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Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease in which the insulin-producing β cells within the pancreas are destroyed. Identification of target Ags and epitopes of the β cell–reactive T cells is important both for understanding T1D pathogenesis and for the rational development of Ag-specific immunotherapies for the disease. Several studies suggest that proinsulin is an early and integral target autoantigen in T1D. However, proinsulin epitopes recognized by human CD4+ T cells have not been comprehensively characterized. Using a dye dilution–based T cell cloning method, we generated and characterized 24 unique proinsulin-specific CD4+ T cell clones from the peripheral blood of 17 individuals who carry the high-risk DR3-DQ2 and/or DR4-DQ8 HLA class II haplotypes. Some of the clones recognized previously reported DR4-restricted epitopes within the C-peptide (C25-35) or A-chain (A1-15) of proinsulin. However, we also characterized DR3-restricted epitopes within both the B-chain (B16-27 and B22-C3) and C-peptide (C25-35). Moreover, we identified DQ2-restricted epitopes within the B-chain and several DQ2- or DQ8-restricted epitopes within the C-terminal region of C-peptide that partially overlap with previously reported DQ-restricted epitopes. Two of the DQ2-restricted epitopes, B18-26 and C22-33, were shown to be naturally processed from whole human proinsulin. Finally, we observed a higher frequency of CDR3 sequences matching the TCR sequences of the proinsulin-specific T cell clones in pancreatic lymph node samples compared with spleen samples. In conclusion, we confirmed several previously reported epitopes but also identified novel (to our knowledge) epitopes within proinsulin, which are presented by HLA class II molecules associated with T1D risk.

Allergen-specific type 2 CD4We sought to characterize at the single-cell level the ex vivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4Dog allergen-specific memory CD4Dog allergen-specific T-cell responses in allergic subjects were dominantly of TOur study demonstrates heterogeneity within allergen-specific T

Four out of six officially recognized dog allergens are members of the lipocalin protein family. So far, a three-dimensional structure has been determined for only one dog allergen, Can f 2, which is a lipocalin protein. We present here the crystal structure of a second lipocalin allergen from dog, a variant of Can f 4. Moreover, we have compared and analyzed the structures of these two weakly homologous (amino acid identity 21%) dog allergens. The size and the amino acid composition of the ligand-binding pocket indicate that Can f 4 is capable of binding only relatively small hydrophobic molecules which are different from those that Can f 2 is able to bind. The crystal structure of Can f 4 contained both monomeric and dimeric forms of the allergen, suggesting that Can f 4 is able to form transient (weak) dimers. The existence of transient dimers in solution was confirmed by use of native mass spectrometry. The dimeric structure of Can f 4 is formed when the ends of four β-strands are packed against the same strands from the second monomer. The residues in the interface are mainly hydrophobic and the formation of the dimer is similar to the major horse allergen Equ c 1. Interestingly, the crystal structure of dog Can f 2 has been reported to show a different type of dimer formation. The capability of these allergens to form dimers may be important for the development of immediate allergic reaction (mast cell activation) because oligomeric allergens can effectively present multivalent epitopes.

Most mammal-derived respiratory allergens belong to the lipocalin family of proteins. Determinants of their allergenic capacity are still unknown. Innate immune cells, in particular dendritic cells, have been shown to be involved in the allergenicity of some proteins. As recognition by dendritic cells is one of the few plausible mechanisms for the allergenicity of proteins, we wanted to investigate their role in the allergenicity of lipocalin allergens. Therefore, we first incubated human monocyte-derived dendritic cells with immunologically functional recombinant allergens mouse Mus m 1, dog Can f 1 and 2, cow Bos d 2, horse Equ c 1 and natural Bos d 2. Then, the surface marker expression and cytokine production of dendritic cells and their capacity to promote T cell proliferation and Th2 immune deviation in naïve CD4(+) T cells were examined in vitro. We found that near to endotoxin-free lipocalin allergens had no effect on the activation, allostimulatory capacity or cytokine production of dendritic cells. The dendritic cells could not induce immune deviation in naïve CD4(+) T cells. In contrast, lipopolysaccharide activated the dendritic cells efficiently. However, lipocalin allergens were not able to modify the lipopolysaccharide-induced responses. We conclude that an important group of mammal-derived respiratory allergens, lipocalins, appear not to be able to activate dendritic cells, a major component involved in the allergenicity of some proteins. It is conceivable that this incapacity of lipocalin allergens to arouse innate immunity may be associated with their poor capacity to induce a strong T cell response, verified in several studies.

