Margarete Focke

Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination

Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross‐reacts with most grass‐, corn‐, and monocot‐derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28‐32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild‐type allergen and group 1 allergens from other grass species. Moreover, peptide‐induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild‐type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE‐ mediated allergies.

Patch testing in children, adults, and the elderly: Influence of age and sex on sensitization patterns

Abstract: Patch testing was done on 2776 consecutive patients (76.5% female) with a locally revised standard series of 34 contact allergens and the results analyzed for age‐ and gender‐specific differences. At least one positive epicutaneous test reaction occurred in 48.9% of patients. Nickel (20.9%), ethylmercuric chloride (13.2%), thimerosal (11.8%), fragrance mix (9.3%), metallic mercury (8.9%), palladium (5.8%), balsam of Peru (3.8%), copper (3.7%), cobalt (3.3%), and chromium (2.3%) were the 10 most important sensitizers. The following tested allergens with sensitization rates of more than 1% were not part of the usual standard series: ethylmercuric chloride, metallic mercury, copper, propolis (1.3%), propylene glycol (1.0%). Reactions to nickel, cobalt, and palladium, but not to chromium, were significantly more abundant in females (p < 0.002, chi‐squared test). The overall sensitization rate was highest in children less than 10 years old (62%) and decreased steadily, to be lowest among patients more than 70 years old (34.9%). The rate of positive reactions to nickel and thimerosal decreased with age, while fragrance mix and metallic mercury stayed at the same level through all age groups.

Identification by immunoblot of venom glycoproteins displaying immunoglobulin E-binding N-glycans as cross-reactive allergens in honeybee and yellow jacket venom.

Background IgE antibodies against carbohydrate epitopes have been identified recently as a major cause of in vitro double positivity to honeybee (HB) and vespid venom in patients with stinging‐insect allergy. As these antibodies possibly have low clinical relevance they may be misleading in the diagnosis of venom allergy.

Generation of an Allergy Vaccine by Disruption of the Three-Dimensional Structure of the Cross-Reactive Calcium-Binding Allergen, Phl p 7

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients’ IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients’ IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen’s three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.

Reassessing the role of hyaluronidase in yellow jacket venom allergy

BACKGROUND Yellow jacket hyaluronidase (YJ-HYA) is considered a major allergen in yellow jacket allergy. It shows 50% homology with the hyaluronidase from honeybee venom, Api m 2. Recently, IgE binding to YJ-HYA and cross-reactivity with Api m 2 has been shown to be due to cross-reactive carbohydrate determinants (CCDs). OBJECTIVE We sought to quantify the importance of YJ-HYA in yellow jacket allergy and the cross-reactivity with Api m 2 by discriminating between carbohydrate and peptide epitopes. METHODS IgE binding to Vespula species venom was studied by means of Western blotting in 136 patients with yellow jacket allergy (31 in vitro single positive to yellow jacket venom and 105 in vitro double-positive to yellow jacket-honeybee). Inhibition studies were carried out with MUXF-BSA (isolated bromelain glycopeptides linked to bovine serum albumin) and purified Api m 2. RESULTS Among yellow jacket single-positive sera, only 1 of 31 bound with YJ-HYA, whereas this was the case in 87% of 105 double-positive sera. Of 83 patients in whom inhibitions were performed, 65% reacted with hyaluronidase through CCDs alone, 27% reacted with both CCDs and peptide epitopes, and 8% reacted only with the hyaluronidase peptide. The protein-specific reactivity with YJ-HYA was cross-inhibited by Api m 2 in 48% (14/29). Antigen 5 and phospholipase A(1) were each recognized by around 90% of sera from both groups, together identifying 97% of patients. CONCLUSION Hyaluronidase is a minor yellow jacket venom allergen, and only 10% to 15% of patients with yellow jacket allergy are estimated to have IgE against the hyaluronidase protein. Peptide-specific cross-reactivity with Api m 2 occurs in half of these sera. Component-resolved diagnosis with antigen 5 and phospholipase would detect virtually all patients with yellow jacket venom allergy.

