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Timothy grass pollen is a source of potent allergens. Among them, Phl p 1 and Phl p 5 are thought to be the most important, as a majority of timothy grass-allergic individuals have IgE antibodies directed against these two allergens. The profilin from timothy grass (Phl p 12) has been registered as a minor allergen, with up to 35% of individuals in populations of grass pollen allergic patients showing IgE binding to Phl p 12. Profilins are primarily minor allergens and are known for a high likelihood of co-sensitization as well as cross-reactivity situations caused by their sequence and structure similarity. The crystal structure of Phl p 12.0101 was determined and it revealed that this allergen may form an unusual dimer not previously observed among any profilins. For example, the Phl p 12 dimer has a completely different geometry and interface when compared with the latex profilin (Hev b 8) dimer that has its crystal structure determined. The structure of Phl p 12.0101 is described in the context of allergenic sensitization and allergy diagnostics. Moreover, the structure of the Phl p 12.0101 dimer is discussed, taking into account the production of recombinant allergens and their storage.

Background: In Portugal, the pollen types most implicated in respiratory allergy are grasses, olive and parietaria. The knowledge of sensitizations to molecular allergens in children and adults can contribute to better diagnosis and treatment of this pathology., Methods: ImmunoCAP singleplex technology was used for molecular allergens and Phadia 250® automatic equipment. g205 (Phl p1); g215 (Phl p5b); g210 (Phl p7); and g212 (Phl p12) allergen determinations were made in 45 patients with positive grass sensitization tests., Results: The majority of patients are sensitized to Phl p1 (91%) and Phl p1+/Phl p5-/Phl p7-/Phl p12- was the most dominant profile (40%). In the adult group, the IgE averages for Phl p1 were approximately 10.46, while they were 8.43 for Phl p5, 0.69 for Phl p7, and 0.06 for Phl p12. In the child group, these values were higher: 22.49, 20.23, 3.89, and 0.35, respectively. For allergens Phl p1, Phl p5, and Phl p7, these differences between the child and adult population were not statistically significant (p=0.754, p=0.806 and p=0.102, respectively), but for Phl p12, a statistically significant difference (p=0.018) was observed., Conclusions: IgE antibodies Phl p1 is the most important allergic marker and sensitivities caused by Phl p12 give rise to higher IgE values in children., (Copyright © 2019 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

Background: It has been suggested that latex-specific IgE analysis may lead to false-positive results, especially in patients with pollen allergy. In the present study, the reasons underlying clinically irrelevant latex-specific IgE positivity were investigated., Methods: Thirty patients with latex allergy (group 1), 89 patients sensitised to aeroallergens (group 2a), and 98 healthy individuals without allergy (group 2b) were enrolled. Participants from all 3 groups were subjected to skin prick tests with aeroallergens including latex, latex-specific IgE analysis (ImmunoCAP), and nasal provocation test with latex. All cases demonstrating positive latex-specific IgE also underwent specific IgE tests (ImmunoCAP) with latex profilin, birch pollen profilin, peach lipid transfer protein, and pineapple bromelain as cross-reactive carbohydrate determinants., Results: Comparison of the atopic and healthy control groups showed that the rate of positive latex-specific IgE was significantly higher in group 2a. Latex profilin-, birch pollen profilin-, and bromelain-specific IgE were remarkably higher in group 2a., Conclusion: False positivity to latex-specific IgE in ImmunoCAP analysis may be observed in approximately 19% of patients with pollen allergy. Profilins and bromelain are the main contributors to clinically irrelevant positive latex-specific IgE., (© 2018 S. Karger AG, Basel.)

In the Mediterranean area, lipid transfer proteins (LTPs) are important causes of plant-food allergies often associated with severe allergic reactions. There, peach LTP (Pru p 3) seems to be the primary sensitizer, whereas in Central Europe, little is known about the importance of LTP sensitization. In this region, allergen extract-based diagnosis is often complicated by co-sensitization to Bet v 1, the major birch pollen allergen, its cross-reactive food allergens, and profilins. We investigated the role of LTP sensitization in Central European patients displaying strong allergic reactions to plant-derived food. Analysis of IgE reactivity revealed that ten of thirteen patients were sensitized to Pru p 3, nine to Bet v 1, and two to profilin. Our results showed that LTP sensitization represents a risk factor for severe allergic symptoms in Central Europe. Furthermore, the strong IgE reactivity detected in immunoblots of plant-food extracts indicated that Pru p 3 can be used as a marker allergen for LTP sensitization also in Central European patients., (© 2017 The Authors Allergy Published by John Wiley & Sons Ltd.)

