Psoriasin, a calcium-binding protein with chemotactic properties is present in the third trimester amniotic fluid.

Psoriasin is a small calcium-binding protein first found in psoriatic lesions and also up-regulated in other inflammatory skin diseases and cancer tissues. Psoriasin is also present in the fetal epithelial cells. Its biological function is unclear, but there is both in vitro and in vivo evidence for its chemokine-like activity. The aim of the present study was to find whether psoriasin could be found in the amniotic fluid and thus could have long-range immunobiological effects. Two recombinant psoriasins were prepared, one in Escherichia coli, the other one in Pichia pastoris. The former was used to produce a rabbit antiserum against psoriasin. Fractionation of full-term amniotic fluids with polyacrylamide gel electrophoresis (PAGE) and gel filtration associated with immunodetection with the antiserum were used to identify a protein compatible with the size of psoriasin. The identity of psoriasin was further verified by mass spectrometric analysis. Expression of psoriasin in cells of the amniotic membranes was detected with nested RT-PCR. Because of its chemokine-like activity, psoriasin present in the amniotic fluid might have consequential immunobiological effects during the fetal development.

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. OBJECTIVE: To produce recombinant dog allergens Can f 1 and Can f 2 in Pichia pastoris yeast and to assess their suitability for the diagnosis of dog allergy. METHODS: Clinically diagnosed dog-allergic patients' and healthy non-atopic dog owners' reactivities against recombinant Can f 1 and Can f 2 and commercial dog epithelial extract were studied by a panel of methods including skin prick test (SPT), ELISA and IgE immunoblotting. RESULTS: Recombinant Can f 1 and Can f 2 were found immunologically functional: they bound dog-allergic patients' IgE in immunoblotting and inhibited specifically the binding of IgE to their natural counterparts in the dog allergen extract. Moreover, patients' IgE reactivity in immunoblotting to natural Can f 1 and their SPT with the recombinant allergen were perfectly concordant (phi coefficient 1.0, P<0.001). The concordance was slightly lower with recombinant Can f 2 (phi coefficient 0.92, P<0.001). A lower number of dog-allergic patients, 52%, reacted against Can f 1 than previously reported. About one-third of the patients reacted to Can f 2. In immunoblotting, the highest prevalence of reactivity, 60%, was directed to an 18 kDa component. Aminoterminal sequencing showed this to be a previously unidentified allergenic protein. CONCLUSIONS: The recombinant allergens can be used reliably to identify Can f 1 and Can f 2-sensitized individuals. However, the two allergens are insufficient as reagents for diagnosing dog allergy.

Probing the mechanisms of low immunogenicity of a lipocalin allergen, Bos d 2, in a mouse model.

BACKGROUND: Bos d 2, a major bovine allergen of the lipocalin family, stimulates very weakly cow dust-asthmatic subjects' peripheral blood mononuclear cells and the spleen cells of several inbred mouse strains immunized with the allergen. OBJECTIVE: To identify the immune mechanisms accounting for the weak stimulatory capacity of Bos d 2. METHODS: The spleen cell responses of BALB/c mice immunized with the allergen and with hen egg lysozyme and tetanus toxoid as control antigens were examined using several in vitro methods. RESULTS: Analysis of the numbers of spleen cells in the antigen-stimulated in vitro cultures with the vital dye 7-amino-actinomycin D showed that Bos d 2 induced a smaller expansion of cells in comparison with the control antigens. Increased cell death in vitro did not account for the weak response against Bos d 2. The number of spleen cells reacting against Bos d 2 also proved to be the lowest when they were analysed by labelling the stimulated cells with 5-6-carboxyfluorescein diacetate succinimidyl ester or by enumerating cytokine-secreting cells by ELISPOT. Eliminating CD8+ cells in the in vitro culture did not enhance the response against Bos d 2. Bos d 2 was also the weakest of the antigens to stimulate the production of soluble cytokines. Adding IL-2, IL-4 or antibody against TGF-beta in the antigen-stimulated spleen cell cultures enhanced the proliferative responses against all the antigens, whereas adding IL-12 or antibody against IL-4 or IL-10 did not enhance the responses. CONCLUSION: The results exclude several mechanisms of peripheral tolerance as an explanation for the poor immune response against Bos d 2, and suggest that the allergen is recognized by a low number of specific T cells. The weak immunogenicity of Bos d 2 may be related to its allergenicity.

