Optimization of the diagnosis and characterization of gibberellin-regulated protein sensitization: An Italian cohort study.

BACKGROUND: Pru p 7 was the first gibberellin-regulated protein (GRP) to be identified as a food allergen as the basis of a pollen food allergy syndrome. OBJECTIVE: To clinically and biologically characterize a group of patients with suspected allergy to Pru p 7 to optimize the diagnostic workup of GRP sensitization. METHODS: Allergy to Pru p 7 was suspected in the presence of a systemic allergic reaction to plant food, positive skin prick test results for cypress pollen and lipid-transfer protein-enriched peach extract, and absence of Pru p 3-specific immunoglobulin E. Controls were patients with food allergies, patients sensitized to Pru p 3, and patients with cypress allergy without food allergy. Diagnostic workup included skin tests, basophil activation test, Western blot, and single and multiplex assays. RESULTS: In total, 23 patients and 14 controls were enrolled. The most implicated food was peach (91.3%). Approximately 70% of patients reacted to multiple foods. Mueller 4 reactions were 8.7%. In 26.1% of cases, a cofactor triggered the reaction. The basophil activation test results were positive for rPru p 7 in 87% of the patients. Specific immunoglobulin E to Pru p 7 was detected in 95.7% by singleplex and in 73.9% by multiplex assays in patients with suspected allergies; 73.9% of them also reacted to cypress pollen GRP (Cup s 7) in Western blot analysis. CONCLUSION: Patients with Pru p 7-Cup s 7 allergy in our cohort confirm a mild-to-severe clinical syndrome characterized by pollen and food allergy. The diagnosis may benefit from the proposed selection criteria that can be used as preliminary steps to further characterize the cross-reactive GRP sensitization.

Sensitisation to peach allergen Pru p 7 is associated with severe clinical symptoms in a Spanish population.

BACKGROUND: Pru p 7 has been reported as a major allergen in peach allergy, associated with severe clinical symptoms and related to IgE sensitisation to cypress pollen. The main objective of this study was to prospectively evaluate the frequency of sensitisation to Pru p 7 and its clinical relevance amongst pediatric patients with peach allergy in Madrid (Spain). METHODS: Patients with a history of IgE-mediated symptoms (oral allergy syndrome, urticaria/angioedema, rhinoconjunctivitis/asthma, gastrointestinal symptoms, or anaphylaxis) occurring within 2 h after peach intake or contact were prospectively recruited from February 2020 to September 2021. Skin tests, sIgE by ImmunoCAP® (Pru p 1, Pru p 3, Pru p 4, Pru p 7, and Cupressus arizonica) and oral food challenge (OFC) were performed. The study was approved by the local Ethics Committee (PI-4513). RESULTS: Ninety-two patients were included (53.3% male); median age, 10 (IQR 6.0-14.75) years. Seventy-four (80.4%) patients had a reaction after ingestion of fresh peach (25.0% from peel, 23.9% from pulp, and 44.6% from both). Fifteen (16.3%) patients were sensitised to Pru p 7. Upper airway symptoms, anaphylaxis, and grade 2 reactions were statistically more frequent in patients sensitised to Pru p 7. Seven (7.9%) patients presented with exercise as a cofactor, four of whom were sensitised to Pru p 7 (p = .001). Patients sensitised to Pru p 7 were significantly more likely to have a positive OFC result than patients who were not (p = .008). Four patients who reacted to peach at OFC were sensitised to Pru p 7. Specific IgE against Cupressus arizonica pollen was positive in 25 (62.5%) patients. CONCLUSIONS: Pru p 7 sensitisation was observed in 16.3% of our population and was related to severe reactions, upper airway symptoms, anaphylaxis, and the presence of an eliciting cofactor.

A European-Japanese study on peach allergy: IgE to Pru p 7 associates with severity.

