Recombinant Mal d 1 facilitates sublingual challenge tests of birch pollen-allergic patients with apple allergy.

It is still unclear whether allergen-specific immunotherapy (AIT) with birch pollen improves birch pollen-related food allergy. One reason for this may be the lack of standardized tests to assess clinical reactions to birch pollen-related foods, for example apple. We tested the applicability of recombinant (r) Mal d 1, the Bet v 1-homolog in apple, for oral challenge tests. Increasing concentrations of rMal d 1 in 0.9% NaCl were sublingually administered to 72 birch pollen-allergic patients with apple allergy. The dose of 1.6 µg induced oral allergy syndromes in 26.4%, 3.2 µg in 15.3%, 6.3 µg in 27.8%, 12.5 µg in 8.3%, 25 µg in 11.1%, and 50 µg in 4.2% of the patients. No severe reactions occurred. None of the patients reacted to 0.9% NaCl alone. Sublingual administration of 50 µg of rMal d 1 induced no reactions in three nonallergic individuals. Our approach allows straight forward, dose-defined sublingual challenge tests in a high number of birch pollen-allergic patients that inter alia can be applied to evaluate the therapeutic efficacy of birch pollen AIT on birch pollen-related food allergy.

Suppression of gastric acid increases the risk of developing immunoglobulin E-mediated drug hypersensitivity: human diclofenac sensitization and a murine sensitization model.

BACKGROUND: Hypersensitivity reactions towards non-steroidal anti-inflammatory drugs (NSAID) are common, although true allergies are detectable only in a subgroup of patients. The current study was prompted by a case observation, where a patient experienced generalized urticaria following his second course of diclofenac and proton pump inhibitor medication, and was found to have diclofenac-specific IgE. During recent years, our group has been investigating the importance of gastric digestion in the development of food allergies, demonstrating anti-acid medication as a risk factor for sensitization against food proteins. OBJECTIVE: Here, we aimed to investigate whether the mechanism of food allergy induction described can also be causative in NSAID allergy, using diclofenac as a paradigm. METHODS: We subjected BALB/c mice to several oral immunization regimens modelled after the patient's medication intake. Diclofenac was applied with or without gastric acid suppression, in various doses, alone or covalently coupled to albumin, a protein abundant in gastric juices. Immune responses were assessed on the antibody level, and functionally examined by in vitro and in vivo crosslinking assays. RESULTS: Only mice receiving albumin-coupled diclofenac under gastric acid suppression developed anti-diclofenac IgG1 and IgE, whereas no immune responses were induced by the drug alone or without gastric acid suppression. Antibody induction was dose dependent with the group receiving the higher dose of the drug showing sustained anti-diclofenac titres. The antibodies induced triggered basophil degranulation in vitro and positive skin tests in vivo. CONCLUSION: Gastric acid suppression was found to be a causative mechanism in the induction of IgE-mediated diclofenac allergy.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Comparison of a two- or three-week regimen and a review of treatment of erythema migrans with phenoxymethylpenicillin.

BACKGROUND: A longer duration treatment is preferred in erythema migrans (EM) to prevent late complaints. OBJECTIVES: To determine whether 20 (20d-pt) or 14 days (14d-pt) of phenoxymethylpenicillin (PenV) have similar efficacy in treating EM and preventing further sequelae. PATIENTS AND METHODS: In a prospective double-centre study, 102 patients with EM were treated with PenV 1.5 million IU thrice daily for either 20 or 14 days and followed up for 12 months. RESULTS: The primary cure rate after treatment with PenV was 91.5% (79.6-97.6) for 20d-pt vs. 91.7% (77.5-98.2) for 14d-pt; p > 0.99). In 7 patients (4/20d-pt and 3/14d-pt), persistent or newly arising symptoms required retreatment. After 1 year, all patients were cured. The immune response showed no statistical difference between the treatment groups in the follow-up period. CONCLUSION: A 2-week treatment regimen of PenV seems to be as effective as a 3-week course with no statistical differences for clinical and serological findings after treatment.

Characterization of the human T cell response to antigen 5 from Vespula vulgaris (Ves v 5).