Threshold levels of purified natural Bos d 2 for inducing bronchial airway response in asthmatic patients.

BACKGROUND: Provocation tests are invaluable in establishing threshold levels and a causal relationship between atopic asthma and a certain allergen source, especially in relation to work-associated exposure. Purified major allergens open possibilities for a more accurate assessment of sensitization. OBJECTIVE: To determine the threshold dose of purified major bovine dander allergen Bos d 2 in bronchial provocation in comparison with the standard allergen and a set of other parameters of allergy. METHOD: Nine consecutive patients referred to hospital for confirming the bovine origin of their occupational asthma were subjected to bronchial provocation tests with purified natural Bos d 2 and a standard bovine dander allergen. Additional tests included bronchial histamine challenge, measurements of total IgE, specific IgE antibody determinations and skin prick tests (SPT) with both allergens. RESULTS: In the provocation tests with Bos d 2, a 15% decrease in the forced expiratory volume in 1 s (FEV1) and/or peak expiratory flow (PEF) values in eight out of nine patients confirmed the predominant role of Bos d 2 in the sensitization. The threshold dose of Bos d 2 varied from 0.1 microg to > 100 microg (median +/- median absolute deviation = 4.5 +/- 3.9 microg). A positive SPT was induced by a median dose of 13.9 +/- 9.8 microg of Bos d 2. Bronchial response to histamine and IgE antibodies against Bos d 2 showed the highest correlations to the provocations results. CONCLUSIONS: The efficacy of Bos d 2 in bronchial provocation in patients with occupational cattle-associated asthma was confirmed and the range of the threshold level was determined. There were individual variations, but the response in provocation remains the reference method for identification of the cause of occupational atopic asthma. SPT and the measurement of specific IgE antibodies, preferably with purified or recombinant major allergens, increase the accuracy of the diagnosis.

Two new variants of the lipocalin allergen Bos d 2.

Allergens from various sources have been shown to comprise several isoforms. In the present study, a series of chromatographic steps was carried out to separate the lipocalin allergen Bos d 2 isoforms present in cow dander. Subsequent HPLC-MS-MS analyses revealed two new Bos d 2 variants. In one of the proteins, tyrosine (Y83) was substituted by aspartic acid, and in the other protein valine (V102) was replaced by alanine. We propose the three Bos d 2 variants be named as Bos d 2.0101 (previously sequenced Bos d 2), Bos d 2.0102 and Bos d 2.0103. Our results suggest that molecular polymorphism is a common property among lipocalin allergens. Since allergen isoforms may show variation in their IgE binding and/or T-cell reactivity, all of the many allergen forms should be taken into account when planning preparations for immunotherapy.

Search for the determinants of allergenicity in proteins of the lipocalin family.

Three different lines of analysis have been applied to approach the problem of the allergenicity of certain proteins: biological functions, molecular structures and immunological properties. It is immediately obvious that these three are interdependent. The lipocalin family of proteins includes a significant number of allergens. A considerable amount of data is already available of lipocalins and some insights about allergenic determinants can now be presented. However, more information on the molecular structures and immunological parameters of lipocalin allergens is required.

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 cow's 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.

Mutant derivatives of the main respiratory allergen of cow are less allergenic than the intact molecule.

