Tolerability and positive efficacy results after subcutaneous immunotherapy with Parietaria judaica depot extract.

AIM: To evaluate tolerability and efficacy of Parietaria judaica subcutaneous immunotherapy on patients with allergic rhinoconjunctivitis. PATIENTS & METHODS: 51 patients were assigned to build-up scheme (six increasing doses) of P. judaica depot native extract, plus three maintenance monthly administrations. RESULTS: Out of 470 administered doses, only 3.8% elicited systemic reactions (1.5% nonspecific and 2.3% grade I). Concerning the exploratory efficacy parameters: cutaneous reactivity at the final visit versus baseline was significantly decreased; specific titers of IgG and IgG4 increased significantly and patients showed a significant decrease in the rhinitis symptoms score. CONCLUSION: P. judaica subcutaneous immunotherapy (Allergovac® depot ROXALL Medicina España S.A., Zamudio, Spain) with an abbreviated up-dosing scheme showed an adequate safety and tolerability profile and induced preliminary efficacy changes.

Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression.

Plant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato (Lycopersicon esculentum) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Following the identification of an immunoglobulin E (IgE)-binding 9-kDa polypeptide in tomato peel, designated Lyc e 3, its partial amino acid sequence was determined by N-terminal protein sequencing. Sequence comparison revealed that Lyc e 3 encodes a nonspecific lipid transfer protein (ns-LTP). In plants, ns-LTPs are encoded by large gene families which differ in primary amino acid sequence, expression and proposed cellular function. To identify Lyc e 3 encoding complementary DNAs (cDNAs), public tomato expressed sequence tag (EST) databases were screened for ns-LTP sequences. Following this strategy, two cDNAs, LTPG1 and LTPG2, with high homology to the N-terminal sequence of Lyc e 3, were identified. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli, followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1- or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein). The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines. These assays revealed a strong (10- to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts. These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition.

Specific immunotherapy for food allergy: basic principles and clinical aspects.

PURPOSE OF REVIEW: Food allergy may be life threatening and its management continues to consist of avoiding relevant allergens and, in the case of accidental ingestion, initiation of appropriate emergency therapy. The aim of this article is to describe current treatment approaches and discuss attempts to use specific immunotherapy for food-allergy treatment. RECENT FINDINGS: A recent study reports the use of sublingual immunotherapy for hazelnut food allergy in hazelnut-allergic patients. A significant increase in tolerance to hazelnuts after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment, have been observed. SUMMARY: The purpose of this review is to highlight the most promising novel approaches for treating food allergy beyond allergen avoidance. Some of these approaches alone, such as traditional Chinese herbal medicine, anti-immunoglobulin E therapy or sublingual immunotherapy for food allergy, or the combination of different approaches, would probably offer the best treatment option for food-allergic patients in the near future.

Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract.

BACKGROUND: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. OBJECTIVE: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. METHODS: This was a randomized, double-blind, placebo-controlled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebo-controlled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebo-controlled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG(4), and serum cytokines before and after treatment. RESULTS: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P = .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG(4) and IL-10 levels after immunotherapy in only the active group. CONCLUSION: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment.

Strong allergenicity of Pru av 3, the lipid transfer protein from cherry, is related to high stability against thermal processing and digestion.

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) have been identified as major fruit allergens in patients from the Mediterranean area. Sensitization to nsLTPs is accompanied by severe reactions, possibly because of specific biophysical and biochemical properties of this allergen family. OBJECTIVE: To assess the protein stability and allergenic potency of nsLTP from fruits in comparison with birch pollen-related allergens from the same allergenic source. METHODS: Stability of natural and recombinant cherry allergens Pru av 3 (nsLTP), Pru av 1 (Bet v 1 homologue), and Pru av 4 (profilin) to pepsin digestion and to thermal processing and stability of allergens in skin prick test reagents was investigated by immunoblotting and/or circular dichroism spectroscopy. Moreover, allergenicity of processed and fresh fruits in regard to Pru av 1 and Pru av 3 was analyzed by histamine release assays. RESULTS: Lipid transfer proteins showed the highest resistance to digestion by pepsin (rPru av 3 > rPru av 1 > rPru av 4). Immunologically active Pru av 3 was detectable after 2 hours of digestion by pepsin, whereas IgE reactivity of Pru av 1 and Pru av 4 was abolished within less than 60 minutes. In contrast with Pru av 1, IgE reactivity to nsLTPs was not diminished in thermally processed fruits, and secondary structures of purified Pru av 3 were more resistant to heating. Moreover, nsLTPs were stable components in skin prick test reagents. Histamine release assays confirmed the strong allergenicity of nsLTPs, which was not affected by protease treatment or thermal processing of fruits. CONCLUSION: In contrast with birch pollen-related allergens, nsLTPs are highly stable to pepsin treatment and thermal processing and show higher allergenic potency. Therefore, nsLTPs have the potential to act as true food allergens, probably eliciting severe systemic reactions by reaching the intestinal mucosa in an intact and fully active form.

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

Component-resolved diagnosis with recombinant allergens in patients with cherry allergy.

BACKGROUND: In pollen-related food allergy, extracts for skin prick tests (SPTs) are often not standardized, and the test reliability is affected by false-negative reactions. OBJECTIVE: We sought to evaluate a panel of recombinant allergens (RAs) derived from one allergenic food for use in component-resolved in vivo diagnosis, taking cherry as a model food. METHODS: Seventy-nine subjects were included in the study: 24 Swiss patients (group 1) with a positive double-blind placebo-controlled food challenge result to cherries, 23 patients with birch pollen allergy but without cherry allergy (group 2), 23 nonatopic subjects (group 3), and 9 Spanish patients with a history of a cherry allergy (group 4). SPTs were performed in duplicate by using recombinant cherry allergens (Bet v 1-related allergen: recombinant (r) Pru av 1; profilin: rPru av 4; and lipid transfer protein: rPru av 3) in concentrations of 10, 50, and 100 microg/mL. Furthermore, IgE reactivity to rPru av 1, rPru av 4, and rPru av 3 was assessed by means of immunoblot analysis. RESULTS: SPT responses with rPru av 1, rPru av 4, and rPru av 3 were positive in 92%, 17%, and 4% of the patients in group 1; in 74%, 30%, and 0% of the patients in group 2; in 0%, 22%, and 89% of the patients in group 4; and negative for all nonatopic subjects (group 3). Thus the sensitivity of a positive SPT response to at least one of the 3 RAs was 96%. The specificities, negative predictive values, and positive predictive values with the 3 RAs were 100%, 96%, and 100% if calculated in relation to the nonatopic control group but 17%, 79%, and 60% when calculated in relation to the control group with birch pollen allergy. The correlation between SPT and immunoblotting results was excellent. Sensitization to rPru av 3 was associated with more severe symptoms than sensitization to rPru av 1. CONCLUSIONS: SPTs with RAs proved to be highly sensitive for diagnosis of cherry allergy. Component-resolved in vivo diagnosis with standardized amounts of stable RAs allows us to determine sensitization patterns directly, to correlate them with severity of clinical symptoms, and to analyze geographic differences.