Sensitization Potency of Sunflower Seed Protein in a Mouse Model: Identification of 2S-Albumins More Allergenic Than SFA-8.

SCOPE: Food allergy to sunflower seed (SFS) protein is not frequent and only non-specific lipid transfert protein (nsLTP) Hel a 3 is officially recognized as a food allergen. Out of the eleven seed storage 2S-albumins (SESA) detected in SFS, only SFA-8 allergenicity has been investigated so far. The study aimed then to evaluate SFS protein allergenicity and particularly, to compare the sensitization potency of SESA in a mouse model. METHODS AND RESULTS: The most abundant SESA and nsLTP were isolated from SFS through a combination of chromatographic methods. Purified proteins were then used to measure specific IgG1 and IgE responses in BALB/c mice orally sensitized to different SFS protein isolates. The study, thus, confirmed the allergenicity of SFA-8 and Hel a 3 but mice were also highly sensitized to other SESA such as SESA2-1 or SESA20-2. Furthermore, competitive inhibition of IgE-binding revealed that SFA-8 IgE-reactivity was due to cross-reactivity with other SESA. 11S-globulins were weakly immunogenic and were rapidly degraded in an in vitro model of gastroduodenal digestion. In contrast, Hel a 3, SESA2-1 and SFA-8 were more resistant to proteolysis and gastroduodenal digestion did not affect their IgE-reactivity. CONCLUSIONS: SESA2-1 or SESA20-2 were more potent allergens than SFA-8 in this mouse model. Allergenicity of SESA must be now confirmed in SFS-allergic patients.

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.

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

Sensitization to Gibberellin-Regulated Protein (Peamaclein) Among Italian Cypress Pollen-Sensitized Patients.

BACKGROUND AND OBJECTIVES: Peach gibberellin-regulated protein (peamaclein) has recently emerged as a relevant food allergen in cypress pollen-hypersensitive patients. Objective: We investigated monosensitization to peamaclein among Italian cypress pollen-allergic patients. MATERIAL AND METHODS: A total of 835 cypress pollen-hypersensitive patients from 28 Italian allergy centers underwent a thorough work-up to determine food-allergic reactions and performed skin prick testing with a commercial peach extract containing peamaclein. IgE to rPru p 3 was measured in peach reactors, and those with negative results were enrolled as potentially monosensitized to peamaclein. IgE reactivity to rPru p 7 was evaluated using immunoblot and an experimental ImmunoCAP with rPru p 7. RESULTS: Skin prick tests were positive to peach in 163 patients (19.5%); however, 127 (77.9%) were excluded because they reacted to Pru p 3. Twenty-four patients (14.7%) corresponding to 2.8% of the entire study population) were considered potentially monosensitized to peamaclein. No geographic preference was observed. Seventeen of the 24 patients (70.8%) had a history of food allergy, mainly to peach (n=15). Additional offending foods included other Rosaceae, citrus fruits, fig, melon, tree nuts, and kiwi. On peach immunoblot, only 3 of 18 putative peamaclein-allergic patients reacted to a band at about 7 kDa; an additional 4 patients reacted at about 50-60 kDa. Ten of 18 patients (56%) had a positive result for Pru p 7 on ImmunoCAP. CONCLUSION: Allergy and sensitization to peamaclein seem rare in Italy. Most patients react to peach, although other Rosaceae fruits and several citrus fruits may also be offending foods. Peach and cypress pollen probably also share cross-reacting allergens other than peamaclein.

Nickel allergy in lipid transfer protein sensitized patients: Prevalence and clinical features.

Nickel (Ni), the main responsible for allergic contact dermatitis worldwide, is also involved in systemic condition called "Systemic Nickel Sulfate Allergy Syndrome (SNAS)." Likewise, IgE-mediated reactivity to Lipid Transfer Protein (LTP) represents the main cause of primary food allergy in adults of Mediterranean countries. We evaluated the prevalence of SNAS in LTP allergic patients and investigated patients' clinical features with double sensitization (LTP and Ni). A retrospective, single-center, observational study was conducted performing a complete allergological work-up including: (1) skin prick tests; (2) serum specific IgE for plant food allergens and rPru p3 (LTP); (3) patch test with 5% Ni sulfate in petrolatum. We enrolled 140 LTP allergic patients of which 36 patients (25.7% of sample) showed additional positivity to Ni patch test. Patients with double sensitization were more frequently females and reported fewer cutaneous symptoms. Higher values of sIgE for peach, apple, peanut, walnut, grain, corn, and garlic were found in LTP allergic patients, while higher values for hazelnut in the other subgroup. The prevalence of SNAS in the LTP allergic population is clinically relevant. Moreover, the clinical and immunological profiles of patients with double sensitization were different from patients monosensitized to LTP.