Neonatal treatment of rat pups with clomipramine (CLI) has been shown to cause long-lasting and persistent depression-related behaviors and changes in sleep architecture and in brain-derived neurotrophic factor (BDNF) signaling in adult animals, producing an animal model of depression. However, the molecular mechanisms which mediate these effects of early-life CLI treatment on adult animals remain largely unknown. In order to characterize these further, we investigated in neonatally CLI-treated rats the sleep architecture as well as the extracellular and cellular levels of sleep regulators (nitric oxide, adenosine) and BDNF, respectively, in the basal forebrain (BF), i.e. the brain area which is implicated in sleep and depression. We found that CLI-treated rats exhibited a disturbed sleep architecture (REM sleep fragmentation was increased and NREM periods preceding REM were shorter) and reduced levels of BDNF and adenosine in the BF, whereas the levels of nitric oxide were elevated. Next, we examined sleep deprivation (SD)-induced homeostatic responses on sleep regulation and brain BDNF levels in CLI-treated rats. Compared to control rats, 3h of SD induced a smaller increase in the amount of NREM sleep during sleep recovery. At the molecular level, the normal homeostatic response was dissociated: the rise in the adenosine level was not accompanied by a rise in the nitric oxide concentration. Moreover, while BF BDNF levels decreased during SD in control rats, such a decline was not observed in CLI rats. Taken together, neonatal CLI treatment produces long-lasting functional changes in the sleep architecture and sleep regulation in adult rats, accompanied by dysregulated BDNF signaling in the BF.

The wake-promoting basal forebrain (BF) is critically involved in sustaining cortical arousal. In the present study, we investigated how aging affects the capacity of the BF to cope with continuous activation during prolonged waking. Increased neuronal activity induces lactate release in the activated brain area, and BF stimulation increases cortical arousal. We usedin vivomicrodialysis to measure lactate levels in the BF, and electroencephalography (EEG) to measure cortical arousal, during 3 h sleep deprivation (SD) in three age groups of rats. Lactate increased during SD in young but not in aged (middle-aged and old) rats. The increase in high-frequency (HF) EEG theta power (7–9 Hz), a marker of cortical arousal and active waking, was attenuated in the aged. Furthermore, a positive correlation between BF lactate release and HF EEG theta increase was found in young but not in aged rats. We hypothesized that these age-related attenuations result from reduced capacity of the BF to respond to increased neuronal activation. This was tested by stimulating the BF with glutamate receptor agonist NMDA. Whereas BF stimulation increased waking in young and old rats, lactate increase and the HF EEG theta increase were attenuated in the old. Also, the homeostatic increase in sleep intensity after SD was attenuated in aged rats. Our results suggest that an age-related attenuation in BF function reduces cortical arousal during prolonged waking. As the quality of waking is important in regulating the subsequent sleep, reduced cortical arousal during SD may contribute to the age-related reduction in sleep intensity.

Summary Lipocalin allergens, which contain most of the important animal-derived respiratory sensitizers, induce T helper type 2 (Th2) deviation, but the reasons for this are not clear. To explore the prospects for peptide-based allergen immunotherapy and to elucidate the characteristics of the immunodominant epitope of Bos d 2, BALB/c mice were immunized with a peptide containing the epitope, peptides containing its analogues, peptides from the corresponding regions of other lipocalin proteins, and peptides with a homologous sequence. We observed that murine spleen cells recognized the immunodominant epitope of Bos d 2, p127–142, in almost the same way as human Bos d 2-specific T cells did. Enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) analyses showed that p127–142 and a corresponding peptide from horse Equ c 1 induced a Th2-deviated cellular response, whereas a homologous bacterial peptide from Spiroplasma citri induced a Th0-type response. Interestingly, the spleen cell response to the bacterial peptide and p127–142 was cross-reactive, that is, able to induce reciprocally the proliferation and cytokine production of primed spleen cells in vitro. More importantly, the peptides were able to skew the phenotype of T cells primed with the other peptide. Our results suggest that modified peptides can be useful in allergen immunotherapy.

Several studies have claimed stress to be a major reason for poor public health in Russia and referred to significant social changes as a reason for the high level of perceived stress among Russians. This article aims to examine how stress and its relation to health are interpreted in the context of everyday life in Russian men's and women's interview talk with a focus on descriptions of recent social changes. The research material consists of 29 thematic interviews of men and women from St. Petersburg aged 15-81. In the analysis of contextual constructions of stress, we found that stress was used not only within a context of an individual's own life as an expression of a strained psycho-physiological state but also denoted larger societal processes and changes. In addition to individual experiences, the whole of Russian society was described as suffering from stress. Throughout the material, most interviewees, whilst outspokenly blaming stress for deteriorating physical health, met difficulties in making concrete these negative influences. Based on analysis, we interpret our interviewees' accounts of stress as a part of the cultural discourse wherein 'stress' serves as a conceptual tool in making interpretations about both the people and their social environment. Stress, as a concept, has emerged in a wide range of different institutional sites, such as the media and public health policy and has become a discursive entity of contemporary social life in Russia. We claim that it has simultaneously become an intermediary concept articulating a shared, cultural experience of the changes in Russian society and their effects on individuals' everyday life and health. Thus, the concept of stress helps people to articulate, make sensible, and cope with the impacts of transition on their individual lives.