Growing evidence indicates that actin cytoskeleton is involved in plant innate immune responses, but the functional mechanism remains largely unknown. Here, we investigated the behavior of a cotton profilin gene (GhPFN2) in response to Verticillium dahliae invasion, and evaluated its contribution to plant defense against this soil-borne fungal pathogen. GhPFN2 expression was up-regulated when cotton root was inoculated with V. dahliae, and the actin architecture was reorganized in the infected root cells, with a clear increase in the density of filamentous actin and the extent of actin bundling. Compared to the wild type, GhPFN2-overexpressing cotton plants showed enhanced protection against V. dahliae infection and the actin cytoskeleton organization in root epidermal cells was clearly altered, which phenocopied that of the wild-type (WT) root cells challenged with V. dahliae. These results provide a solid line of evidence showing that actin cytoskeleton reorganization involving GhPFN2 is important for defense against V. dahliae infection.

Summary: Introduction. Phleum pratense (Timothy grass) is the most frequent cause of grass allergy in Europe. Molecular-based allergy diagnostics have been recently introduced in the clinical practice, allowing to define and characterize exactly the sensitization profile. Aim of the study. The present study was aimed to investigate the possible relationships between Graminaceae pollen data and the pattern of IgE reactivity to different allergen components across Italy. Methods. Pollen data, including duration and quantity, were recorded over a 10-year period in 24 Italian centres located along the Italian peninsula. Phl p molecular patterns (Phl p 1, 5, 7, 12) were evaluated in 4 different Italian geographical areas. Results. There were significant differences about pollen count and sensitization prevalence across Italy. Different molecular patterns were defined considering the different Italian locations. Conclusion. This study demonstrates that Phleum pratense sensitization is relevant in Italy, but there are significant geographical variations variations as far as pollen exposure and pattern of IgE reactivity to the considered allergen components are concerned. This information may have clinical relevance in managing patients allergic to grass pollen.

Gibberellin-regulated protein (GRP) is a new allergen in peach allergy, with an amino acid sequence very well conserved through several botanical species. We investigated the allergenicity of GRP in fruit allergies other than peaches and identified the clinical characteristics of fruit allergy patients with GRP sensitization. One hundred consecutive Japanese patients with fruit allergies were enrolled in the present study. To identify the features of GRP sensitization, we selected patients with negative ImmunoCAP results for Bet v 1 homologs and profilin, which are marker allergens for pollen-food allergy syndrome (PFAS), or lipid transfer protein. These patients underwent specific immunoglobulin E measurements by enzyme-linked immunosorbent assay (ELISA) and skin prick tests (SPT) using purified nPru p 7. Twenty of 100 consecutive patients with fruit allergies had negative ImmunoCAP results for Bet v 1 homologs and profilin. Thirteen (65.0%) of the 20 patients had positive ELISA and/or SPT results using nPru p 7, whereas one of the 20 patients had positive ImmunoCAP results for Pru p 3. In 13 nPru p 7-sensitized patients, the causative foods were peaches (92.3%), apricots (61.5%), oranges (46.2%) and apples (30.8%). Ten patients (76.9%) had multiple causative fruits. Frequent symptoms included facial edema (92.3%) and laryngeal tightness (66.7%). In eight patients (61.5%), exercise or aspirin intake enhanced the allergic reaction onset as cofactors. The prevalence of GRP sensitization was high in Japanese fruit allergy patients except for PFAS patients. In conclusion, GRP-sensitized patients may have allergies to multiple fruits and may show peculiar characteristics such as facial swelling and cofactor dependence., (© 2017 Japanese Dermatological Association.)