BACKGROUND: The T cell reactivity to the major allergen of bee venom, phospholipase A2, has been thoroughly characterized. In contrast, only little is known about the human cellular response to major allergens from wasp venom. OBJECTIVE: To characterize the human T cell response to antigen 5 from Vespula vulgaris, Ves v 5. METHODS: Recombinant Ves v 5 was used to establish allergen-specific T cell lines (TCL) and T cell clones (TCC) from the peripheral blood of vespid-allergic and non-allergic individuals. Ves v 5-specific TCL were mapped for T cell epitopes using overlapping synthetic peptides representing the complete amino acid sequence of Ves v 5. Ves v 5-specific TCC were analysed for antigen-induced secretion of IL-4, IFN-gamma and IL-10. RESULTS: Seventeen distinct T cell epitopes were recognized by allergic individuals among which Ves v 5(181-192) was identified as a dominant T cell epitope. Partially different epitopes were observed in TCL from non-allergic subjects and the dominant epitope Ves v 5(181-192) was not prevalent in these cultures. Ves v 5-specific TCC isolated from allergic individuals did not show the typical T helper type 2 (Th2)-like cytokine profile in response to specific stimulation, i.e. high amounts of IL-4 and low IFN-gamma. TCC from non-allergic individuals showed a Th1-like cytokine pattern. CONCLUSIONS: Our findings provide evidence that the allergic T cell response to Ves v 5 is not Th2-dominated and that different immunogenic sites on this major wasp venom allergen are recognized by allergic and non-allergic individuals.

Type I allergy to elderberry (Sambucus nigra) is elicited by a 33.2 kDa allergen with significant homology to ribosomal inactivating proteins.

BACKGROUND: Patients suffering from allergic rhinoconjunctivitis and dyspnoea during summer may exhibit these symptoms after contact with flowers or dietary products of the elderberry tree Sambucus nigra. OBJECTIVE: Patients with a history of summer hayfever were tested in a routine setting for sensitization to elderberry. Nine patients having allergic symptoms due to elderberry and specific sensitization were investigated in detail. We studied the responsible allergens in extracts from elderberry pollen, flowers and berries, and investigated cross-reactivity with allergens from birch, grass and mugwort. METHODS: Sera from patients were tested for IgE reactivity to elderberry proteins by one-dimensional (1D) and 2D electrophoresis/immunoblotting. Inhibition studies with defined allergens and elderberry-specific antibodies were used to evaluate cross-reactivity. The main elderberry allergen was purified by gel filtration and reversed-phase HPLC, and subjected to mass spectrometry. The in-gel-digested allergen was analysed by the MS/MS sequence analysis and peptide mapping. The N-terminal sequence of the predominant allergen was analysed. RESULTS: 0.6% of 3668 randomly tested patients showed positive skin prick test and/or RAST to elderberry. IgE in patients' sera detected a predominant allergen of 33.2 kDa in extracts from elderberry pollen, flowers and berries, with an isoelectric point at pH 7.0. Pre-incubation of sera with extracts from birch, mugwort or grass pollen rendered insignificant or no inhibition of IgE binding to blotted elderberry proteins. Specific mouse antisera reacted exclusively with proteins from elderberry. N-terminal sequence analysis, as well as MS/MS spectrometry of the purified elderberry allergen, indicated homology with ribosomal inactivating proteins (RIPs). CONCLUSION: We present evidence that the elderberry plant S. nigra harbours allergenic potency. Independent methodologies argue for a significant homology of the predominant 33.2 kDa elderberry allergen with homology to RIPs. We conclude that this protein is a candidate for a major elderberry allergen with designation Sam n 1.

The oak processionary caterpillar as the cause of an epidemic airborne disease: survey and analysis.

