Tuomas Virtanen

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.

Recombinant allergen fragments as candidate preparations for allergen immunotherapy

BACKGROUND Lately, renewed interest has arisen in the new forms of allergen immunotherapy because they may offer alternatives for drug treatment. OBJECTIVE The purpose of this study was to develop a well-characterized preparation of the main respiratory cow dander allergen, Bos d 2, with attenuated allergenic activity. METHODS The immunologic characteristics of Bos d 2 preparations were studied by indirect IgE ELISA, ELISA inhibition, Western blotting, histamine release, skin prick tests, and the proliferation tests of allergen-specific T-cell clones. RESULTS The complete recombinant Bos d 2 was observed to bind effectively, IgE of cow-allergic patients in indirect ELISA. In other experiments, the IgE-binding capacity of recombinant Bos d 2 proved to be lower compared with native Bos d 2. When the two overlapping recombinant fragments of Bos d 2 (corresponding amino acids 1-131 and 81-172, respectively) covering the whole molecule were compared with the complete recombinant Bos d 2 with several methods, only a low level of residual reactivity was observed. For example, recombinant fragments could not bind antibody at all in ELISA inhibition tests retaining, however, some reactivity in skin prick tests. In contrast, the fragments were able to stimulate vigorously Bos d 2-specific T-cell clones. CONCLUSION The approach we have taken may offer a simple and reproducible way to produce hypoallergenic preparations for immunotherapy, circumventing simultaneously some of the problems of other experimental methods such as individual T-cell epitope recognition in peptide-based immunotherapy.

Complementary DNA cloning of the predominant allergen of bovine dander: A new member in the lipocalin family

BACKGROUND A number of allergenic proteins in animal danders have been characterized at the molecular level, but little is known of their biologic functions. We have found that the prevalence of IgE antibodies among patients with cattle-associated asthma is highest against a dander protein referred to as BDA20. OBJECTIVE The study was performed to characterize the molecular structure of BDA20,* the predominant allergen in bovine dander. METHODS Clones encoding allergens were identified and isolated from a complementary DNA library by immunoblotting and DNA hybridization and sequenced. Recombinant proteins were produced in Escherichia coli. Immunoreactivity of the recombinant proteins and amino acid sequences of peptides obtained from native BDA20 after Lys-C cleavage were used to identify clones coding for BDA20. RESULTS In this article we report the cDNA and amino acid sequences of BDA20. Homology comparisons showed that BDA20 belongs to the family of lipocalins. CONCLUSIONS The results link a dander allergen to a group of functionally important proteins. Lipocalins are present in various body fluids and secretions of several animal species in which they function as carriers of small hydrophobic molecules, such as retinoids and pheromones. If allergenicity proves to be a property shared by lipocalins, our results will have considerable implications for allergen research.

Important Animal Allergens Are Lipocalin Proteins: Why Are They Allergenic?

Major respiratory allergens of dogs, mice, rats, horses and cows belong to the lipocalin group of proteins. The sequence identity of lipocalins is often less than 20%, but they contain between one and three structurally conserved regions and their three–dimensional structures are similar. Lipocalins share common biological functions, predominantly related to the transport of small hydrophobic molecules, such as vitamins and pheromones. Immune reactivity to lipocalin allergens is not well known. In Bos d 5, the IgE–binding epitopes are spread along the molecule, whereas in Bos d 2, the C terminus appears to contain the human B cell epitopes. Bos d 5 contains several murine T cell epitopes. No information is available on human T cell epitopes. The maximal number of epitopes an allergic patient’s T cells could recognize in Bos d 2 was five. Three of the epitopes were colocalized in the structurally conserved regions of lipocalins. Interestingly, one of the epitopes was recognized by the T cells of all patients and the computer predictions suggested that there would be an epitope in the corresponding parts of human endogenous lipocalins. The proliferative responses of peripheral blood mononuclear cells of Bos d 2–allergic subjects to Bos d 2 were weak. The T cell response was Th2–dominated. To explain these observations, we have proposed that the allergenicity of lipocalins may be a consequence of molecular mimicry between lipocalin allergens and endogenous lipocalins at the T cell level.

Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy

Background The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts.