Glutathione Counteracts the Effects of Japanese Cedar (Cryptomeria japonica) Pollen Allergen Cry j1.

Japanese cedar (Cryptomeria japonica) pollen allergen Cry j1 increases the intracellular calcium concentration in human keratinocytes, and also impairs the epidermal barrier function. Here, we show that reduced glutathione (GSH) blocks both thrombin activation and the Cry j1-induced intracellular calcium elevation in cultured human keratinocytes, and also prevents the Cry j1-induced decrease of barrier function in ex vivo human skin.

The clinical impact of cross-reactions between allergens on allergic skin diseases.

PURPOSE OF REVIEW: The route of allergen sensing via the skin appears to influence the immune system towards mounting a type 2 response, especially in genetically predisposed individuals. Allergens recognized this way may derive from microbial, animal, food, or other plant sources and trigger atopic dermatitis. Allergens can be grouped into families depending on their structure and function, harboring significant structural and sequence similarities. Cross-reactivity between allergens is believed to arise as a consequence, and to underlie the development of further atopic diseases. RECENT FINDINGS: Especially for the plant allergens of the families of PR10-related proteins and profilins, immune cross-reactions have been described. Actual studies support that food and pollen allergens can aggravate skin lesions in patients suffering from atopic dermatitis. Further on, allergens derived from air-borne or skin-borne fungi belong to common allergen families and bear cross-reactivity potential. Cross-reactivity to human homologous proteins, so-called autoallergens, is discussed to contribute to the chronification of atopic dermatitis. SUMMARY: Due to high evolutionary conservation, allergic reactions can be triggered by highly homologous members of allergen families on the humoral as well as on the cellular level.

LTP allergy/sensitization in a pediatric population.

Plant lipid transfer proteins (LTPs) are widespread plant food allergens, highly resistant to food processing and to the gastrointestinal environment, which have been described as the most common food allergens in the Mediterranean area. LTP allergy is widely described in adults, but it represents an emerging allergen also in the pediatric population. Little is known about the real prevalence and the clinical features of this allergy in children and it still often remains underdiagnosed in these patients. An early identification and a deeper knowledge of this allergy in childhood can avoid severe systemic reactions and improve the child's quality of life. Pediatricians should always consider the possibility of LTP involvement in cases of plant-derived food allergy.

Identification of the linear immunodominant epitopes in the ß subunit of ß-conglycinin and preparation of epitope antibodies.

ß-conglycinin is one of the major allergens in soybean protein. The purpose of this study was to predict and to identify the major linear epitopes of the ß subunit of ß-conglycinin. Potential linear epitopes were predicted and confirmed by three immunoinformatics tools combined with the Immune Epitope Database (IEDB). Ten potential epitope peptides were synthesized by Fmoc (9-fluorenylmethoxycarbonyl) solid phase peptide synthesis and were validated by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using sera from soybean allergic patients. Polyclonal antibodies, which were prepared by immunizing rabbits with synthesized peptides, were used to confirm their binding ability with ß-conglycinin through western blot and dot blot assays. The results showed that 10 peptides were screened as the main epitopes for the ß subunit of ß-conglycinin. All 10 peptides (P1-P10) presented IgG binding activity, and P2 and P6 were also validated as IgE binding peptides. Moreover, the results of dot blot showed that P5 and P8 might be located inside the protein molecule. Western blot indicated that most of polyclonal antibodies were bound effectively to the ß subunit of ß-conglycinin. In addition, few polyclonal antibodies exhibited an immune cross-reaction with the α and α' subunits.