BACKGROUND: Pru p 3 and Pru p 7 have been implicated as risk factors for severe peach allergy. This study aimed to establish sensitization patterns to five peach components across Europe and in Japan, to explore their relation to pollen and foods and to predict symptom severity. METHODS: In twelve European (EuroPrevall project) and one Japanese outpatient clinic, a standardized clinical evaluation was conducted in 1231 patients who reported symptoms to peach and/or were sensitized to peach. Specific IgE against Pru p 1, 2, 3, 4 and 7 and against Cup s 7 was measured in 474 of them. Univariable and multivariable Lasso regression was applied to identify combinations of parameters predicting severity. RESULTS: Sensitization to Pru p 3 dominated in Southern Europe but was also quite common in Northern and Central Europe. Sensitization to Pru p 7 was low and variable in the European centers but very dominant in Japan. Severity could be predicted by a model combining age of onset of peach allergy, probable mugwort, Parietaria pollen and latex allergy, and sensitization to Japanese cedar pollen, Pru p 4 and Pru p 7 which resulted in an AUC of 0.73 (95% CI 0.73-0.74). Pru p 3 tended to be a risk factor in South Europe only. CONCLUSIONS: Pru p 7 was confirmed as a significant risk factor for severe peach allergy in Europe and Japan. Combining outcomes from clinical and demographic background with serology resulted in a model that could better predict severity than CRD alone.

Peach extract induces systemic and local immune responses in an experimental food allergy model.

Peach allergy is among the most frequent food allergies in the Mediterranean area, often eliciting severe anaphylactic reactions in patients. Due to the risk of severe symptoms, studies in humans are limited, leading to a lack of therapeutic options. This study aimed to develop a peach allergy mouse model as a tool to better understand the pathomechanism and to allow preclinical investigations on the development of optimized strategies for immunotherapy. CBA/J mice were sensitized intraperitoneally with peach extract or PBS, using alum as adjuvant. Afterwards, extract was administered intragastrically to involve the intestinal tract. Allergen provocation was performed via intraperitoneal injection of extract, measuring drop of body temperature as main read out of anaphylaxis. The model induced allergy-related symptoms in mice, including decrease of body temperature. Antibody levels in serum and intestinal homogenates revealed a Th2 response with increased levels of mMCPT-1, peach- and Pru p 3-specific IgE, IgG1 and IgG2a as well as increased levels of IL-4 and IL-13. FACS analysis of small intestine lamina propria revealed increased amounts of T cells, neutrophils and DCs in peach allergic mice. These data suggest the successful establishment of a peach allergy mouse model, inducing systemic as well as local gastrointestinal reactions.

Risk Factors in Severe Anaphylaxis: Which Matters the Most, Food or Cofactors?

BACKGROUND AND OBJECTIVE: The prevalence of anaphylactic shock, the most severe manifestation of anaphylaxis, remains unknown. Risk factors and biomarkers have not been fully identified. Objective: To identify risk factors in patients who experience anaphylactic shock. METHODS: Using lipid transfer protein (LTP) allergy as a model, we compared the characteristics of patients who developed anaphylaxis and anaphylactic shock. We recorded demographics, pollen sensitization, foods ingested up to 2 hours before onset of the reaction, and the presence of cofactors. Culprit foods were identified through a compatible clinical history and positive allergology work-up (skin prick test and/or sIgE). RESULTS: We evaluated 150 reactions in 55 patients with anaphylaxis (134 reactions) and 12 with anaphylactic shock (16 reactions). Patients in the anaphylaxis group experienced twice as many reactions (mean [SD], 2.4 [2.5] for anaphylaxis vs 1.3 [1.5] for anaphylactic shock; P<.02). No relationship was found between any food group and severity of the reaction. The most frequent food involved in both groups of patients was the combination of several plant-derived foods (plant food mix), followed by peach and nuts. Indeed, in the reactions caused by plant food mix, the presence of a cofactor was observed more often than in other food groups. On the other hand, cofactors were not present in peach- and nut-related reactions. Exercise was the most frequent cofactor in all groups. CONCLUSION: In our series, the severity of the reactions was not determined by the kind of food or presence of a cofactor. Anaphylactic shock seems to be an infrequent presentation that may be associated with other individual-related factors requiring further evaluation.