Summary 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. Objective The aim of this study was to characterize IgE cross-reactivities between lipocalin proteins, including five animal-derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL). Methods The recombinant proteins were validated by chromatography and mass spectrometry. The IgE-binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross-reactivities between the lipocalin proteins were determined by ELISA inhibition. Results ELISA inhibition revealed IgE cross-reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients of whom six were IgE-positive for Can f 1. Conclusion Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three-dimensional structures. The clinical significance of the findings needs to be elucidated. Low-level IgE cross-reactivity can play a role in regulating immune response to lipocalin allergens.

Background Peptide immunotherapy is a promising alternative for treating allergic diseases. One way to enhance the efficacy of peptide immunotherapy is to use altered peptide ligands (APLs) that contain amino acid substitutions compared with the natural peptide. Objective To evaluate the potential of an APL of the immunodominant epitope of lipocalin allergen Bos d 2 for peptide immunotherapy. Methods Peripheral blood CD4 + T-cell responses of 8 HLA-DR4–positive subjects to the natural ligand of Bos d 2 (p127-142) or to an APL (pN135D) were analyzed by MHC class II tetramer staining after in vitro expansion with the peptides. Long-term T-cell lines (TCLs) were induced with the peptides, and the cytokine production, cross-reactivity, and T-cell receptor Vβ subtype expression of the TCLs were analyzed. Results CD4 + T cells specific for both p127-142 and pN135D were readily detected in peripheral blood after a single in vitro stimulation. Whereas the TCLs induced with p127-142 were T H 2/T H 0-deviated, those induced with pN135D were T H 1/T H 0-deviated and highly cross-reactive with p127-142. Moreover, the pN135D-induced TCLs appeared to use a broader repertoire of T-cell receptor Vβ subtypes than those induced with p127-142. Conclusion An APL of an immunodominant allergen epitope was able to induce a novel T H 1-deviated T-cell population cross-reactive with the natural epitope in vitro . This cell population could have a therapeutic immunomodulatory function in vivo through bystander suppression. Clinical implications These results support the idea that altered peptide ligands may be used to enhance the efficacy of peptide immunotherapy.

We have previously shown that the major dog allergen Can f 1 contains seven T cell epitope regions, none of which was preferentially recognized. To identify the immune characteristics of Can f 1 epitopes and to verify their suitability for peptide-based allergen immunotherapy, short-term T cell lines were generated with epitope-containing peptides from peripheral blood mononuclear cells of Can f 1 skinprick test-positive allergic and healthy control subjects. The lines were examined for their proliferative capacity and cytokine production upon stimulation with the allergen peptide, a homologous peptide from human tear lipocalin (TL) and Can f 1 and TL proteins. Can f 1 peptides induced proliferation of T cells and gave rise to T cell lines with comparable efficiencies. In particular, the T cell lines of allergic subjects induced with p33–48 and p107–122 favoured the production of interferon-γ and interleukin-10, respectively. A greater number of Can f 1-specific T cell lines were generated from allergic than from healthy individuals. Two p107–122-induced Can f 1-specific T cell lines also reacted to a homologous peptide of human TL. Our results suggest that several T cell epitope-containing peptides should be used in combination for specific immunotherapy in Can f 1 allergy.

SummaryBackground Human CD4+ T cell responses to important animal allergens are still insufficiently understood. Objective To comprehensively characterize in vitro and ex vivo the peripheral blood memory CD4+ T cell responses of subjects with and without allergy to the major dog allergen Can f 5, the only known animal allergen in the kallikrein family of proteins. Methods Can f 5-specific memory CD4+ T cell lines (TCLs) were established from the peripheral blood of 12 subjects with and 12 subjects without allergy to Can f 5 and characterized for their functional and phenotypic properties. The results were evaluated with those obtained ex vivo with a novel CD154 enrichment method. The epitopes recognized by the Can f 5-specific TCLs were determined with 72 overlapping 16-mer peptides covering the sequence of the allergen. Results Can f 5-specific TCLs were obtained at about tenfold higher frequency from allergic than from non-allergic subjects. Functionally, the TCLs of allergic subjects displayed a Th2-biased cytokine phenotype and increased T cell receptor avidity, whereas the TCLs of non-allergic subjects displayed a Th1-/Th0-biased cytokine phenotype and lower TCR avidity. The higher frequency and the Th2 phenotype of Can f 5-specific memory CD4+ T cells in allergic subjects were confirmed by the CD154 enrichment method ex vivo. Six distinct T cell epitope regions of Can f 5 were predominantly recognized by the TCLs from allergic subjects. Conclusions and Clinical Relevance Can f 5-specific memory CD4+ T cell responses differ considerably between subjects with and without allergy, as assessed by both in vitro and ex vivo approaches. Peptides containing the dominant T cell epitopes of Can f 5 can be employed for developing peptide-based immunotherapy for dog allergy.