BACKGROUND: Allergen immunotherapy offers an alternative for drug treatment in the management of allergic diseases. Because immunotherapy often induces side-effects, less allergenic preparations would be beneficial. OBJECTIVE: The purpose of this study was to examine whether the allergenicity of a cow-derived lipocalin allergen, Bos d 2, could be diminished by substituting or deleting carboxy-terminal amino acids including the cysteine which forms a disulphide bond with a cysteine inside the molecule. METHODS: Four recombinant mutants of Bos d 2 were created by substituting or deleting the four most carboxy-terminal amino acids. The immunological characteristics of the mutant preparations were compared with the unmodified rBos d 2 by Western blotting, ELISA inhibition, skin prick tests, and the proliferative responses of allergen-specific T-cell clones. RESULTS: In Western blot, one of the two monoclonal antibodies showed reduced binding to the preparations without the terminal cysteine. In contrast, the other monoclonal antibody, human IgE and rabbit immune serum bound equally well to all the preparations. ELISA inhibition analyses revealed, however, that the preparations without the terminal cysteine bound antibody less efficiently. They were needed 15-38 times more than the unmodified rBos d 2 to cause the same level of inhibition. Surprisingly, one of the mutants with the terminal cysteine but a mutated adjacent amino acid turned out to be the weakest in inducing skin reactivity. All the preparations stimulated well allergen-specific T-cell clones. CONCLUSIONS: The results show that the allergenicity of a lipocalin allergen, Bos d 2, can be diminished by modifying the carboxy-terminal end of the molecule. Modifications in the area which encompasses a disulphide bond impaired the antibody binding without affecting the T-cell stimulatory capacity. It was also shown that in vivo tests are necessary for determining the allergenicity of a modified allergen.

Mycobacterium malmoense-specific nested PCR based on a conserved sequence detected in random amplified polymorphic DNA fingerprints.

Mycobacterium malmoense is an opportunistic human pathogen of increasing clinical importance. Since it is difficult to detect and identify the organism by conventional techniques, it was decided to seek a nucleic acid amplification method specific for M. malmoense. The method was based on detection of a conserved band in random amplified polymorphic DNA (RAPD) fingerprints of 45 M. malmoense strains. This band was a 1,046-bp product which was proven to be M. malmoense specific in dot blot hybridization analysis with a panel of mycobacterial strains belonging to 39 other species. The fragment was sequenced, and oligonucleotide primers were synthesized to evaluate the specificity of the PCR. Two primer pairs were found to be specific and sensitive in the nested PCR that was developed. All 49 M. malmoense strains analyzed produced a PCR product of the expected size. In contrast, no strains belonging to the other mycobacterial species tested produced amplicons with these primers under specified reaction conditions. The results of the electrophoresis were confirmed by the hybridization with the M. malmoense-specific oligonucleotide probe. This method could be applied to the analysis of clinical or environmental samples, permitting the rapid detection of M. malmoense.

T cell epitopes of a lipocalin allergen colocalize with the conserved regions of the molecule.

In this study we characterized the human T cell-reactive sites of the major cow dander allergen, Bos d 2, a member of the lipocalin protein family. We showed that Bos d 2 contains only a limited number of epitopes. This is in contrast to many other allergens, which usually contain multiple T cell epitopes throughout the molecule. The epitopes of Bos d 2 were primarily concentrated in the conserved regions of the molecule. One of the epitopes was recognized by all the cow-asthmatic individuals regardless of their HLA phenotype. Computer-predicted T cell epitopes on Bos d 2, other lipocalin allergens, and human endogenous lipocalins were situated in similar locations on these molecules and corresponded to experimentally identified epitopes on Bos d 2. The results suggest that human endogenous lipocalins could be involved in the modulation of immune responses against exogenous lipocalin allergens. In addition, our findings are likely to facilitate the development of new forms of immunotherapy against allergies induced by the important group of lipocalin allergens.

Probing the molecular basis of allergy. three-dimensional structure of the bovine lipocalin allergen Bos d 2.

The three-dimensional structure of the major bovine allergen Bos d 2 has been determined by using x-ray diffraction at 1.8-A resolution. Structurally Bos d 2 is a member of the lipocalin family comprising proteins with transport functions. There is a flat small cavity inside the Bos d 2 protein core suitable for ligand binding, and it is possible that Glu115 and Asn37 inside the core are able to make hydrogen bonds with the ligand. Many allergens from different animals belong to the lipocalin family. The amino acid residue similarities between these lipocalins indicate putative regions for IgE binding. Comparison with the available allergen structures from other sources suggests that these allergens are roughly the same size and that their shape is more spherical than elliptical.

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.

A bovine dander allergen, comparative modeling, and similarities and differences in folding with related proteins.