Gibberellin-regulated protein allergy: Clinical features and cross-reactivity.

Gibberellin-regulated proteins (GRPs)/GASA proteins are members of cysteine-rich antimicrobial peptide families and are conserved in a broad range of plants. Some GRPs in fruits and pollens have been identified as allergens including peach Pru p 7, Japanese apricot Pru m 7, orange Cit s 7, pomegranate Pun g 7, and cypress pollen GRP. The clinical features of fruit-derived GRP allergies frequently include systemic reactions, multiple fruit allergies regardless of plant kingdom classifications and, less frequently, cofactor-dependence. Multiple fruit allergies might be related to cross-reactivity between GRPs. Clinical cross-reactivity, at least between the four fruit-derived GRPs, has been proven. In addition, GRP allergy induces peculiar clinical symptoms, such as laryngeal tightness and facial swelling, especially eyelid edema, which was proposed to be a predictive factor for Pru p 7 allergy. Fruit-derived GRPs have an unusually high content of cysteine, resulting in high stability to heat and resistance to digestive enzymes. Therefore, GRPs are considered "true" food allergens that induce severe allergic reactions. As an alternative mechanism of fruit-derived GRP allergies, cross-reactivity between fruit GRP and cypress pollen GRP, which might play a role as a sensitizer, is suspected. Taken together, these characteristics indicate GRPs are clinically relevant plant allergens. This review article summarizes our current knowledge of the clinical features and important aspects of GRP sensitization and allergy.

Soybean ß-conglycinin caused intestinal inflammation and oxidative damage in association with NF-κB, TOR and Nrf2 in juvenile grass carp (Ctenopharyngodon idella): varying among different intestinal segments.

The current study aimed to investigate the effects and mechanisms of dietary soybean ß-conglycinin in immune function and oxidative damage among different intestinal segments of juvenile grass carp (Ctenopharyngodon idella). 240 fish (13.77 ±â€¯0.10 g) were fed control or 8% ß-conglycinin diet for 7 weeks. Dietary ß-conglycinin caused inconsistent suppression effects on the innate immune by decreasing complement component, lysozyme, antimicrobial peptide and acid phosphatase among different intestinal segments. Meanwhile, dietary ß-conglycinin caused inflammation in the mid and distal intestine by raising pro-inflammatory cytokines and declining anti-inflammatory cytokines mRNA levels, while more serious in the distal intestine than in the mid intestine. Furthermore, dietary ß-conglycinin regulating inflammatory cytokines might be associated with transcription factors nuclear factor-κB P65 (NF-κB P65) nucleus translocation and target of rapamycin (TOR) phosphorylation in the distal intestine but only related to TOR phosphorylation in the mid intestine. Interestingly, in the proximal intestine, dietary ß-conglycinin decreased both pro-inflammatory and anti-inflammatory cytokines mRNA level, and did not affect NF-κB P65 nucleus translocation and TOR phosphorylation. For oxidative damage, dietary ß-conglycinin exposure elevated both malondialdehyde (MDA) and protein carbonyl (PC) contents in the distal intestine, which might be attributed to the suppression of the Mn-SOD, catalase (CAT) and glutathione peroxidase (GPx) activities. In the mid intestine, dietary ß-conglycinin only increased PC content in association with the low activities of CAT, GPx and glutathione peroxidase (GR). Unexpectedly, in the proximal intestine, dietary ß-conglycinin did not significantly change MDA and PC contents while decreased antioxidant enzyme activities. Furtherly, dietary ß-conglycinin affect the antioxidant enzyme activity might be regulated by the varying pattern of nuclear factor-erythroid 2-related factor 2 (Nrf2) nucleus translocation among these three intestinal segments. In summary, dietary ß-conglycinin caused intestinal inflammation and oxidative damage in association with NF-κB, TOR and Nrf2 signaling molecules, which were varying among the three intestinal segments of grass carp.

Characterization of Patients with Allergic Rhinitis to Ragweed Pollen in Two Distinct Regions of Romania.