Profilins are small actin-binding proteins found in eukaryotes and involved in cell development, cytokinesis, membrane trafficking, and cell motility. From an allergenic point of view, profilins are panallergens usually involved in allergic polysensitization, although they are generally recognized as minor allergens. The objectives of this study were to identify and characterize the profilin from Plantago lanceolata pollen and to investigate the cross-reactivity between profilins from different pollen allergenic sources. Profilins from P. lancelolata (Pla l 2) and palm tree pollen (Pho d 2) were purified by affinity chromatography, deeply characterized and identified by mass spectrometry. Pla l 2 allergenicity was confirmed by immunoblot with serum samples from a patient population sensitized to profilin. Immunoblot inhibition was performed to study IgG reactivity between different pollen profilins. IgE cross-reactivity was demonstrated by ImmunoCAP inhibition. Pla l 2 is the second P. lanceolata allergen included in the IUIS Allergen Nomenclature database. Four peptides from purified Pla l 2 were identified with percentages of homology with other pollen profilins between 73 and 86%. Eighty-six percent (21/24) of the patient population recognized Pla l 2. The allergenic relatedness between Pla l 2, Pho d 2 and six pollen profilins was confirmed, and IgE cross-reactivity of Pla l 2 with rBet v 2 and rPhl p 12 was demonstrated. Pla l 2 is the profilin from P. lanceolata. The demonstrated allergenicity of this protein and its cross-reactivity with other pollen profilins support its use in profilin diagnostic assays., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tomatoes (Lycopersicon esculentum Mill.) are a widely consumed vegetables and contain many health beneficial micronutrients. Unfortunately, they may also cause adverse allergic reactions in sensitized people. Many studies, conducted in recent years, indicate that organically produced vegetables have higher nutritional value, improved sensory quality and contain more health-enhancing bioactive compounds than vegetables grown under the conventional system. However, the relation between organic methods of cultivation and allergenic potential of tomatoes has received little scientific attention. This study analyzed samples of five tomato cultivars taken from organic and conventional systems over three consecutive years. The content of profilin, Bet v 1 and lipid transfer protein (LTP) analogues in tomato samples was determined using an indirect ELISA assay. Substantial quantities of these proteins were found in certain cultivars across all three years of cultivation. On the basis of these findings, organically grown tomatoes appear to offer little advantage over conventionally cultivated plants in terms of reduced allergenic potential.

Background: Pollens from mesquite (Prosopis juliflora) are potent allergen responsible in causing immediate hypersensitivity reactions in susceptible people in tropical countries., Objective: This study aimed to clone, express and purify the mesquite pollen profilin (Pro j 2) as well as evaluating its nucleotide sequence homology in order to predict allergenic cross-reactivity with profilins of common allergenic plants., Methods: Immunoblotting assay and specific ELISA were applied to determine the immunoreactivity of sera from 35 patients who were allergic to mesquite pollen. The mesquite profilin-coding sequence was cloned into PTZ57R/T vector and amplified. The cDNA of mesquite pollen profilin was then expressed in Escherichia coli using pET-21b (+) vector and puri?ed by one-step Ni2+ a?nity chromatography. IgE binding capacity of the recombinant mesquite profiling (rPro j 2) was analyzed by specific ELISA, immunoblotting, and inhibition assays., Results: cDNA nucleotide sequencing revealed an open reading frame of 399bp encoding for 133 amino acids which belongs to the profilin family. Seventeen patients (17/35, 48.57%) had significant specific IgE level for rPro j 2. Immunodetection and inhibition assays indicated that puri?ed rPro j 2 might be similar as that in the crude extract., Conclusion: Pro j 2, as a new allergen from mesquite pollen, was produced in E. coli with an IgE-reactivity similar to that of its natural counterpart. The amino acid sequences homology analysis of mesquite profilin and several profilin molecules from other plants showed high degree of cross-reactivity among plant-derived profilins from unrelated families.