The most important allergenic protein in cow dander and urine is Bos d 2. It is proposed to belong to the family of lipocalins, which are proteins capable of binding small hydrophobic molecules. The allergenic properties of Bos d 2 indicate an interaction between the accessible regions of the native protein and IgE. In this work, a three-dimensional model was created for Bos d 2 by comparative modeling, and features characteristic of outlier lipocalins were observed. The protruding regions of the surface were characterized and used in predicting the possible B-cell epitopes. There is a pocket inside the core and its size is appropriate for small molecules. The model shows a hydrophilic amino acid side chain of glutamic acid 115 on the inner surface of the hole and a phenylalanine as the "gatekeeper" instead of tyrosine, which is common in experimentally modeled lipocalins.

Molecular and crystal properties of Bos d 2, an allergenic protein of the lipocalin family.

The relationship between the molecular structure of allergenic proteins and the allergenic determinants is one of the central issues in allergology. We report here that the natural preparation of Bos d 2, a mammalian lipocalin allergen, comprises three molecular variant proteins of 17,829, 17,781, and 17,800 Da. When cDNA of Bos d 2 (Genome Sequence Data Base No. L42867) was recloned and expressed in Pichia pastoris, two proteins were produced. One of the proteins (17,831 Da) and the proteins in the natural preparation had pyroglutamate as the N-terminal residue; in the other (17,849 Da) the N-terminal residue was glutamine. Recombinant Bos d 2 protein was crystallized and the native data set was collected at 1.8 A resolution.

Tissue localization of bovine dander allergen Bos d 2.

BACKGROUND: Domestic mammals are important sources of indoor allergens. However, the origin at the tissue level and the biologic function of mammalian allergens are largely unknown. OBJECTIVE: The aim of this study was to localize the source of the major bovine dander allergen, Bos d 2, in bovine tissues. METHODS: Samples from several organs were tested for the presence of mRNA encoding Bos d 2 and Bos d 2 protein by using the reverse transcriptase polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: Skin proved to be the only tissue where mRNA encoding Bos d 2 was detected. This observation was confirmed by immunohistochemistry with a monoclonal anti-Bos d 2 antibody as the primary antibody. In the skin sections, Bos d 2 was found in the secretory cells of apocrine sweat glands and the basement membranes of the epithelium and hair follicles. Bos d 2 belongs to the family of lipocalins comprising a number of pheromone carrier proteins that are present, for example, in the secretions of the apocrine sweat glands. CONCLUSION: Together with earlier data, our findings suggest that Bos d 2 is produced in sweat glands and transported to the skin surface as a carrier of the pheromone ligand. Because dander allergens of a number of mammalian species are lipocalins, the common biologic function of being pheromone carriers seems to be a common feature of an important group of aeroallergens.

Humoral responses to bovine dust in dairy farmers with allergic rhinitis.

During the indoor cattle feeding season, dairy farmers are continuously exposed to allergenic bovine-derived materials for a long time every year. Limited information is available about the dynamics of exposed people's antibody responses under the influence of this kind of exposure. In this study, the level of antibodies to bovine epithelial antigen (BEA) was found to reflect the level of clinical allergy. On the other hand, the level of immunoglobulin (Ig)E, IgA and IgG antibodies to bovine urinary antigen (BUA) was lower in the group of bovine-allergic farmers than among other farmers. It was concluded that antibodies to different allergen extracts reflect different phenomena. Antibodies to BEA seem to be associated more with allergic symptoms, where as anti-BUA antibodies might reflect the level of bovine exposure. The indoor cattle feeding season did not cause any major changes in dairy farmers' humoral responses. The continued allergen exposure did not increase the level of sensitization as determined by antibody responses.

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.

Molecular analysis of allergenic proteins in bovine dander.

An analytic procedure was established to characterize bovine dander proteins with allergenic properties. The proteins from dander extract were separated by size-exclusion gel filtration, and the fractions were studied with SDS-PAGE followed by immunoblotting. An 11-kDa allergen was found in the same gel filtration fractions as 20- and 22-kDa allergens, and this suggests that the 11-kDa allergen is a dimer in its native form. Our method also detected two separate 22-kDa allergens. The primary structure of the major bovine dander allergen (BDA20) was also studied. A protein sequencer was used to determine the amino acid sequences of enzymatically cleaved peptides. The homology searches revealed that BDA20 is not a previously known bovine protein.