antiphospholipid syndrome. OBJECTIVE: We undertook a study of four patients with thrombosis associated with the antiphospholipid syndrome after each patient experienced anaphylaxis attributable to ingestion of vegetal foods. METHODS: IgE antibody levels to various foods were determined in serum specimens from the study patients, and skin prick tests with the same allergens were conducted to determine their in vivo responses. Hematologic, cardiopulmonary, vascular, and rheumatologic studies were also performed. IgG anticardiolipin antibody levels were determined by ELISA. RESULTS: All four patients fulfilled the criteria for antiphospholipid syndrome and had high levels of specific IgE antibodies for certain food allergens. By immunoblot analysis, the presence of serum IgE specific for a 45-kD protein band in an almond extract was detected in these four patients who experienced food-related anaphylaxis. No specific IgE was detected in sera from normal subjects. No IgE antibodies specific for the food panallergen lipid transfer proteins were detected. CONCLUSIONS: This is the first report of severe food-precipitated anaphylaxis associated with the antiphospholipid syndrome and the first description of a patient with allergy to blackberry. The possible involvement of food panallergens distinct from lipid transfer proteins is also discussed.