Background: Caryota mitis is a common plant in tropical and subtropical areas. It produces larger amount of pollen, which has great potential for allergenicity in the pollination season., Objective: This study aims to identify the components of Caryota mitis pollen contributing to human allergic diseases., Methods: The sera from 20 patients with a positive skin prick test to Caryota mitis pollen crude extract (CmPCE) were collected; the allergic components were determined by the forming of immune complexes in the sera and CmPCE. The results were confirmed by the immune competitive inhibitory assay., Results: Sixteen out of 20 collected serum samples reacted to a 14 kDa protein fractioned from CmPCE; this 14 kDa antigen also had positive reactions to CmPCE in an ELISA assay. 12 samples from 20 collected serums positively reacted to recombinant CmProfilin (rCmProfilin), as shown by Western blotting, and also showed positive reactivity in ELISA. Preincubation of sera with rCmProfilin eliminated the reactivity of the patients' sera to this 14 kDa band., Conclusion: A 14 kDa protein from the CmPCE was identified as the major allergic component of CmPCE.

Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767 bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability. Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species. Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases.

Background: Peach allergy is the main cause of vegetable food allergy in the Mediterranean area. Pru p 3 is the major allergen, and it is mainly found in the peel., Objective: We sought to calculate the frequency of tolerance to peach pulp in Spanish children suffering from allergic reactions after eating or having contact with peach and to analyze the sensitization pattern to peach allergens., Methods: Fifty-seven children (32 boys; median age, 7.4 yr) were included in the study. A systematized questionnaire on allergic reactions to peach was administered. Prick tests with peach peel and pulp, peach lipid transfer protein (LTP), and profilin were performed. Serum-specific IgE to peach, rPru p 1, rPru p 3, and rPru p 4 was determined by ImmunoCAP. Oral food challenges (OFCs) with peach pulp were performed on all but one child., Results: Eighty-eight percent of the children had positive prick tests with peach peel, 35% with peach pulp, 88% with peach LTP, and 9% with profilin. Serum-specific IgE to peach was demonstrated in 100% of the patients; rPru p 3-specific IgE in 96%; rPru p 1-specific IgE in 11%; and rPru p 4-specific IgE in 10%. Oral food challenge was negative in 52 (93%) children., Conclusions: More than 90% of the study population tolerated peeled peach. Pru p 3 was the major allergen. Pru p 1 and 4 showed low prevalence., (© 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Background: Pollen allergy is the most common allergic disease. However, tropical pollens, such as those of Palmae, have seldom been investigated compared with the specific immunotherapy studies done on hyperallergenic birch, olive, and ragweed pollens. Although poly(lactic-co-glycolic acid) (PLGA) has been extensively applied as a biodegradable polymer in medical devices, it has rarely been utilized as a vaccine adjuvant to prevent and treat allergic disease. In this study, we investigated the immunotherapeutic effects of recombinant Caryota mitis profilin (rCmP)-loaded PLGA nanoparticles and the underlying mechanisms involved., Methods: A mouse model of allergenic asthma was established for specific immunotherapy using rCmP-loaded PLGA nanoparticles as the adjuvant. The model was evaluated by determining airway hyperresponsiveness and levels of serum-specific antibodies (IgE, IgG, and IgG2a) and cytokines, and observing histologic sections of lung tissue., Results: The rCmP-loaded PLGA nanoparticles effectively inhibited generation of specific IgE and secretion of the Th2 cytokine interleukin-4, facilitated generation of specific IgG2a and secretion of the Th1 cytokine interferon-gamma, converted the Th2 response to Th1, and evidently alleviated allergic symptoms., Conclusion: PLGA functions more appropriately as a specific immunotherapy adjuvant for allergen vaccines than does conventional Al(OH)3 due to its superior efficacy, longer potency, and markedly fewer side effects. The rCmP-loaded PLGA nanoparticles developed herein offer a promising avenue for specific immunotherapy in allergic asthma.