BACKGROUND: Thaumetopoea processionea L. (order Lepidoptera), the oak processionary moth, is found in oak forests in most European countries. Its third to sixth larval instars are armed with poisonous hairs (setae) containing an urticating toxin (thaumetopoein) potentially harmful to humans. Because T. processionea infests trees at the edges of forests or standing alone people frequently come into contact with its setae. In the woodland bordering on the western suburbs of Vienna conditions favouring its increase have led to frequent outbreaks of lepidopterism. OBJECTIVES: To determine the incidence of lepidopterism in a suburban environment with three separate caterpillar-infested oak trees and to ascertain the frequency of the various symptoms of lepidopterism and the manner of contact with setae. METHODS: We conducted a telephone survey of all the households/institutions located within 500 m of the infested trees. To gain more information on patients' symptoms and on situations likely to lead to increased contact with setae we asked those who reported cutaneous reactions to complete a questionnaire. As part of the environmental study we described the outbreak site, examined patients and, with tape-strip samples taken from the surface of the soil, looked for setae persisting in the environment. RESULTS: Of 1025 people surveyed 57 (5.6%) reported one or more symptoms of lepidopterism: 55 (96%) reported pruritus, 54 (95%) dermatitis, eight (14%) conjunctivitis, eight (14%) pharyngitis and two (4%) respiratory distress. The questionnaire was returned by 37 (69%) of the individuals with dermatitis. Of those, 16% had reacted with weal formation, 49% with papular rash and 22% with toxic irritant dermatitis. In 13% of respondents it was not possible to define the reaction. The risk factor analysis showed that airborne contamination was the most important cause: 97% of people had frequently passed an infested tree, 57% lived near a tree (in a neighbouring garden) and 32% had a tree in their own garden. Direct contact with larvae was of minor importance (38%). In four of the tape-strip samples intact setae were identified 1 year after the infestation. CONCLUSIONS: Lepidopterism caused by T. processionea is a public health problem of increasing significance. In years with outbreaks of the pest it can reach epidemic proportions in communities located near infested trees. Contact with airborne setae was mainly responsible for the occurrence of the disease.

[Sensitization to tea tree oil in Germany and Austria. A multicenter study of the German Contact Dermatitis Group]. / Sensibilisierung auf Teebaumöl in Deutschland und Osterreich--Eine multizentrische Studie der Deutschen Kontaktallergiegruppe.

BACKGROUND AND AIM: Tea tree oil, a distillation product of the Australian tea tree (Melalence alternitolia) is increasingly used as an alternative remedy for various dermatological diseases. Tea tree oil contains several allergenic monoterpenes and sesquiterpenes. In this multicenter study it was evaluated, whether the increasing use of tea tree oil has lead to an increased frequency of sensitization in Germany and Austria which would justify its inclusion into the standard series. PATIENTS AND METHOD: For patch testing a standardized tea tree oil was used, dissolved 5% in diethylphtalate (DEP). Consecutive patients of 11 dermatological departments in Germany and Austria were tested. Readings were taken on day 2 and 3 according to the guidelines of the German Contact Dermatitis Research Group (DKG). RESULTS: 5% tea tree oil was positive in 36/3375 patients (1.1%). Sensitization frequencies showed great regional variations and ranged from 2.3% (Dortmund), 1.7% (Buxtehude), 1.1% (Essen), 0.7% (Graz), to 0% (Berlin, Vienna). 14/36 patients (38.9%) also showed a positive patch test reaction to oil of turpentine. CONCLUSION: Our results show that tea tree oil is an important contact allergen for some centers. It should be tested, if medical history suggests its previous use. Considering the great regional differences in frequencies of sensitization its inclusion into the standard series is not recommended yet.

Allergologic exploration of germins and germin-like proteins, a new class of plant allergens.

BACKGROUND: Germins and the related germin-like proteins (GLPs) are glycoproteins expressed in many plants in response to biotic and abiotic stress. To test the potential impact of germins and GLPs, recombinant germin from Triticum aestivum (tGermin) and GLPs from Arabidopsis thaliana (tGLP), both produced in transformed tobacco plants, were used. METHODS: Sera from 82 patients with type I allergy to birch, grass or mugwort pollen and/or wheat were tested in immunoblot for IgE binding to tGermin and tGLP, and the IgE reactivity after chemical and enzymatic deglycosylation was analysed. The biological activity of tGermin and tGLP was determined in a histamine release assay and in skin prick testing (SPT). RESULTS: In an immunoblotting assay, 24 out of 82 tested sera (29.26%) from allergic patients showed IgE-binding to tGermin, and 18 of these sera (21.95%) displayed also IgE-binding to tGLP. The deglycosylation experiments indicated that glycan moieties contribute significantly to the IgE-binding of tGermin and tGLP. Both tGermins and tGLP induced specifically histamine release in an in vitro assay as well as in SPT. CONCLUSION: Our in vitro and in vivo findings demonstrate that germin and GLPs are capable to bind IgE most likely via carbohydrate determinants, and represent allergenic molecules.