Background and objectives: Ragweed pollen is a major source of allergen, which has rarely been observed in Romania until now. In this study, we evaluated the symptoms and associated factors in patients with allergic rhinitis to ragweed pollen in two distinct regions of Romania. Materials and Methods: We evaluated the records of patients newly diagnosed with allergic rhinitis induced by ragweed pollen in two allergological centers from North-West (NW) and Central parts of Romania between 2013 and 2015. The patients were clinically evaluated regarding disease length, presence, and severity of the allergic rhinitis symptoms and the association with other allergic manifestations (asthma and conjunctivitis). Results: The sensitization to ragweed was significantly higher in the NW part compared to the Central part (18.27% vs 4.1%, p < 0.001). More patients with monosensitization to ragweed pollen were observed in the NE center (27%) compared to the Central one (20.7%). Patients with monosensitization to ragweed pollen presented more severe forms of rhinitis (70% vs 31.5%, p = 0.02) in the NW part compared to polysensitized patients. The total symptoms score was significantly higher in patients from the Central part compared to the NW part (9.21 ± 2.01 vs 5.76 ±1.96, p < 0.001). Bronchial asthma was associated at a similar frequency to allergic rhinitis in both centers, but it was more frequently observed in monosensitized patients in the NW center. Allergic conjunctivitis was more frequently reported by patients from the Central part (75.86 vs 41.9, p = 0.02), while in the NW region it was noticed more commonly in monosensitized patients (65% vs 33.33, p = 0.02). Conclusions: Allergic rhinitis to ragweed pollen has been more frequently reported in the NW part of Romania. Patients with severe forms of rhinitis were observed in the central part, while in the NW the severe forms of disease were reported by patients with monosensitization. Ragweed pollen is intensely allergogenic and determines association of ocular and asthma symptoms. Co-sensitization increases the risk of asthma association.

Estimated risk reduction to packaged food reactions by epicutaneous immunotherapy (EPIT) for peanut allergy.

BACKGROUND: Peanut allergy is a generally persistent, sometimes life-threatening food allergy. With no treatments demonstrating the ability to cure a food allergy, the focus of drugs in development has been on providing a level of protection against accidental exposure reactions. However, no study has estimated the relative risk reduction of a food-allergic population receiving a specific immunotherapeutic treatment for their allergies. OBJECTIVE: To estimate the relative risk reduction when consuming peanut-contaminated packaged food products in a double-blind, placebo-controlled Phase 3 study population of children treated with epicutaneous immunotherapy (EPIT) for 12 months with either a patch containing 250 µg peanut protein (250-µg patch) or a placebo patch. METHODS: The probability of an allergic reaction due to the unintended presence of peanut protein in packaged food products was modeled per study group and food category combination using Monte Carlo simulations. Risks per eating occasion of a contaminated packaged food product and the number of individuals per study population predicted to react on a yearly basis were investigated. RESULTS: The population treated with the 250-µg patch demonstrated a significantly increased dose-response distribution after 12 months of treatment, which resulted in a relative risk reduction of 73.2% to 78.4% when consuming peanut-contaminated packaged food products. In contrast, no statistically significant change was observed for the placebo group at the 12-month point. CONCLUSION: Our study estimates a substantial relative risk reduction for allergic reactions among peanut-allergic children after 12 months of EPIT with the 250-µg patch, supporting the potential real-world clinical relevance of this investigational immunotherapy and its possible role as a future therapy for peanut-allergic children. ClinicalTrials.gov Identifier: NCT02636699.

Ragweed pollen and allergic symptoms in children: Results from a three-year longitudinal study.