Allergy to citrus fruits is often associated with pollinosis and sensitization to other plants due to a phenomenon of cross-reactivity. The aims of the present study were to highlight the cross-reactivity among citrus and the major allergenic pollens/fruits, throughout clinical and molecular investigations, and to evaluate the sensitization frequency to citrus fruits in a population of children and adults with pollinosis. We found a relevant percentage of sensitisation (39%) to citrus fruits in the patients recruited and in all of them the IgE-mediated mechanism has been confirmed by the positive response to the prick-to-prick test. RT-PCR experiments showed the expression of Cit s 1, Cit s 3 and a profilin isoform, already described in apple, also in Citrus clementine pollen. Data of multiple sequence alignments demonstrated that Citrus allergens shared high percentage identity values with other clinically relevant species (i.e. Triticum aestivum, Malus domestica), confirming the possible cross-allergenicity citrus/grasses and citrus/apple. Finally, a novelty of the present work has been the expression of two phospholipaseA2 isoforms (PLA2 α and β) in Citrus as well as in Triticum pollens; being PLA2 able to generate pro-inflammatory factors, this enzyme could participate in the activation of the allergenic inflammatory cascade.

Background: Grasses belong to major sources of inhaled allergens. The knowledge of particular molecules responsible for hypersensitivity is of crucial importance for better understanding of individual differences among single allergic subjects and allergic populations living in various world-areas., Methods: Specific-IgE-antibodies against Phl p 1, Phl p 5, Phl p 7, Phl p 12 were detected in a group of 130 Phleum-allergic-subjects (82 children, 48 adults)., Results: Phl p 1 antibodies were detected in most pediatric and adult patients, however, the children were associated with higher RAST classes more often. Anti-Phl p 5-antibodies were found more frequently in adults. An increase was observed in the number of pediatric patients reacting to Phl p 7 and Phl p 12. There were no differences in concentrations of specific-IgE against Phl p 5, Phl p 7 and Phl p 12 depending on age. Almost 10% of allergic children produced antibodies directed exclusively against minor allergens or did not produce specific-IgE-antibodies against tested molecules. Part of the patients reacted to profilin and calcium-binding protein originating from only one source (Phl p 12/Bet v 2 and Phl p 7/Bet v 4)., Conclusions: Antibodies against Phl p 1 and Phl p 5 can be used as a marker of allergy to grasses in adult patients. Children reacted exclusively to minor allergens more frequently than adults. Prolonged allergen exposure is evidently necessary to induce sensitization to Phl p 5. A high level of homology between profilins and calcium-binding proteins enables only one allergen to be used for diagnostic purposes but a possibility of a reaction to species-bound epitopes should be taken into account.

Banana fruit allergy is well known, but neither immunoglobulin E recognition patterns to purified plant food allergens nor true prevalences of putative banana allergens have been established. This study aimed to characterize β-1,3-glucanase and thaumatin-like protein (TLP) as banana allergens, testing them, together with other plant food allergens, in 51 children with allergic reactions after banana ingestion and both positive specific IgE and skin prick test (SPT) to banana. Banana β-1,3-glucanase and TLP were isolated and characterized. Both banana allergens, together with kiwifruit TLP Act d 2, avocado class I chitinase Pers a 1, palm pollen profilin Pho d 2 and peach fruit lipid transfer protein (LTP) Pru p 3, were tested by in vitro and in vivo assays. Banana β-1,3-glucanase (Mus a 5) was glycosylated, whereas banana TLP (Mus a 4) was not, in contrast with its homologous kiwi allergen Act d 2. Specific IgE to both banana allergens, as well as to peach Pru p 3, was found in over 70% of sera from banana-allergic children, and Mus a 4 and Pru p 3 provoked positive SPT responses in 6 of the 12 tested patients, whereas Mus a 5 in only one of them. Both peptidic epitopes and cross-reactive carbohydrate determinants were involved in the IgE-binding to Mus a 5, whereas cross-reactivity between Mus a 4 and Act d 2 was only based on common IgE protein epitopes. Profilin Pho d 2 elicited a relevant proportion of positive responses on in vitro (41%) and in vivo (58%) tests. Therefore, Mus a 4 and LTP behave as major banana allergens in the study population, and profilin seems to be also a relevant allergen. Mus a 5 is an equivocal allergenic protein, showing high IgE-binding to its attached complex glycan, and low in vivo potency., (© 2011 John Wiley & Sons A/S.)