Lipocalins are one of the most important groups of inhalant animal allergens. The analysis of structural features of these proteins is important to get insights into their allergenicity. We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen. The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the β-sheet in dimer formation. According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution. Native mass spectrometry was employed to characterize quantitatively transient dimerization of two lipocalin allergens, Bos d 2 and Bos d 5, in solution.

Peptide-based allergen immunotherapy is a novel alternative for conventional allergen immunotherapy. Here, we have characterized the immunomodulatory potential of heteroclitic peptide analogs of the immunodominant epitope of lipocalin allergen Bos d 2 on specific human T-cell clones. The TCR affinity of Bos d 2-specific T-cell clones for the natural peptide ligand and its heteroclitic analogs was assessed with fluorescent-labeled MHC class II tetramers. The activation and cytokine production of the clones were analyzed upon stimulation with the different ligands. Moreover, the capacity of the heteroclitic analogs to induce hyporesponsiveness and cell death was examined. The T-cell clones F1-9 and K3-2 bound MHC class II tetramers loaded with the heteroclitic peptide analogs of the immunodominant epitope of Bos d 2 with increased affinity. At similar peptide concentrations, stimulation of the clones with the heteroclitic analogs favored increased IFN-gamma/IL-4 and IFN-gamma/IL-5 ratios in comparison with stimulation with the natural peptide ligand. Moreover, the T-cell clones stimulated with the heteroclitic analogs exhibited an increased susceptibility to cell death or hyporesponsiveness upon re-stimulation. Our results suggest that heteroclitic analogs of a T-cell epitope of an allergen may enhance the efficacy of peptide-based allergen immunotherapy.

One prerequisite for developing peptide-based allergen immunotherapy is knowing the T cell epitopes of an allergen. In this study, human T cell reactivity against the major dog allergen Can f 1 was investigated to determine peptides suitable for immunotherapy. Seven T cell epitope regions (A–G) were found in Can f 1 with specific T cell lines and clones. The localization of the epitope regions shows similarities with those of the epitopes found in Bos d 2 and Rat n 1. On average, individuals recognized three epitopes in Can f 1. Our results suggest that seven 16-mer peptides (p15–30, p33–48, p49–64, p73–88, p107–122, p123–138, and p141–156), each from one of the epitope regions, show widespread T cell reactivity in the population studied, and they bind efficiently to seven HLA-DRB1 molecules (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominant in Caucasian populations. Therefore, these peptides are potential candidates for immunotherapy of dog allergy.

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.

Summary 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 (φ coefficient 1.0, P

Summary 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 µg to > 100 µg (median ± median absolute deviation = 4.5 ± 3.9 µg). A positive SPT was induced by a median dose of 13.9 ± 9.8 µg 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.

The immunological characteristics of an important group of animal-derived allergens, lipocalins, are poorly known. To explore the immunology of the lipocalin allergen Bos d 2, several mouse strains with different H-2 haplotypes were immunized with the allergen. Only the BALB/c mouse mounted a distinct humoral response against Bos d 2. The proliferative spleen cell responses of all mouse strains remained very weak. Further experiments with BALB/c mice confirmed that Bos d 2 is a weak inducer of both humoral and cellular responses, and that the responses were weaker than with the control antigens hen egg lysozyme (HEL) and tetanus toxoid. IgG subclass analyses showed that Bos d 2 was prone to favor the T(h)2 response. Although s.c. immunization using complete Freund's adjuvant favored the T(h)1-deviated immune response by lymph node cells, Bos d 2 was able to induce the production of IL-4 while the control antigen HEL did not. Epitope mapping revealed that BALB/c mice recognized one immunodominant epitope in Bos d 2, almost identical to that recognized by humans. The epitope was shown to be immunogenic in subsequent experiments. However, further studies are needed to clarify the significance of priming and stimulation doses of the immunodominant and other epitopes in Bos d 2 for the outcome of immune response against the allergen. The murine immune response against Bos d 2 closely resembled that observed in humans. The weak immunogenicity of Bos d 2 may be associated with its allergenicity.

Background The recently identified dog lipocalin allergen Can f 4 is an important respiratory allergen. Objective We sought to comprehensively characterize the memory CD4 + T-cell responses of allergic and nonallergic subjects to Can f 4. Methods Can f 4–specific CD4 + CD45RO + T-cell lines (TCLs) from allergic and healthy subjects were established and characterized by their functional and phenotypic properties. The epitope specificity of the TCLs was tested with 48 overlapping 16-mer peptides spanning the sequence of Can f 4. HLA restriction of the specific TCLs and the binding capacity of the epitope-containing peptides to common HLA class II molecules were studied. Results Can f 4–specific memory CD4 + TCLs were obtained at an 8-fold higher frequency from allergic than from nonallergic subjects. Functionally, the TCLs of allergic subjects exhibited a higher T-cell receptor avidity and expression of CD25 and predominantly produced IL-4 and IL-5. The TCLs of nonallergic subjects mostly secreted IFN-γ and IL-10, with high CXCR3 expression. Several distinct T-cell epitope regions along the allergen were identified. Importantly, the peptides from the region between amino acids 43 and 67 showed promiscuous HLA-binding capacity and induced memory CD4 + T-cell responses in 90% of the allergic donors. Conclusion Productive T H 2-deviated memory T-cell responses to Can f 4 are observed in allergic but not nonallergic subjects. A 19-mer peptide sequence covering the core of the immunodominant region of the allergen is a potential target for the development of peptide-based allergen immunotherapy.