Cloning of the patatin-like latex allergen Hev b 7, its expression in the yeast Pichia pastoris and its immunological characterization.

The 43-kD latex allergen Hev b 7 was purified from the latex of Hevea brasiliensis and identified by N-terminal and internal peptide sequences as highly homologous to patatins. Patatins are storage proteins encoded by a multigene family found in plants such as potato and tomato. We have obtained a cDNA clone coding for a cytoplasmic form of Hev b 7. The recombinant protein was expressed in the methylotrophic yeast Pichia pastoris at 10 mg/l culture supernatant. Both natural Hev b 7 and rHev b 7 were recognized by IgE in 11% of the latex-allergic patients. rHev b 7 inhibited binding to its counterpart in natural rubber latex extracts. Purified rHev b 7 used at concentrations of 10 micrograms/ml in skin prick tests produced wheal-and-flare reactions of sizes equal to those produced by nHev b 7. Furthermore, we were able to show that rHev b 7 possessed esterase activity. A plant expression system for the production of larger quantities of recombinant latex allergens as an alternative to the preparation from H. brasiliensis sap is discussed.

cDNA cloning of the 43-kDa latex allergen Hev b 7 with sequence similarity to patatins and its expression in the yeast Pichia pastoris.

IgE-mediated hypersensitivity to latex proteins present in health care products, particularly in latex gloves, has become an important public health problem in recent years. We purified natural Hev b 7, a 43-kDa patatin-like allergen from the latex of Hevea brasiliensis and determined several internal peptide sequences. A heterologous hybridization probe of a patatin gene of potato, to which these peptides could be aligned best, was used to screen a latex cDNA library. The cDNA encoded an acidic protein of 388 amino acids with a molecular mass of 42.9 kDa. The deduced amino acid sequence had 39-42% identity to patatins from Solanum tuberosum. The purified recombinant Hev b 7 expressed in the yeast Pichia pastoris displayed, similarly to patatins from S. tuberosum, esterase activity. Both natural and recombinant Hev b 7 were recognized by IgE from sera of latex-sensitized allergic individuals. In contrast to patatins from S. tuberosum and Nicotiana tabacum, natural Hev b 7 lacked an N-terminal leader peptide for targeting to the endoplasmatic reticulum and was not glycosylated. These results establish the 43-kDa patatin-like protein as a latex allergen and raise the possibility of different cellular localization and function compared to S. tuberosum patatins.

Comparison of narrow-band (311 nm) UVB and broad-band UVA after oral or bath-water 8-methoxypsoralen in the treatment of psoriasis.

BACKGROUND: There is a disparity between the absorption spectrum of 8-methoxypsoralen and the action spectrum for psoralen-sensitized erythema. In an action spectrum corrected for unsensitized reaction 313 and 365 nm have similar efficacies. OBJECTIVE: We evaluated the relative erythemogenic and antipsoriatic efficacy of narrow-band (311 nm) UVB with and without prior psoralen exposure. We also compared the effects of narrow-band UVB and broad-band UVA after oral and bath-water psoralen exposure. METHODS: Patients with psoriasis underwent half-side comparison studies. In one group the therapeutic efficacy of 311 nm UVB with and without oral psoralen was assessed. The second group received UVA and 311 nm UVB after oral psoralen. The third group was exposed to both radiation sources after bath-water exposure. RESULTS: The erythemogenic, pigmentogenic, and therapeutic efficacy of 311 nm was increased by oral psoralen. With systemic 8-methoxypsoralen, UVA was comparable to 311 nm UVB. After bath-water exposure, 311 nm was clearly superior to broad-band UVA. CONCLUSION: The efficacy of narrow-band 311 nm UVB can be enhanced by psoralen. Narrow-band 311 nm UVB is also effective after psoralen bath-water delivery.