Common ragweed is a highly allergenic invasive species in Europe, expected to become widespread under climate change. Allergy to ragweed manifests as eye, nasal and lung symptoms, and children may retain these throughout life. The dose-response relationship between symptoms and pollen concentrations is unclear. We undertook a longitudinal study, assessing the association between ragweed pollen concentration and allergic eye, nasal and lung symptoms in children living under a range of ragweed pollen concentrations in Croatia. Over three years, 85 children completed daily diaries, detailing allergic symptoms alongside daily location, activities and medication, resulting in 10,130 individual daily entries. The daily ragweed pollen concentration for the children's locations was obtained, alongside daily weather and air pollution. Parents completed a home/lifestyle/medical questionnaire. Generalised Additive Mixed Models established the relationship between pollen concentrations and symptoms, alongside other covariates. Eye symptoms were associated with mean daily pollen concentration over four days (day of symptoms plus 3 previous days); 61 grains/m3/day (95%CI: 45, 100) was the threshold at which 50% of children reported symptoms. Nasal symptoms were associated with mean daily pollen concentration over 12 days (day of symptoms plus 11 previous days); the threshold for 50% of children reporting symptoms was 40 grains/m3/day (95%CI: 24, 87). Lung symptoms showed a relationship with mean daily pollen concentration over 19 days (day of symptoms plus 18 previous days), with a threshold of 71 grains/m3/day (95%CI: 59, 88). Taking medication on the day of symptoms showed higher odds, suggesting responsive behaviour. Taking medication on the day prior to symptoms showed lower odds of reporting, indicating preventative behaviour. Different symptoms in children demonstrate varying dose-response relationships with ragweed pollen concentrations. Each symptom type responded to pollen exposure over different time periods. Using medication prior to symptoms can reduce symptom presence. These findings can be used to better manage paediatric ragweed allergy symptoms.

Cashew Tree Pollen: An Unknown Source of IgE-Reactive Molecules.

Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.

Subpollens delivery of Platanus acerifolia pollen allergen Pla a3 and nucleic acid into lungs and cells.

Pollen allergy is a very serious seasonal respiratory disease. However, there has been a lack of understanding how pollen allergens enter the body and act on cells. This study focused on the release, transport and characteristic of Pla a3 allergen of the Platanus acerifolia pollen. Pla a3 protein was purified by prokaryotic expression system for preparation of polyclonal antibody. The distribution and release of Pla a3 protein in pollen were observed by immunohistochemistry. Mice were immunized with purified Pla a3 protein and SPPs, respectively. The pathological examination of mouse lung tissue proved that SPPs, as a fine particle in the range of 0.1-1µm, can enter the deep part of the lung directly through the respiratory tract and led to inflammation. Furthermore, DAPI staining confirmed a certain amount of nucleic acids in SPPs. After incubation with SPPs for 6 h, the Pla a3 mRNA could be detected in A549 cells by PCR. This suggests that nucleic acid wrapped in SPPs could be delivered into A549 cells. These results could provide a new clue and experimental data accumulation for further study on the mechanism of pollen sensitization.

NFκB- and MAP-Kinase Signaling Contribute to the Activation of Murine Myeloid Dendritic Cells by a Flagellin A:Allergen Fusion Protein.

Fusion proteins incorporating the TLR5-ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. We recently reported a flagellin:allergen fusion protein containing the TLR5-ligand flagellin A (FlaA) from Listeriamonocytogenes and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to prevent allergic sensitization in an experimental mouse model. This study analyzes the signaling pathways contributing to rFlaA:Betv1-mediated pro- and anti-inflammatory cytokine secretion and cell metabolism in myeloid dendritic cells (mDCs) in vitro. The influence of mammalian target of rapamycin (mTOR)-, NF?B-, and MAP kinase (MAPK)-signaling on cytokine secretion and metabolic activity of bone marrow (BM)-derived mDCs stimulated with rFlaA:Betv1 were investigated by pre-treatment with either mTOR- (rapamycin), NF?B- (dexamethason, BMS-345541, TPCA-1, triptolide, or BAY-11) or MAPK- (SP600125, U0126, or SB202190) inhibitors, respectively. rFlaA:Betv1-mediated IL-10 secretion as well as activation of mDC metabolism, rather than pro-inflammatory cytokine secretion, were inhibited by rapamycin. Inhibition of NFκB-signaling suppressed rFlaA:Betv1-induced IL-12, while inhibition of MAPK-signaling dose-dependently suppressed rFlaA:Betv1-induced IL-10 as well as pro-inflammatory IL-6 and TNF-α production. Notably, with the exception of a partial JNK-dependency, rFlaA:Betv1-mediated effects on mDC metabolism were mostly NF?B- and MAPK-independent. Therefore, MAPK-mediated activation of both NFκB- and mTOR-signaling likely is a key pathway for the production of pro- and anti-inflammatory cytokines by flagellin fusion protein vaccines.

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.