Allergy to Gibberellin-Regulated Proteins (Peamaclein) in Children.

BACKGROUND: Gibberellin-regulated proteins (GRPs, Peamaclein) are allergens recently identified in plant-derived food allergy (FA), and little is known about the clinical manifestations of this allergic condition in the European population, especially in children. OBJECTIVE: Our study aimed to identify and characterize pediatric patients with pollen-FA due to GRP sensitization. METHODS: We retrospectively analyzed the charts of patients referred to the Allergy Unit of the Meyer Children's Hospital in Florence for suspected FA. Three main eligibility criteria based on the actual knowledge of GRP allergy were used to select patients deserving further investigations: (1) systemic reactions after consumption of fruit or an unknown culprit food, (2) positive skin prick tests to both cypress pollen and Pru p 3-enriched peach peel extracts, (3) negative in vitro test results for Pru p 3 serum-specific Immunoglobulin E (sIgE). We performed the in vitro test to determine the anti-rPru p 7 (Peamaclein) sIgE levels in the selected patients. RESULTS: We identified 10 pediatric patients with Pru p 7 allergy and described their characteristics. The use of our eligibility criteria showed a high accuracy in identifying these patients: 100% of the selected patients had positive in vitro results for Pru p 7. We therefore proposed a diagnostic algorithm for Pru p 7 allergy. CONCLUSION: This is the first case series of European pediatric patients with a demonstrated Peamaclein allergy. These findings broaden our knowledge on GRP allergy in pediatric populations and could help clinicians to suspect, diagnose, and manage this recently discovered plant-derived FA.

Energy Landscapes of Ligand Motion Inside the Tunnel-Like Cavity of Lipid Transfer Proteins: The Case of the Pru p 3 Allergen.

Allergies are a widespread problem in western countries, affecting a large part of the population, with levels of prevalence increasingly rising due to reasons still not understood. Evidence accumulated in recent years points to an essential role played by ligands of allergen proteins in the sensitization phase of allergies. In this regard, we recently identified the natural ligand of Pru p 3, a lipid transfer protein, a major allergen from peach fruit and a model of food allergy. The ligand of Pru p 3 has been shown to play a key role in the sensitization to peach and to other plant food sources that provoke cross-reactivity in a large proportion of patients allergic to peach. However, the question of which is the binding pose of this ligand in its carrier protein, and how it can be transferred to receptors of the immune system where it develops its function as a coadjuvant was not elucidated. In this work, different molecular dynamics simulations have been considered as starting points to study the properties of the ligand⁻protein system in solution. Besides, an energy landscape based on collective variables that describe the process of ligand motion within the cavity of Pru p 3 was obtained by using well-tempered metadynamics. The simulations revealed the differences between distinct binding modes, and also revealed important aspects of the motion of the ligand throughout its carrier protein, relevant to its binding⁻unbinding process. Our findings are potentially interesting for studying protein⁻ligand systems beyond the specific case of the allergen protein dealt with here.

Glycosylated nanostructures in sublingual immunotherapy induce long-lasting tolerance in LTP allergy mouse model.

An effective specific immunotherapy should contain elements to generate specific recognition (T-cell peptides) and to modulate the immunological response towards a Th1/Treg pattern by enhancing dendritic cells (DCs). We propose a novel sublingual immunotherapy for peach allergy, using systems, that combine Prup3-T-cell peptides with mannose dendrons (D1ManPrup3 and D4ManPrup3). Peach anaphylactic mice were treated 1, 2 and 5 nM concentrations. Tolerance was assessed one/five weeks after finishing treatment by determining in vivo/in vitro parameters after challenge with Prup3. Only mice receiving D1ManPrup3 at 2 nM were protected from anaphylaxis (no temperature changes, decrease in Prup3-sIgE and -sIgG1 antibody levels, and secreting cells) compared to PBS-treated mice. Moreover, an increase of Treg-cells and regulatory cytokines (IL-10+/IFN-γ+) in CD4+-T-cells and DCs were found. These changes were maintained at least five weeks after stopping treatment. D1ManPrup3 is an effective new approach of immunotherapy inducing protection from anaphylaxis which persists after finishing treatment.

Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy.

BACKGROUND: Peach is a common elicitor of food allergic reactions. Peach-induced immediate reactions may occur as benign pollen-food syndromes, usually due to birch pollen-related PR-10 cross-reactivity in temperate climates, and as potentially severe primary food allergies, predominantly related to nsLTP Pru p 3 in Mediterranean regions. The newly described peach allergen Pru p 7 has gained recent attention as a potential peach allergy severity marker. Sensitization to Pru p 7 and its allergenic homologues of the gibberellin-regulated protein family occurs in areas with high Cupressaceae tree pollen exposure. OBJECTIVE: We sought to investigate the distribution, clinical characteristics and molecular associations of Pru p 7 sensitization among subjects with suspected peach allergy in different regions of France. METHODS: Subjects with suspected peach allergy (n = 316) were included. Diagnostic work-up was performed according to current guidelines, including open food challenge when required. IgE antibody measurements and competition experiments were performed using the ImmunoCAP assay platform. RESULTS: Sensitization to Pru p 7 was present in 171 (54%) of all subjects in the study and in 123 of 198 (62%) diagnosed as peach allergic, more than half of whom were sensitized to no other peach allergen. Frequency and magnitude of Pru p 7 sensitization were associated with the presence of peach allergy, the clinical severity of peach-induced allergic reactions and the level of cypress pollen exposure. Cypress pollen extract completely outcompeted IgE binding to Pru p 7. Pru p 7 was extremely potent in basophil activation tests. CONCLUSION AND CLINICAL RELEVANCE: A subtype of Cupressaceae pollinosis, characterized by Pru p 7 sensitization, can be an underlying cause of severe peach allergy.

Food-dependent, exercise-induced anaphylaxis in a patient allergic to peach.

Determining the single factor that triggered anaphylactic shock can be challenging. We present an interesting case of a 25-year-old female patient with recurrent anaphylactic reactions developing after eating various foods, particularly in presence of co-factors of allergic reactions. Symptoms occurred after consumption of various kinds of foods - peach, pancakes with cottage cheese and fruit, a meal from a Chinese restaurant - all eaten on other occasions without symptoms. During diagnosis, skin prick tests were negative for all tested allergen extracts (both inhalatory and food) from Allergopharma. Prick by prick tests were positive for the peach - wheal diameter - 6 mm, nectarine - 4 mm (histamine 4 mm, negative control 0 mm). Increased levels of asIgE were found for allergens of peach (0.55 kU/L).Open challenge test with one mid-size peach combined with the physical exercise challenge test was positive. ImmunoCAP ISAC test indicated increased levels of IgE specific for the lipid transfer protein (LTP) for walnut (nJug r 3), peach (Pru p 3), wheat (rTri a 14) and plane tree (rPla a 3). The patient was diagnosed with food-dependent, exercise-induced anaphylaxis associated with an allergy to lipid transport proteins (LTPs).

Evaluation of serum IgE in peach-allergic patients with systemic reaction by using recombinant Pru p 7 (gibberellin-regulated protein).

BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n=27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n=17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P<0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction.

Genome-wide association study of self-reported food reactions in Japanese identifies shrimp and peach specific loci in the HLA-DR/DQ gene region.

Food allergy is an increasingly important health problem in the world. Several genome-wide association studies (GWAS) focused on European ancestry samples have identified food allergy-specific loci in the HLA class II region. We conducted GWAS of self-reported reactivity with common foods using the data from 11011 Japanese women and identified shrimp and peach allergy-specific loci in the HLA-DR/DQ gene region tagged by rs74995702 (P = 6.30 × 10-17, OR = 1.91) and rs28359884 (P = 2.3 × 10-12, OR = 1.80), respectively. After HLA imputation using a Japanese population-specific reference, the most strongly associated haplotype was HLA-DRB1*04:05-HLA-DQB1*04:01 for shrimp allergy (P = 3.92 × 10-19, OR = 1.99) and HLA-DRB1*09:01-HLA-DQB1*03:03 for peach allergy (P = 1.15 × 10-7, OR = 1.68). Additionally, both allergies' associated variants were eQTLs for several HLA genes, with HLA-DQA2 the single eQTL gene shared between the two traits. Our study suggests that allergy to certain foods may be related to genetic differences that tag both HLA alleles having particular epitope binding specificities as well as variants modulating expression of particular HLA genes. Investigating this further could increase our understanding of food allergy aetiology and potentially lead to better therapeutic strategies for allergen immunotherapies.