Inconsistent Results of Diagnostic Tools Hamper the Differentiation between Bee and Vespid Venom Allergy

Background Double sensitization (DS) to bee and vespid venom is frequently observed in the diagnosis of hymenoptera venom allergy, but clinically relevant DS is rare. Therefore it is sophisticated to choose the relevant venom for specific immunotherapy and overtreatment with both venoms may occur. We aimed to compare currently available routine diagnostic tests as well as experimental tests to identify the most accurate diagnostic tool. Methods 117 patients with a history of a bee or vespid allergy were included in the study. Initially, IgE determination by the ImmunoCAP, by the Immulite, and by the ADVIA Centaur, as well as the intradermal test (IDT) and the basophil activation test (BAT) were performed. In 72 CAP double positive patients, individual IgE patterns were determined by western blot inhibition and component resolved diagnosis (CRD) with rApi m 1, nVes v 1, and nVes v 5. Results Among 117 patients, DS was observed in 63.7% by the Immulite, in 61.5% by the CAP, in 47.9% by the IDT, in 20.5% by the ADVIA, and in 17.1% by the BAT. In CAP double positive patients, western blot inhibition revealed CCD-based DS in 50.8%, and the CRD showed 41.7% of patients with true DS. Generally, agreement between the tests was only fair and inconsistent results were common. Conclusion BAT, CRD, and ADVIA showed a low rate of DS. However, the rate of DS is higher than expected by personal history, indicating that the matter of clinical relevance is still not solved even by novel tests. Furthermore, the lack of agreement between these tests makes it difficult to distinguish between bee and vespid venom allergy. At present, no routinely employed test can be regarded as gold standard to find the clinically relevant sensitization.

Levels of histamine and other biogenic amines in high-quality red wines

Biogenic amines in wine may impair sensory wine quality and cause adverse health effects in susceptible individuals. In this study, histamine and other biogenic amines were determined by HPLC after amine derivatisation to dansyl chloride conjugates in 100 selected high-quality red wines made from seven different cultivars. Amine levels varied considerably between different wines. The most abundant amines were putrescine (median = 19.4 mg l−1, range = 2.9–122), histamine (7.2 mg l−1, 0.5–26.9), and tyramine (3.5 mg l−1, 1.1–10.7), whereas lower levels were found for isoamylamine (median = 0.25 mg l−1), phenylethylamine (0.16 mg l−1), cadaverine (0.58 mg l−1), spermidine (1.8 mg l−1) and tryptamine (0.06 mg l−1). Positive correlations were observed between isoamylamine and phenylethylamine, and between histamine, putrescine and tyramine levels. Amine concentrations were similar in all wine cultivars except Pinot noir and St. Laurent wines, which showed significantly higher tryptamine and cadaverine levels. The results indicate that levels of histamine and other biogenic amines may vary considerably between red wines independent of grape variety and that high amounts can also be found in high-rated wines. Adopting a legal histamine threshold level of 10 mg l−1 in the European Union, as formerly introduced in other countries, would have excluded 34% of the investigated wines from the market.

A cream containing the chelator DTPA (diethylenetriaminepenta‐acetic acid) can prevent contact allergic reactions to metals

Chelating agents in protective barrier creams have often been used in the prevention of allergic contact dermatitis to nickel. In a pilot study, we demonstrated the preventive effect of 10% diethylenetriaminepentaacetic acid (DTPA) in an oil‐in‐water emulsion in nickel‐sensitized patients. Now we reproduced these results in a randomized, double‐blind study. Additionally, we investigated the efficacy of the barrier cream in other clinically relevant metal allergies. Individuals sensitized to various metals had a significant decrease in positive patch test reactions after pre‐treatment with the DTPA‐cream: 2.5% nickel sulfate (24/28 positive without pre‐treatment versus 1/28 with pre‐treatment; p<0.0001), 5% nickel sulfate (30/32 versus 15/32; p=0.0003), 1% cobalt chloride (19/20 versus 6/20; p=0.001) and 5% copper sulfate (13/14 versus 5/14; p=0.02). However, the cream had no protective effect with 1% palladium chloride (17/23 versus 16/23) and with 0.5% potassium dichromate (9/13 versus 7/13). We conclude that the DTPA‐cream clearly abrogates positive patch test reactions in nickel‐, cobalt‐ and copper‐sensitized subjects and that it may therefore be helpful in the management of allergic contact dermatitis.

Genetic Engineering of the Major Timothy Grass Pollen Allergen, Phl p 6, to Reduce Allergenic Activity and Preserve Immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1–33, 1–57, and 31–110 of the major timothy grass pollen allergen Phl p 6 aa 1–110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical α-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the α helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31–110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31–110 inhibited patients’ IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.

Anaphylaxis induced by horsefly bites: Identification of a 69 kd IgE-binding salivary gland protein from Chrysops spp. (Diptera Tabanidae) by western blot analysis

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