Purpose Divergent results on the IgE reactivity of dog-allergic subjects to Can f 4 have been reported. The aim of this study was to evaluate the significance of Can f 4 in dog allergy and to develop an immunochemical method for measuring Can f 4 content in environmental samples. Methods We purified the natural dog allergen Can f 4 from a dog dander extract by monoclonal antibody-based affinity chromatography and generated its variant in a recombinant form. Sixty-three dog-allergic patients and 12 nonallergic control subjects were recruited in the study. The IgE-binding capacity of natural Can f 4 and its recombinant variant was assessed by ELISA, immunoblotting, and skin prick tests (SPT). Results Eighty-one percent of the dog-allergic patients showed a positive result to the immunoaffinity-purified natural Can f 4 in IgE ELISA, but only 46% in IgE immunoblotting. Respective results with the recombinant Can f 4 variant were 54% and 49%. SPT results reflected those obtained in ELISA and immunoblotting. The overall IgE reactivity of the immunoaffinity-purified natural Can f 4 was found to depend strongly on the integrity of the allergen's conformation. A sandwich ELISA based on monoclonal antibodies was found to be functional for measuring Can f 4 in environmental samples. Conclusions Can f 4 is a major allergen of dog together with Can f 1 and Can f 5. In combination with other dog allergens, it improves the reliability of allergy tests in dog allergy., published version, peerReviewed

Lipocalin allergens form a notable group of proteins, as they contain most of the significant respiratory allergens from mammals. The basis for the allergenic capacity of allergens in the lipocalin family, that is, the development of T-helper type 2 immunity against them, is still unresolved. As immunogenicity has been proposed to be a decisive feature of allergens, the purpose of this work was to examine human CD4+ T cell responses to the major dog allergen Can f 1 and to compare them with those to its human homologue, tear lipocalin (TL). For this, specific T cell lines were induced in vitro from the peripheral blood mononuclear cells of Can f 1-allergic and healthy dog dust-exposed subjects with peptides containing the immunodominant T cell epitopes of Can f 1 and the corresponding TL peptides. We found that the frequency of Can f 1 and TL-specific T cells in both subject groups was low and close to each other, the difference being about two-fold. Importantly, we found that the proliferative responses of both Can f 1 and TL-specific T cell lines from allergic subjects were stronger than those from healthy subjects, but that the strength of the responses within the subject groups did not differ between these two antigens. Moreover, the phenotype of the Can f 1 and TL-specific T cell lines, determined by cytokine production and expression of cell surface markers, resembled each other. The HLA system appeared to have a minimal role in explaining the allergenicity of Can f 1, as the allergic and healthy subjects' HLA background did not differ, and HLA binding was very similar between Can f 1 and TL peptides. Along with existing data on lipocalin allergens, we conclude that strong antigenicity is not decisive for the allergenicity of Can f 1.

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.

Background About one in every four cases of occupational rhinitis recorded in Finland is animal-induced. Bovine allergens are the most important in this respect and the largest patient group consists of dairy farmers. Allergen immunotherapy, if proven effective, safe and feasible, would be ideal for their treatment. The development of recombinant allergens has offered new potential therapeutic prospects. Fragments of recombinant Bos domesticus (Bos d 2) allergen could be suitable for this purpose because they are recognized by T cells but their IgE-binding capacity is attenuated. Objective The aim of this study was to verify whether the potential of the two fragments of recombinant Bos d 2 (corresponding to amino acids 1–131 and 81–172) to induce immediate allergic reaction in a shock organ (nose) was decreased compared to the complete recombinant allergen, which would be an advantageous property for a preparation intended for allergen immunotherapy. Methods The study group consisted of 10 dairy farmers with cow-induced allergic rhinitis. We used the IgE titres against native Bos d 2 measured by indirect IgE ELISA to characterize the level of sensitization and compared the IgE titres in the rhinitis patients with 12 cow-sensitized asthmatic farmers and 12 healthy students. In vitro reactivity against recombinant Bos d 2 and its two fragments was studied by indirect IgE ELISA and in vivo reactivity by nasal provocation tests. Results The IgE titres against native Bos d 2 of patients with rhinitis tended to be lower than the titres of asthmatics. The healthy students did not exhibit any detectable IgE reactivity to native Bos d 2. In the patients with rhinitis, there was no statistically significant difference between IgE responses against native and recombinant Bos d 2, whereas with both in vitro and in vivo, the reactivity to both fragments of recombinant Bos d 2 was lower than the reactivity to the complete recombinant allergen. Conclusions Due to the decreased in vivo capacity to induce immediate allergic reactions, the fragments may be better tolerated in allergen immunotherapy than the complete allergen.