Background: IgE recognition of panallergens having highly conserved sequence regions, structure, and function and shared by inhalant and food allergen sources is often observed., Methods: We evaluated the IgE recognition profile of profilins (Bet v 2, Cyn d 12, Hel a 2, Hev b 8, Mer a 1, Ole e 2, Par j 3, Phl p 12, Pho d 2), PR-10 proteins (Aln g 1, Api g 1, Bet v 1.0101, Bet v 1.0401, Cor a 1, Dau c 1 and Mal d 1.0108) and tropomyosins (Ani s 3, Der p 10, Hel as 1, Pen i 1, Pen m 1, Per a 7) using the Immuno-Solid phase Allergen Chip (ISAC) microarray system. The three panallergen groups were well represented among the allergenic molecules immobilized on the ISAC. Moreover, they are distributed in several taxonomical allergenic sources, either close or distant, and have a route of exposure being either inhalation or ingestion., Results: 3,113 individuals (49.9% female) were selected on the basis of their reactivity to profilins, PR-10 or tropomyosins. 1,521 (48.8%) patients were reactive to profilins (77.6% Mer a 1 IgE(+)), 1,420 (45.6%) to PR-10 (92.5% Bet v 1 IgE(+)) and 632 (20.3%) to tropomyosins (68% Der p 10 IgE(+)). A significant direct relationship between different representative molecules within each group of panallergens was found. 2,688 patients (86.4%) recognized only one out of the three distinct groups of molecules as confirmed also by hierarchical clustering analysis., Conclusions: Unless exposed to most of the allergens in the same or related allergenic sources, a preferential IgE response to distinct panallergens has been recorded. Allergen microarray IgE testing increases our knowledge of the IgE immune response and related epidemiological features within and between homologous molecules better describing the patients' immunological phenotypes.

Background: Lipid transfer proteins (LTPs) and profilins are the most important panallergens in the management of patients who are allergic to pollen and plant food in our area. LTPs are highly stable proteins that can induce systemic symptoms after ingestion. Profilins are labile proteins that are present in pollens and vegetables. Considered markers of several types of pollen sensitization, they are responsible for cross-reactivity between pollens and vegetables. The objective of this study was to assess the frequency of sensitization to LTP and profilin using skin prick tests (SPTs) in patients referred to our allergy unit for any complaint (not only pollen and plant food allergy)., Methods: The study sample comprised 430 consecutive patients who were evaluated using their medical history and SPTs with pollen, date palm profilin, and peach extract enriched in Pru p 3 (30 g/mL) as an LTP marker., Results: We found that 52 (12.1%) patients were sensitized to profilin and 53 (12.3%) to LTP. Pollen allergy was diagnosed in 53% and plant food allergy in 11%. In the LTP-sensitized group and the profilin-sensitized group, 37.7% and 34.6% of the patients had plant food allergy, respectively. Thirty-three patients (62.3%) were sensitized to LTP but had no symptoms after eating vegetables., Conclusions: To the best of our knowledge, this is the first study to analyze the real rate of sensitization to profilin and LTP in a population sensitized to allergens other than pollens and plant foods. Twelve percent of patients were sensitized to both profilin and LTP. A large proportion of LTP-sensitized patients had no symptoms at the time of the study.

