Spatial clustering of the IgE epitopes on the major timothy grass pollen allergen Phl p 1: importance for allergenic activity

BACKGROUND: The major timothy grass pollen allergen Phl p 1 is one of the most potent and frequently recognized environmental allergens. OBJECTIVE: We sought to study at a molecular and structural level the IgE recognition of Phl p 1 and its relation to allergenic activity. METHODS: Monoclonal human IgE antibody fragments specific for Phl p 1 and group 1 allergens from various grasses were isolated from a combinatorial library made of lymphocytes from patients with grass pollen allergy. Recombinant Phl p 1 fragments and the 3-dimensional structure of Phl p 1 were used to localize the major binding site for the IgE antibodies. A rPhl p 1 fragment containing this binding site was expressed in Escherichia coli, purified, and tested for IgE reactivity and allergenic activity with sera and basophils from patients with grass pollen allergy. RESULTS: Monoclonal antibodies, as well as polyclonal serum IgE, from patients with grass pollen allergy defined a C-terminal fragment of Phl p 1 that represents a sterically oriented portion on the Phl p 1 structure. This Phl p 1 portion bound most of the allergen-specific IgE antibodies and contained the majority of the allergenic activity of Phl p 1. CONCLUSION: IgE recognition of spatially clustered epitopes on allergens might be a general factor determining their allergenic activity. CLINICAL IMPLICATIONS: Geographic distribution of IgE epitopes on an allergen might influence its allergenic activity and hence explain discrepancies between diagnostic test results based on IgE serology and provocation testing. It might also form a basis for the development of low allergenic vaccines.     

Influence of drug content,type of semi-solid vehicle and rheological properties on the skin penetration of the model drug fludrocortisone acetate

Throughout Europe, topical creams containing corticosteroids are diluted with various neutral cream bases to meet the specific needs of patients. Even though this practice has been common for years, its effect has not been thoroughly investigated and so the effectiveness of the diluted topical steroidal creams is difficult to predict. In the present study, the model drug fludrocortisone acetate was incorporated into three cream bases of different hydrophilicity that are commonly used in Austria. Different final drug concentrations were chosen for comparative studies. Additionally, a semi-solid preparation developed by our group was investigated for comparison. These formulations were tested in diffusion and tape stripping experiments. Diffusion cell studies showed that changes in drug concentration do not necessarily change the skin permeation behaviour in vitro. The tape stripping protocol was successfully optimised for investigation of semi-solid preparations to provide reproducible and accurate results despite the challenges of investigating semi-solid formulations. The results showed that tape stripping experiments are more suitable to elucidate subtle differences between formulations. The composition of the cream bases exhibited stronger effects on the skin penetration of the steroidal drug irrespective of its concentration than the rheological properties. No correlation between formulation viscosity and skin penetration was found.     

Characterization of Der p 21, a new important allergen derived from the gut of house dust mites

Background: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. Methods: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. Results: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an α‐helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X‐ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients’ IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti‐Der p 21 IgG antibodies inhibited mite‐allergic patients’ IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross‐inhibition studies identified it as a new mite allergen. Conclusions: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.     

Rapid cardiac hybrid imaging with minimized radiation dose for accurate non-invasive assessment of ischemic coronary artery disease

Background

Ischemic coronary artery disease (CAD) is a major cause for morbidity and mortality resulting in a continuously increasing number of diagnostic interventions. We have validated a new hybrid imaging method using minimized radiation dose for rapid non-invasive prediction of invasive coronary angiography (CA) findings with regard to coronary lesion detection and revascularization.

Methods

Forty patients referred for elective invasive coronary angiography (CA) due to suspected CAD were prospectively enrolled to undergo a low-dose CTCA with prospective ECG-triggering and a stress-only SPECT-MPI scan administering half of the standard low-dose stress ^99mTc-tetrofosmin activity. The latter was acquired immediately after adenosine stress (omitting the standard 30-60 min waiting time). After fusing CTCA and SPECT-MPI decisions towards conservative management versus revascularization strategy based on hybrid images were compared to the decisions taken by the interventional operator in the catheterization laboratory based on CA. The latter served as standard of reference.

Results

Hybrid images yielded sensitivity, specificity, positive and negative predictive values and accuracy of 100%, 96.0%, 100%, 93.8% and 97.5% for predicting coronary revascularization. The estimated mean effective radiation doses were significantly lower for hybrid imaging (4.7 ± 1.0 mSv) than for invasive CA (8.7 ± 4.2 mSv; P < 0.001 vs. hybrid). Total non-invasive protocol time was below 60 min, comparing favourably to standard SPECT protocols.

Conclusions

Rapid cardiac hybrid imaging allows accurate prediction of invasive CA findings and of treatment decision despite minimized radiation dose and protocol time.     

Identification of cross-reactive and genuine Parietaria judaica pollen allergens

BACKGROUND: The weed Parietaria judaica is one of the most important pollen allergen sources in the Mediterranean area. OBJECTIVE: We sought to identify P judaica pollen allergen, which might be used to serologically distinguish genuine Parietaria sensitization and cross-reactivity to allergens from other weed species (eg, mugwort and ragweed). METHODS: The allergen profile of P judaica IgE-reactive sera from weed pollen-sensitized allergic individuals from the Mediterranean region (n = 36) with high Parietaria pollen exposure and from weed pollen-allergic patients with little or no Parietaria exposure (Austria, n = 42; Scandinavia, n = 8; United States, n = 19) was established by CAP FEIA measurements and by IgE immunoblot inhibition experiments with recombinant allergens. RESULTS: The majority (83%) of the Mediterranean weed pollen-allergic patients mounted high IgE antibody levels (mean specific IgE, 20.89 kUA/L) against recombinant (r) Par j 2, whereas only 7% of the non-Mediterranean weed-allergic patients showed low IgE reactivity to rPar j 2 (mean specific IgE, 1.03 kUA/L). The cytoskeletal protein profilin and a 2-EF-hand calcium-binding allergen were identified as cross-reactive Parietaria allergens, which were recognized preferentially by Parietaria -positive, non-Mediterranean weed pollen-allergic patients. CONCLUSION: rPar j 2 might be used as a diagnostic marker allergen to identify weed pollen-allergic patients who are genuinely sensitized against Parietaria pollen and thus would be particularly suited for specific immunotherapy with Parietaria pollen extract.