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.

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.

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. (J Allergy Clin Immunol 1998;101:349-53.)

The initial T cell receptor (TCR) alpha gene rearrangements were analyzed in fetal and neonatal thymocyte hybridomas by Southern blotting. Interestingly, in 30% of all thymocyte hybridomas and in all fetal day 16 thymocyte hybridomas the most proximal J alpha 50 (psi J alpha) gene was rearranged. This rearrangement was found on one chromosome only and mostly in association with a delta rearrangement on the homologous chromosome. J alpha 50 was rearranged to multiple target genes based on the variable size of the restriction fragments. In addition, delta rearrangement was found with a concomitant alpha rearrangement in the majority of hybridomas and it was not only associated with J alpha 50 but with several other rearranged J alpha genes as well. Our results clearly demonstrate that T cell precursors are not pre-committed to either delta or alpha rearrangement but a flexible progenitor responds to multiple regulatory signals during T cell differentiation and they do not support the notion that delta rec-psi J alpha rearrangement is required for cell commitment to TCR alpha gene rearrangement.

A two-site immunometric assay based on monoclonal antibodies (mAb) was developed for the measurement of a 20-kDa major allergen of cow. The sensitivity of this assay was (BDA20) 1 ng/ml. It was used to measure airborne allergen concentrations in 10 Finnish cowsheds. The mean concentration of the BDA20 measured at two stationary sites was 280 ng/m3. Concentrations varied more than 10-fold among cowsheds (54–804 ng/m3). The mean intertest coefficient of variation was 8.2%, and the mean intratest variation 4.1 %.

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.

Allergic sensitization results from a complex interaction between genetic and environmental factors. Earlier studies have shown that highly polymorphic HLA genes are associated with a variety of allergies. Several important respiratory allergens belong to the family of lipocalin proteins. These include occupational sensitizers, such as cow Bos d 2 or rat Rat n 1, and prevalent indoor sensitizers, such as dog Can f 1 or cockroach Bla g 4. HLA associations with sensitization to lipocalin allergens are incompletely known. In the present study we have investigated an association between HLA alleles and sensitization to the major cow allergen Bos d 2. The HLA-DR/DQ genotypes of 40 Bos d 2-sensitized subjects having occupational asthma were determined by polymerase chain reaction (PCR) and the results were compared with the genotypes of 151 unrelated Finnish subjects. The frequencies of HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501 were significantly higher among Bos d 2-sensitized than among control subjects. In addition, the allergic subjects expressed significantly lower frequencies of HLA-DRB1*0301 and DQB1*0201 alleles than did the control subjects. These data suggest that the HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501, and the haplotypes that include them, are associated with sensitization to the major cow allergen Bos d 2, whereas HLA-DRB1*0301 and DQB1*0201 are dissociated with it. Amino acid analysis provides a biologically plausible explanation for the HLA associations.

Although allergen-specific CD4(+) T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naive repertoires are incompletely known. Here, we analyzed the DRB1*0401-restricted responses of peripheral blood-derived memory (CD4(+)CD45RO(+)) and naive (CD4(+)CD45RA(+)) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2(127-142)-specific memory T cells in the peripheral blood-derived cultures appeared to be higher in subjects with allergy than those without, whereas naive Bos d 2(127-142)-specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2(127-142)-specific T cells of naive origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long-term Bos d 2(127-142)-specific T-cell lines generated from both memory and naive T-cell pools from individuals with allergy proliferated more strongly, produced more IL-4 and IL-10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T-cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naive allergen-specific T-cell repertoires differ between individuals with or without allergy.

We have previously proposed that mammalian lipocalin allergens are recognized suboptimally by the human immune system due to their homology with endogenous lipocalins. Here, we have characterized in detail the human T cell recognition of one of the previously identified T cell epitopes of the major dog allergen Can f 1, contained in peptide p105–120. A panel of peptide analogues (altered peptide ligands, APLs) of p105–120 was tested on two specific T cell clones restricted by different human leukocyte antigen (HLA) alleles. Interestingly, we identified for both of the clones several heteroclitic APLs that were capable of stimulating them at 10–30-fold lower concentrations than the natural peptide. Moreover, one of the heteroclitic APLs identified with the T cell clones, L115F, was observed to induce a stronger polyclonal T cell response than the natural allergen peptide from the peripheral blood mononuclear cells (PBMCs) of six Can f 1-allergic subjects studied. The heteroclitic APLs bound with the same affinity as p105–120 to common HLA-DR- and HLA-DP-alleles, suggesting that their improved stimulatory capacity is attributable to a more efficient T cell receptor (TCR) recognition rather than increased HLA binding. Collectively, our data suggest that p105–120 is recognized suboptimally by human T cells. This may contribute to the allergenicity of Can f 1.