Plant profilins are known pan-allergens involved in the cross-reactions between pollen and plant foods. Peanut profilin, Ara h 5, is one of the important peanut allergens. Presently, most immunological, biochemical and structural studies on peanut allergens have focused on the three major allergens (Ara h 1, 2 and 3). Here Ara h 5 was cloned, expressed in Escherichia coli, Rosetta2(DE3) (Novagen), purified using a combination of ammonium sulfate fractionation and size-exclusion chromatography and yielded a total of 29 mg/l of culture. IgE reactivity was assayed using multiplexed microarray with other peanut allergens (Ara h 1, 2, 3, and 8) and birch (Bet v 2) and timothy (Phl p 2) profilin using sera from peanut allergic Swedish patients. Using homology modeling, Ara h 5 structure was also generated, compared against other profilins and utilized to predict surface-exposed residues potentially forming epitopes. The allergen was recognized by 3 out of 33 sera (9.1%). IgE reactivity to Ara h 5 also coincided with that of two other profilins, Phl p 12 and Bet v 2, confirming cross-reactivity. Interestingly, IgE reactivity to Ara h 5 was higher than above-mentioned profilins which may be indicating specificity of sera towards peanut profilin. Eight surface-exposed epitopes were predicted and verified against experimentally validated sequential epitopes. Three epitopes (#1, 5 and 7) mostly located at the accessible loops and neutral to relatively electropositive sites were found common among profilins, which should be involved in cross-reactivity. A specific putative epitope (#4) was also found which may explain the relative high IgE reactivity to Ara h 5 as compared to the other profilins. Due to its close relation to other allergenic profilins, Ara h 5 could be used as a model and allergen of choice for profilin allergy diagnosis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Pollen food syndrome results from cross-reactivity between pollen-specific IgE and homologous proteins found in fruits and vegetables. These proteins can be grouped into several categories based on structure and include profilins, pathogenesis-related proteins, and cross-reactive carbohydrate determinants. Although cooking the reactive fruits and vegetables has been shown to destroy IgE-binding epitopes, evidence suggests that the remaining linear epitopes can bind cross-reactive T cells and enhance T-cell activation in vitro. Several methods of diagnosing food allergies exist, including skin prick tests and double-blind food challenges; however, diagnosing pollen food syndrome depends almost exclusively on clinical history. Immunotherapy has been studied as a treatment for pollen food syndrome, with highly variable results.

Plant profilins form a well-known panallergen family responsible for cross-sensitization between plant foods and pollens. We sought to map T and B-cell epitopes on the Iranian Crocus sativus profilin by bioinformatics tools. The predicted peptides are useful for further vaccine development.

Background: Plant profilins are actin-binding proteins that form a well-known panallergen family responsible for cross-sensitization between plant foods and pollens. Melon profilin, Cuc m 2, is the major allergen of this fruit., Objective: We sought to map IgE epitopes on the 3-dimensional structure of Cuc m 2., Methods: IgE binding to synthetic peptides spanning the full Cuc m 2 amino acid sequence was assayed by using a serum pool and individual sera from 10 patients with melon allergy with significant specific IgE levels to this allergen. Three-dimensional modeling and potential epitope location were based on analysis of both solvent exposure and electrostatic properties of the Cuc m 2 surface., Results: Residues included in synthetic peptides that exerted the strongest IgE-binding capacity defined 2 major epitopes (E1, consisting of residues 66-75 and 81-93, and E2, consisting of residues 95-99 and 122-131) that partially overlapped with the actin-binding site of Cuc m 2. Two additional epitopes (E3, including residues 2-10, and E4, including residues 35-45) that should show weaker putative antigen-antibody associations and shared most residues with synthetic peptides with low IgE-binding capacity were predicted on theoretical grounds., Conclusions: Strong and weak IgE epitopes have been uncovered in melon profilin, Cuc m 2., Clinical Implications: The different types of IgE epitopes located in the 3-dimensional structure of melon profilin can constitute the molecular basis to explain the sensitization and cross-reactivity exhibited by this panallergen family.

Background: Profilins are cross-reactive plant allergens responsible for multiple pollen sensitization and pollen-associated food allergy. While it is assumed that profilins from different species are immunologically equivalent, some studies suggest partial or even lacking IgE cross-reactivity between certain profilins., Objective: We aimed to obtain a semi-quantitative assessment of the contributions of conserved and species-specific epitopes to IgE binding of plant profilins., Methods: We compared model structures of profilins from timothy, mugwort, celery and bell pepper with crystal structures of birch and latex profilins. We predicted potential conformational epitopes that consisted of contiguous patches of at least 20% surface-exposed residues. Celery and timothy profilins were purified from their natural sources, and profilins from birch, mugwort, bell pepper and latex were expressed in Escherichia coli. The structural integrity of all purified proteins was confirmed by circular dichroism spectroscopy. IgE ELISAs and ELISA inhibitions using sera from 22 profilin-sensitized allergic patients were carried out., Results: Peptide backbone conformations of all six profilins were highly similar. Nine variable epitopes and two containing high proportions of conserved residues were predicted. IgE from all sera bound to all tested profilins and the amounts were highly correlated. However, IgE inhibition experiments revealed that up to 60% of total IgE binding was mediated by species-specific epitopes. The extent of cross-reactivity among profilins from timothy, birch, latex and celery was greater than cross-reactivity to mugwort and bell pepper profilins. This pattern was mirrored by sequence similarities among one of the predicted variable epitopes. Patients with IgE to cross-reactive epitopes displayed allergic reactions to a greater number of plant foods than patients having IgE directed to species-specific epitopes., Conclusion: The large extent of cross-reactivity among plant profilins justifies using a single profilin for diagnosis. However, the fine specificity of IgE directed to variable epitopes may influence the clinical manifestation of profilin sensitization.

