Marianne van Hage-Hamsten

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment

Type I allergy is an immunoglobulin E (IgE)‐mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen‐containing sources but cannot identify the disease‐eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients’ IgE reactivity profiles to large numbers of disease‐causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

Storage mite allergy is common in a farming population

The prevalence of storage mite allergy was investigated in an epidemiological study of respiratory symptoms in farmers on Gotland, an island in the Baltic Sea. A questionnaire concerning work‐related and chronic symptoms from the airways and eyes was completed by 2578 farmers. A sample of 440 farmers subsequently underwent examination comprising skin‐prick tests (animal danders, pollens, moulds and house dust mite), blood sampling for RAST against four storage mites (Acarus siro, Lepidoglyphus destructor. Tyrophagus putrescentiae and Glycyphagus domesticus) and total IgE analyses. Immediate onset hypersensitivity reactions in the airways and eyes were very common among active farmers on Gotland, the prevalence being as high as 40.0%. The prevalence of atopic allergy was 15.6%. Allergy to storage mites was diagnosed, from the case history and a positive RAST to at least one of the four storage mites, in fifty‐two of 440 studied farmers (12%), corresponding to a calculated prevalence of storage mite allergy in the whole farming population of Gotland of 6.2%. The corresponding prevalence among farmers with hypersensitivity symptoms was 15.4%and among those with possibly IgE‐mediated symptoms it was as high as 37.8%. The present study clearly demonstrates a high prevalence of allergy to storage mites among farmers with respiratory symptoms.

Influence of interaction of environmental risk factors and sensitization in young asthmatic children

BACKGROUND The increasing prevalence of asthma and allergy in many countries demands evaluation of potential risk factors to improve the possibility of prevention. OBJECTIVE We studied the association between exposure to cat and dog allergen and allergic sensitization in young children with asthma and interactions with potential environmental risk factors. METHODS One hundred eighty-nine young children with asthma were evaluated. IgE antibodies to cat and dog were analyzed. Questionnaires were filled in focusing on exposure to cats and dogs, environmental tobacco smoke (ETS), and signs of home dampness as indicated by window pane condensation (WPC) during the first years of life. House dust was analyzed for content of cat (Fel d 1) and dog (Can f 1) allergen. RESULTS There was a strong association between the degree of reported exposure to cat and dog and the concentration of the respective allergens in floor dust. A dose-response relationship was found between cat exposure, measured as either reported degree of cat exposure or cat allergen levels in dust, and sensitization both to cat and dog. No such relationship was found between exposure and sensitization to dog. WPC increased the risk for sensitization to cat (odds ratio = 2.6, 95% confidence interval 1.2-5.8), whereas ETS strongly tended to do so both to cat and dog. Interaction was found between exposure to ETS, WPC, and high levels of cat allergen (>8 microg/g dust). The presence of all 3 risk factors revealed a multiplicative interaction with a high risk of sensitization to cat (odds ratio = 42.0, 95% confidence interval 3.7-472.8). CONCLUSIONS Keeping cats indoors may be a health hazard for infants and young children at risk for development of asthma, particularly when they live in a damp house and their parents smoke.

β-Tryptase measurements post-mortem in anaphylactic deaths and in controls

The reliability of measuring mast cell tryptase in post-mortem blood to diagnose anaphylactic deaths has been questioned because moderate elevation of tryptase can be seen also in control cases. Very high tryptase concentrations have been recorded even in a few control cases with known cause of death such as myocardial infarction or trauma. Aiming to compare findings we measured tryptase in 193 cases: 176 with known cause of death, 10 unexplained deaths and seven anaphylactic or anaphylactoid deaths (AADs). Using binary logistic regression we calculated the sensitivity and specificity of the tryptase test at different cut-off values and found 10 micrograms/l to be optimal, the sensitivity being 86% and the specificity 88%. Traumatic deaths (n = 23), sudden infant death syndrome (n = 40) or deaths after heroin-injection (n = 22) showed elevated tryptase values in 35%, 35% and 32%, respectively, and in 40% of the unexplained deaths (n = 10), which was higher than expected (12%). Heart blood tryptase level was elevated in 22% of the controls and femoral blood tryptase in 10%. No correlations were seen with age or post-mortem delay. It is concluded that tryptase measurements are useful in confirming death from AAD, and that blood should be sampled from the femoral vessels. In unexplained deaths tryptase measurement is a useful indicator, but the diagnosis is not to be based on the test alone.

The crystal structure of the major cat allergen Fel d 1, a member of the secretoglobin family.

The domestic cat (Felis domesticus) is one of the most important causes of allergic asthma worldwide. The dominating cat allergen, Fel d 1, is composed of two heterodimers. Recently, it has been shown that recombinant Fel d 1, consisting of chain 2 and chain 1 fused together without additional linker, has immunological properties indistinguishable from the natural heterodimeric protein. Herein, we report the crystal structure of recombinant monomeric Fel d 1 at 1.85-Å resolution, determined by multi-wavelength anomalous diffraction using selenomethionine substituted protein. Fel d 1 is an all-helical protein and consists of eight helices. The two halves of the recombinant Fel d 1 molecule, corresponding to the wild-type Fel d 1 chains, are very similar in three-dimensional structure, despite the lack of significant sequence identity. The structure of the Fel d 1 presents a striking similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties. An internal, asymmetric cavity is formed in the Fel d 1 that could bind an endogenous ligand. The distribution of residues lining this cavity suggests that such a ligand must be amphipathic. The structure of Fel d 1 displays the localization of three previously defined Fel d 1 IgE epitopes on the surface of the protein. The three-dimensional structure provides a framework for rational design of hypoallergenic mutants aimed for treatment of cat allergy.