The clinical and immunological effects of Pru p 3 sublingual immunotherapy on peach and peanut allergy in patients with systemic reactions.

BACKGROUND: The peach non-specific lipid transfer protein, Pru p 3, is the primary sensitizer in fruits and responsible for severe reactions in the Mediterranean area. Peach allergy is frequently associated with other allergies such as peanut. Therefore, it is important to assess how specific immunotherapy to Pru p 3 could affect both peach and peanut tolerance. OBJECTIVES: To evaluate peach and peanut desensitization and immunological changes after 1 year of Pru p 3 sublingual immunotherapy (SLIT) in patients with systemic allergic reactions to peach and/or peanut. METHODS: Forty-eight peach allergic patients, 36 treated with SLIT and 12 non-treated, were monitored for 12 months. Treated patients were subclassified as peanut allergic (Group A), sensitized (Group B) or tolerant (Group C). SLIT effect was evaluated by skin prick test (SPT) reactivity and food challenge. Immunological changes were evaluated by monitoring sIgE and sIgG4 levels and basophil reactivity. RESULTS: After 1 year of SLIT, the weal area in SPT significantly decreased and a significant increase in peach threshold in treated patients was observed (P < 0.001). Patients in Group A showed a significant decrease in peanut SPT weal area and an increase in peanut threshold (P < 0.001). Immunological changes were observed in treated patients only, with a significant decrease in sIgE and a parallel increase in sIgG4, sIgG4/sIgE and basophil reactivity for both Pru p 3 and Ara h 9. CONCLUSIONS AND CLINICAL RELEVANCE: After 1 year, Pru p 3 SLIT induces both desensitization and immunological changes not only for peach but also for other food allergens relevant in the induction of severe reactions such as peanut.

Targeted temperature management after cardiac arrest with anaphylaxis.

Fatal anaphylaxis is uncommon but not rare. Extrapolated mortality rates are 0.52% of total anaphylaxis patients Bock et al. (Jan. 2001) [1]. Nevertheless, compared with the incidence of the other cardiac arrest events, the incidence of cardiac arrest due to anaphylaxis is relatively small. As a result, the effect using targeted temperature management after anaphylaxis is not clearly understood. We report the case of a 63-year-old man who developed cardiac arrest after ingestion of two pieces of peach. He was resuscitated and his circulation returned spontaneously after approximately 11min of cardiopulmonary resuscitation, but he was unresponsive and had fixed dilated pupils. We initiated therapeutic hypothermia on the basis of protocol for 24h. The patient was gradually and successfully cooled and rewarmed. The patient opened his eyes spontaneously on day 5, obeyed commands on day 6, and was discharged on day 18. At the time of discharge, he had no neurologic deficiencies or other complications.

Lettuce Allergy Is a Lipid Transfer Syndrome-Related Food Allergy With a High Risk of Severe Reactions.

BACKGROUND AND OBJECTIVE: Lipid transfer protein (LTP) sensitization is the most common cause of food allergy in the Mediterranean area, with peach allergy acting as the primary sensitizer in most cases. Lettuce has been described as a common offending food in patients with LTP syndrome. The aim of the study was to investigate the frequency and clinical expression of LTP syndrome in a sample of lettuceallergic patients. METHODS: We determined specific IgE to Pru p 3 and lettuce in a sample of 30 patients with a diagnosis of lettuce allergy. Symptoms elicited by other LTP-containing plant-derived foods and the presence of cofactors were assessed. RESULTS: The clinical symptoms of lettuce allergy were frequently severe, with 18 of the 30 patients experiencing anaphylaxis. All the patients had allergic reactions to other plant foods. Cofactors were involved in the clinical reactions of 13 of the 30 patients. Sensitization to pollens was found in 90% of patients. CONCLUSIONS: Lettuce allergy is found not as an isolated condition but in the context of LTP syndrome and it is characterized by severe reactions and frequent cofactor association.