The substantial gender difference in life-expectancy is among the key characteristics of current health crisis in Russia. Despite a relatively large body of epidemiological literature on gender difference, there is little empirical research on the gendered meanings of health among Russian lay people. This study aims to enhance understanding of gendered meanings of health by analysing lay accounts of the gender gap in life-expectancy on the basis of 29 interviews with Russians aged 15-81. The analysis showed that gender difference was largely attributed to structural conditions and changes in Russian society and, to a lesser extent, to behavioural factors. Another important conclusion drawn from the analysis was that talk about gender included very few alternatives to conventional gender relations, or negotiation of their effects on health and illness. We interpret these findings to reflect, first, the culturally weak role of the individual in Russian discourses of health that are still largely focused on the role of government as primarily responsible for public health. Secondly, it seems that there are few alternatives to conventional discourses of gender in post-Soviet Russia; the gender relations in people's understanding appear to be static and persistent despite recent profound social changes.

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in the cholinergic basal forebrain (BF) during sleep deprivation (SD) is implicated in adenosine (AD) release and induction of recovery sleep. Aging is associated with impairments in sleep homeostasis, such as decrease in non-rapid eye movement sleep (NREM) intensity following SD. We hypothesized that age related changes in sleep homeostasis may be induced by impairments in NO-mediated sleep induction. To test this hypothesis we measured levels of NO and iNOS in the BF during SD as well as recovery sleep after SD and NO-donor (DETA/NO) infusion into the BF in three age groups of rats (young, 4 months; middle-aged, 14 months; old, 24 months). We found that in aged rats as compared to young (1) recovery NREM sleep intensity was significantly decreased, (2) neither iNOS nor NO increased in the BF during SD, and (3) DETA/NO infusion failed to induce sleep. Together, these results support our hypothesis that aging impairs the mechanism through which NO in the BF induces sleep.

Background Despite the fact that most significant mammalian respiratory allergens are lipocalin proteins, information on the human T cell reactivity to these allergenic proteins is largely missing. Objective Knowing the T cell epitopes in allergens is a prerequisite for developing novel preparations for allergen immunotherapy. Methods Specific T cell lines were generated with recombinant Equ c 1 from the peripheral blood mononuclear cells (PBMCs) of 10 horse-allergic subjects. For determining T cell epitopes, the lines were stimulated with 16mer synthetic Equ c 1 peptides overlapping by 14 amino acids. The binding capacity of Equ c 1 peptides to human leucocyte antigen class II molecules was determined by the competitive ELISA. Results The major horse allergen Equ c 1 resembles two other lipocalin allergens, the major cow allergen Bos d 2 and the major dog allergen Can f 1, in that it is weakly stimulatory for the PBMCs of sensitized subjects. Moreover, the T cell epitopes of Equ c 1 are clustered in a few regions along the molecule, as is the case with Bos d 2 and Can f 1. Similar to Bos d 2, Equ c 1 contains one immunodominant epitope region at the carboxy-terminal end of the molecule. The T cell lines of eight horse-allergic subjects out of 10 showed strong reactivity to one or both of the two overlapping peptides, p143-158 and p145-160, in this region. The region probably contains two overlapping epitopes. Conclusion The 18mer peptide p143-160 from the immunodominant region of Equ c 1 is a potential candidate for the peptide-based immunotherapy of horse-sensitized subjects.

Summary Background The significance of specific T cell receptor (TCR) Vβ subtypes and human leucocyte antigen (HLA) class II alleles for the development of allergy to lipocalin allergens such as the major dog allergen Can f 1 is not clear at present. Objective To characterize the TCR Vβ usage in the Can f 1-specific T cell lines and the HLA class II genotypes of Can f 1-allergic and non-allergic subjects. Methods T cell lines were induced with recombinant Can f 1 from the peripheral blood mononuclear cells of 12 non-atopic dog owners and 26 dog-allergic patients. Thirteen of the dog-allergic subjects were sensitized to Can f 1. Expression of the TCR Vβ subtypes on CD4+ T cells in the T cell lines was measured by flow cytometry. The subjects were HLA genotyped for DRB1, DQB1 and DPB1 loci. Results Can f 1-specific T cell lines were obtained from 18 subjects, with either positive (n=8) or negative (n=10) skin prick tests (SPTs) to recombinant Can f 1. The frequency of TCR Vβ5.1+ T cells was significantly higher in the T cell lines of subjects with negative SPTs to the allergen. Moreover, DR4–DQ8 haplotype was over-represented among these subjects. Conclusion The DR4–DQ8 haplotype and the TCR Vβ5.1+ CD4+ T cells may be protective against allergy to Can f 1.