Lipocalins as allergens

The term allergy refers to clinical conditions caused by an inappropriate immune response to innocuous proteins in genetically predisposed persons. Allergens of animal origin are responsible for a significant proportion of allergies. In recent years, it has become evident that practically all respiratory animal allergens characterized at the molecular level belong to the lipocalin family of proteins. The current list comprises the major allergens of horse, cow, dog, mouse, rat and cockroach as well as beta-lactoglobulin of cows milk. While the molecular structure of all these allergens is known, far less information is available regarding their immunological characteristics. Knowing the way the immune system recognizes these allergens and reacts to them might, however, be the key for discovering the common denominator of the allergenicity of lipocalins. The human body contains numerous endogenous lipocalins, and the immune system has to adapt to their presence. We have proposed that under these conditions the immune response against the lipocalin allergens which are structurally related to endogenous lipocalins might be the pathway to allergy in genetically predisposed persons. The same might well apply also to other allergens with homologous endogenous counterparts.

Animal‐derived lipocalin allergens exhibit immunoglobulin E cross‐reactivity

Background Although knowledge of the IgE cross‐reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross‐reactivity of animal allergens is poorly known.

IgG and IgE antibody responses to cow dander and urine in farmers with cow‐induced asthma

Cow‐asthmatic farmers ‘and negative control subjects’ IgG and IgE antibody responses to bovine epithelial antigen (BEA) and urinary antigen (BUA) were studied by enzyme‐linked immunosorbent assay (ELISA) and Western blotting. The anti‐BEA IgE responses of 10 highly reactive sera were also studied by crossed radioimmunoelectrophoresis (CRIE). The relative amount of allergens common to both BEA and BUA was measured by IgE ELISA inhibition and found to be 3%. In immunoblotting the IgG reactivity of the asthmatic farmers to BEA and BUA declined along their anti‐BEA IgE ELISA titres. Control subjects had IgG antibodies mainly to high molecular weight components (50–70 kD) but lacked detectable IgE responses. The IgE reactivity of the asthmatic farmers was directed to only a few components. A total of two main allergens were found in cow dander (20 and 22 kD) and one in cow urine (20 kD). The 20 kD component was shown to be the most important allergen in cow antigen extracts. In CRIE, seven reactive arcs were detected. Arcs 1, 2 and 5 were detected by all 10 sera and are 3 by six and arc 7 by seven sera.

Allergy to lipocalins: a consequence of misguided T-cell recognition of self and nonself?

Abstract The molecular mimicry between lipocalin allergens and endogenous lipocalins at the T-cell level may explain why the immune response against lipocalins is Th2-dominated and results in allergy. This view is supported by recent studies of autoimmune and parasitic diseases and peptide analogues.

Mammalian lipocalin allergens – insights into their enigmatic allergenicity

Most of the important mammal‐derived respiratory allergens, as well as a milk allergen and a few insect allergens, belong to the lipocalin protein family. As mammalian lipocalin allergens are found in dander, saliva and urine, they disperse effectively and are widely present in the indoor environments. Initially, lipocalins were characterized as transport proteins for small, principally hydrophobic molecules, but now they are known to be involved in many other biological functions. Although the amino acid identity between lipocalins is generally at the level of 20–30%, it can be considerably higher. Lipocalin allergens do not exhibit any known physicochemical, functional or structural property that would account for their allergenicity, that is, the capacity to induce T‐helper type 2 immunity against them. A distinctive feature of mammalian lipocalin allergens is their poor capacity to stimulate the cellular arm of the human or murine immune system. Nevertheless, they induce IgE production in a large proportion of atopic individuals exposed to the allergen source. The poor capacity of mammalian lipocalin allergens to stimulate the cellular immune system does not appear to result from the function of regulatory T cells. Instead, the T cell epitopes of mammalian lipocalin allergens are few and those examined have proved to be suboptimal. Moreover, the frequency of mammalian lipocalin allergen‐specific CD4+ T cells is very low in the peripheral blood. Importantly, recent research suggests that the lipocalin allergen‐specific T cell repertoires differ considerably between allergic and healthy subjects. These observations are compatible with our hypothesis that the way CD4+ T‐helper cells recognize the epitopes of mammalian lipocalin allergens may be implicated in their allergenicity. Indeed, as several lipocalins exhibit homologies of 40–60% over species, mammalian lipocalin allergens may be immunologically at the borderline of self and non‐self, which would not allow a strong anti‐allergenic immune response against them.