Eyelid edema as a predictive factor for sensitization to Pru p 7 in peach allergy.

The clinical and laboratory factors related to Pru p 7 sensitization in peach allergy, compared with those associated with sensitization to Pru p 1, which is related to pollen-food allergy syndrome (PFAS), have still not been clarified. The aim of the present study was to identify the clinical and laboratory features associated with sensitization to Pru p 7. Fifty patients (male : female, 14:36; mean age, 31.8 years) with peach allergy were enrolled in this study. Specific immunoglobulin E (sIgE) against the purified nPru p 7 was measured by enzyme-linked immunoassay, whereas using ImmunoCAP, sIgE levels against peach, rPru p 1, rPru p 3 and rPru p 4 were measured. Correlations between the clinical characteristics of peach allergy and sensitization to the allergens were statistically analyzed. In 50 patients with peach allergy, 13 and 33 patients were sensitized with nPru p 7 and rPru p 1, respectively. Laryngeal tightness, conjunctival injection, facial edema, eyelid edema and urticaria were significantly associated with Pru p 7 sensitization. In contrast, oropharyngeal symptoms were strongly related to Pru p 1 sensitization but were significantly less common in the Pru p 7-positive group. Co-sensitization to the representative pollens related to PFAS, such as alder pollens, was significantly related to Pru p 1 sensitization, but not Pru p 7 sensitization. Along with the absence of co-sensitization to pollens related to PFAS, facial edema and especially eyelid edema after the ingestion of peaches could be a predictive factor for sensitization to Pru p 7.

Molecular cloning of plane pollen allergen Pla a 3 and its utility as diagnostic marker for peach associated plane pollen allergy.

BACKGROUND: Non-specific lipid transfer proteins (nsLTP) are considered to provoke allergic symptoms to plane tree pollen, which are frequently associated with peach allergy. OBJECTIVE: The objective was to clone the cDNA of plane pollen nsLTP Pla a 3, to characterize IgE-binding and allergenic potency of recombinant Pla a 3 in comparison to its natural counterpart and peach nsLTP Pru p 3. METHODS: Natural Pla a 3 was purified from plane pollen and analysed by mass spectrometry (MS). Recombinant Pla a 3 was characterized by SDS-PAGE and CD spectroscopy. Specific IgE to extract, components of plane pollen and Pru p 3 was measured by ImmunoCAP in sera of patients allergic to either plane pollen (n = 10), peach (n = 15) or both (n = 15). Biological potency of the proteins was investigated by in vitro mediator release assays and IgE cross-reactivity by competitive ELISA. RESULTS: Two Pla a 3 isoforms were identified. Recombinant Pla a 3 showed high purity, structural integrity, IgE-binding capacity comparable to nPla a 3 and biological potency. Sensitization to plane pollen extract was confirmed in 24/25 plane pollen allergics. The frequency of sensitization to Pla a 3 was 53% among patients allergic to both plane pollen and peach and 10% among plane pollen allergics tolerating peach where most patients were sensitized to Pla a 1. Pla a 3 and Pru p 3 showed strong bi-directional IgE cross-reactivity in patients allergic to peach and plane pollen, but not in peach allergics tolerating plane pollen. Levels of IgE-binding were generally higher to Pru p 3 than to Pla a 3. CONCLUSION: Sensitization to Pla a 3 is relevant in a subgroup of plane pollen allergics with concomitant peach allergy. IgE testing with Pla a 3 may serve as a marker to identify plane pollen allergic patients at risk of LTP-mediated food reactions and thereby improve in vitro diagnostic procedures.