We have proposed earlier that the poor capacity of the lipocalin allergen Bos d 2 to stimulate highly allergic subjects' peripheral blood mononuclear cells could be ascribed to endogenous lipocalins and could be related to the allergenic potential of the molecule. Here, we have characterized the proliferative and cytokine responses of human T cell clones against the immunodominant epitope of Bos d 2. We observed, for clone F1-9, that a substitution of aspartic acid for asparagine in the core region of the epitope increased the stimulatory capacity of the peptide about 100-fold in comparison with the natural peptide. For clone K3-2, from a different patient, the substitution of lysine for glutamine or isoleucine for leucine in the core region resulted in about 30-fold and 10-fold increases in the stimulatory capacity of the peptides, respectively. The clones also recognized self-protein-derived peptides but not the peptides derived from other lipocalins. We suggest that the poor recognition of the immunodominant epitope of Bos d 2 can be a factor accounting for Bos d 2-allergic subjects' weak cellular responses. Suboptimal recognition of self and allergen epitopes by T cells may be of significance for the allergenicity of proteins.

We have previously demonstrated that the onset of TCR alpha gene rearrangement is mainly restricted to the J alpha50 gene in fetal day 1delta thymocyte hybridomas. Now, J alpha50 rearrangements from fetal thymocyte hybridomas and from day 15.5 fetal thymus have been isolated and sequenced. We demonstrate that J alpha50 is rearranged to the rearranged Vdelta1 Ddelta2 gene segment. This indicates that the TCR alpha rearrangement process is initiated in fetal thymocytes far earlier than previously thought. These thymocytes have their delta genes still accessible for rearrangement and therefore, are controlled by the TCR delta enhancer (Edelta) (and/or another TCR delta specific cis-acting element). Our results further suggest that both Edelta and the TCR alpha enhancer (Ealpha) are active at the onset of alpha rearrangements or alternatively, the initial activation of the J alpha locus is controlled by Edelta. In addition, Vdelta1 Ddelta2 J alpha50 gene segments are replaced by secondary alpha rearrangements, indicating that thymocytes with the early alpha rearrangement are committed to the alphabeta lineage.

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.

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. (J Allergy Clin Immunol 1997;100:721-7.)

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. (J ALLERGY CLIN IMMUNOL 1996;97:1297-1303.)

Summary Background Cow dust is one of the most important inducers of occupational allergic diseases in Finland. For example, in 1991 it accounted for almost 40% of the new occupational asthma cases. Objective This study compares the performance of the purified major cow allergen (BDA20) and crude bovine epithelial extract (BEA) in diagnostic tests and examines the role of milk allergy-associated bovine proteins (bovine serum albumin, α-lactalbumin, β-lactoglobulin, casein) in respiratory cow allergy. Methods The humoral responses of cow-asthmatic and healthy farmers to the various components of BEA were analysed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The levels of specific Ige and IgG antibodies were quantifieated with enzyme-linked inimunosorbent assays (FLISAs). The cellular responses were analysed with antigen-specific lymphoeyte proliferation tests. Results The specific anti-BDA20 IgE measurement was found to be best in distinguishing between the asthmatic farmers and their healthy colleagues. It proved possible to determine a cut-off value that gave the analysis a specificity and sensitivity of 100%; the distinction between the two groups was highly significant (P 0.0001). In the lymphocyte proliferation analysis, cow asthma was more closely associated with reactivity to BDA20 than to BEA. In the measurement of anti-BDA20 and anti-BEA IgG antibody levels, considerable overlap between the groups was observed, suggesting that these antibodies are not directly involved in cow allergy. When proteins associated with milk allergy were used as test reagents, no statistically significant differences could be observed between the groups, except for anti-casein IgE antibodies the level of which, however, overlapped considerably between the farmer groups. Conclusion These findings suggest that purified BDA20 is better than BEA for diagnosing cow asthma and that proteins associated with milk allergy are of only marginal significance in this disease.

We have characterized bovine allergens by constructing and analyzing a complementary DNA library from bovine skin. Clones producing proteins that reacted with IgE antibodies from persons with allergy were purified and sequenced. One set of the allergen-coding clones showed an almost complete homology with the bovine oligomycin sensitivity–conferring protein of the mitochondrial adenosine triphosphate synthase complex. The IgE antibodies adsorbed with the recombinant allergen reacted with an 11 kd protein in the cow dander extract. Binding of the IgE from patients allergic to the recombinant allergen expressed in Escherichia coli confirmed the allergen-coding ability of the complementary DNA sequence. The prevalence of the IgE-positive sera among patients with cow allergy and control subjects suggests that the recombinant allergen represents one of the minor allergens in cow dander. This is the first time a mammalian allergen has been identified as a protein with a known function. ( J Allergy Clin Immunol 1995;95:1255-60.)