Background: In contrast to other Rosaceae fruit, only few cases of patients with adverse reactions to strawberry are listed in literature. OBJECTIVE To identify allergenic proteins in strawberry and to express and characterize recombinant strawberry lipid transfer protein (LTP; rFra a 3)., Methods: Established apple-allergic patients were recruited on the basis of a reported allergic reaction to strawberry (n=28, confirmed by double-blind placebo-controlled food challenge in four patients) or on the basis of IgE reactivity to LTP (n=34). Sensitization to purified natural and recombinant allergens was assessed by RAST, immunoblot (inhibition) and basophil histamine release (BHR). A strawberry cDNA library was screened for genes homologous to known fruit allergens. Fra a 3 was cloned and expressed in the yeast Pichia pastoris and compared with peach and apple LTP by RAST, immunoblot-inhibition and BHR tests., Results: Genes homologous to Bet v 1, Bet v 6, profilin and LTP were identified in a strawberry cDNA library. In BHR the rFra a 3 induced histamine release at a 100-fold higher concentration than peach LTP. RAST inhibition showed high cross-reactivity to peach and apple LTP, although IgE reactivity was lower by a factor 5. On strawberry immunoblot, patients' IgE showed reactivity to a Bet v 1 homologue, profilin, LTP and high-molecular weight bands., Conclusion: In addition to a Bet v 1 homologue, strawberry also contains IgE-binding profilin and LTP. The rFra a 3 has less allergenic potency than peach and apple LTP, and therefore is an interesting tool for future immunotherapy. Fra a 3 does not seem to be clinically relevant.

Background: The allergenicity of the ornamental tree Robinia pseudoacacia, or black locust, is unknown., Objective: To evaluate the prevalence of sensitization to R. pseudoacacia pollen, its possible allergenic cross-reactivity with other common pollens, and the potential implication of pollen panallergens (profilin, polcalcin, and 1,3-beta-glucanase) as a cause of sensitization to R. pseudoacacia pollen., Methods: Skin prick testing with R. pseudoacacia pollen was performed in 149 patients with pollinosis. Nasal challenge with R. pseudoacacia pollen was performed in 10 patients. The prevalence of sensitization to the recombinant forms of profilin (rChe a 2), polcalcin (rChe a 3), and the N-terminal of the 1,3-beta-glucanase (rNtD of Ole e 9) was investigated. Immunoblotting, enzyme-linked immunosorbent assay, and competitive inhibition assays were performed with R. pseudoacacia pollen and recombinant pollen allergens., Results: Sixty-four patients (43%) had positive skin prick test reactions to R. pseudoacacia pollen. Nasal challenge results were positive in 5 sensitized patients and negative in 4 controls and 1 sensitized patient. The allergenic profile of R. pseudoacacia pollen comprises at least the panallergen profilin, a calcium-binding protein, and a 1,3-beta-glucanase. The prevalence of sensitization to rChe a 2, rChe a 3, and rNtD of Ole e 9 was 60%, 33%, and 87%, respectively, among patients sensitized to R. pseudoacacia pollen. Binding of IgE to R. pseudoacacia extract was completely inhibited by Robinia, Chenopodium, Olea, Cupressus, and Lolium extracts., Conclusions: The high prevalence of R. pseudoacacia pollen sensitization in patients with pollinosis is likely to be due to cross-sensitization to panallergens (profilin, polcalcin, and 1,3-beta-glucanase) from other common pollens. This phenomenon may lead to a diagnosis of "allergy mirages."

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.