Lack of allergenic cross‐reactivity between storage mites and Dermatophagoides pteronyssinus

The allergenic cross‐reactivity between storage mites (Lepidoglyphus destructor, Glycyphagus domesticus, Tyrophagus putrescentiae and Acarus siro) and the house dust mite Dermatophagoides pteronyssinus was studied with the radio‐allergo‐sorbent test (RAST) and the RAST inhibition technique. RAST‐positive sera were obtained from fifty‐three farmers who were positive to at least one of the four storage mites. Sera from twelve of these farmers, eight of whom were also positive to D. pteronyssinus, were investigated with the RAST inhibition technique. No significant correlations were found between IgE‐antibody levels to any single storage mite and D. pteronyssinus. There was no correlation between the RAST results to A. siro and G. domesticus, whereas a significant correlation was found between L. destructor, G. domesticus and T. putrescentiae. The RAST inhibition studies confirmed the greater allergenic similarity between L. destructor, G, domesticus and T. putrescentiae than between A. siro and the other three storage mites. The results of our studies support the hypothesis that each of the storage mites and D. pteronyssinus possess their own unique allergen or allergens. Furthermore, L. destructor, G. domesticus and T. putrescentiae seem to be allergenically more closely related to each other than to A. siro.

Formation of Disulfide Bonds and Homodimers of the Major Cat Allergen Fel d 1 Equivalent to the Natural Allergen by Expression in Escherichia coli

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

Allergenic characterization of Acarus siro and Tyrophagus putrescentiae and their crossreactivity with Lepidoglyphus destructor and Dermatophagoides pteronyssinus

Allergenic components in extracts of Acarus siro and Tyrophagus putrescentiae were identified by sodium dodecyl sulphate‐polyacrylamide (SDS‐PAGE) and immuno‐blotting. Five and four allergenic components, respectively, were visualized with sera from farmers sensitized to storage mites. The highest frequency of IgE‐binding was to a 15kDa component of A. siro (7/9 sera) and a 16kDa component of T. putrescentiae (23/29 sera). The allergenic crossreactivity of A. siro and T. putrescentiae with Lepidoglyphus destructor and Dermatophagoides pteronyssinus was studied with an immunoblotting inhibition technique. Crossreactivity between D. pteronyssinus on the one hand and A. siro and T. putrescentiae on the other, was shown, as the IgE reactivity to a 25 kDa component of D. ptertonyssinus was inhibited to the same degree by extracts of A. siro, T. putrescentiae and D. pteronyssinus. However, D. pteronyssinus was a poor inhibitor of the allergenic components of A. siro and T. putrescentiae. Strong cross‐reactivity was also shown between L. destructor and the allergenic components of A. siro and T. putrescentiae, while the latter mite species only to a very low degree inhibited the allergenic components of L. destructor.

Immunoblot multi‐allergen inhibition studies of allergenic cross‐reactivity of the dust mites Lepidoglyphus destructor and Dermatophagoides pteronyssinus

The allergenic similarity of the pyroglyphid mite D. pteronyssinus and the glycyphagid mite L. destructor was investigated with a new immunoblotting inhibition technique allowing simultaneous comparison of several allergens. Extracts of D. pteronyssinus and L. destructor were separated by SDS‐PAGE and electroblotted to nitrocellulose (NC). A serum pool containing IgE specific to the major allergens in both mites was mixed with serially diluted extracts of D. pteronyssinus and L. destructor and incubated with the mite allergens of NC. The inhibition of the IgE binding to NC was evaluated by densitometric scanning and percentage inhibition was calculated. The IgE antibodies to the 25‐kD component in D. pteronyssinus, were inhibited to the same degree by extracts of D. pteronyssinus and L. destructor. Another major allergen component in D. pteronyssinus (16 kD) was also inhibited by L. destructor extract but to a lesser degree: 400 times more of the heterologous than of the homologous extract was needed for 50% inhibition. To produce 50% of heterologous inhibition of the two major allergen components at 15 and 53 kD of L. destructor, 2000 and 10000 times more, respectively, of D. pteronyssinus than of L. destructor extract were needed. Two minor allergen components of L. destructor showed some cross‐reactivity with D. pteronyssinus. However, L. destructor was a stronger inhibitor of D. pteronyssinus than vice versa, probably because the sera were obtained from persons more sensitized to L. destructor than to D. pteronyssinus.

Predominance of mite allergy over allergy to pollens and animal danders in a farming population

The prevalence of IgE‐mediated hypersensitivity to respiratory allergens, including Lepidoglyphus destructor (a storage mite) and Dermatophagoides pteronyssinus, was studied in a rural population of Gotland, a Swedish island in the Baltic Sea. A sample of 440 farmers underwent examination, comprising skin‐prick tests (animal danders, pollens, moulds, house dust mite and wheat) and blood sampling for radio‐allergosorbent tests (RAST) (birch, Timothy grass, dog, D. pteronyssinus, Cladosporium and L. destructor). The highest prevalence of positive skin‐prick tests was noted for D. pteronyssinus (6.0%). Low prevalences of positive skin‐prick tests, 0.7‐2.7%, were found for pollens, animal danders and moulds. Among the tested allergens, D. pteronyssinus and L. destructor yielded the highest prevalences of positive RAST results, namely 5.2 and 6.8%, respectively. Most farmers with allergy to pollens and animal danders had symptoms both from the upper and lower airways and from the eyes. Among farmers with both asthma and rhinoconjunctivitis, the prevalence of positive RAST results for L. destructor and D. pteronyssinus were similar. The present study clearly demonstrates that mites are by far the most important allergens in the farming population on Gotland, whereas otherwise common allergens such as pollen and animal danders are clearly less significant.