Major Cat Allergen Research Articles

Phenotypic analysis of perennial airborne allergen‐specific CD4+ T cells in atopic and non‐atopic individuals

Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4+ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4+ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Using HLA class II tetrameric complexes based on a HLA-DPB1*0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. Ex vivo Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. Circulating Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals.

Two Newly Identified Cat Allergens: The von Ebner Gland Protein Fel d 7 and the Latherin-Like Protein Fel d 8

Introduction: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. Materials and Methods: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. Results: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. Discussion: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.

Prediction of the secondary structure and B cell epitopes on major cat allergen Fel d 1

Objective To predict and analyze the secondary structure and B cell epitopes of cat major allergen Fel d 1.Methods Based on the amino acid sequence of chain Ⅰ and chain Ⅱ of Fel d 1.the secondary structure,hydrophilicity,flexible regions,surface probability,and antigenic index were predicted by the methods of Chou-Fasman,Kyte-Doolittle,Karplus-Schulz,Emini and Jameson-Wolf respectively,and DNAStar Protean software,Rtault The potential B cell epitopes of Fel d 1 were localized in the regions of 15～21,48～57,103-111,138～143,and 151～161.Conclusion These findings are helpful for the identification of the dominant B cell epitopes.The results can provide the antibodies design and antigenieity modification of Fel d 1 with theoretical foundation. Key words: Cat major allergen; Fel d 1; Secondary structure; B cell epitopes

Recognition of the Major Cat Allergen Fel d 1 through the Cysteine-rich Domain of the Mannose Receptor Determines Its Allergenicity

Dendritic cells are professional antigen-presenting cells that are specialized in antigen uptake and presentation. Allergy to cat has increased substantially in recent years and has been shown to be positively associated with asthma. We have recently shown that the mannose receptor (MR), a C-type lectin expressed by dendritic cells, recognizes various glycoallergens from diverse sources and is involved in promoting allergic responses to a major house dust mite allergen in vitro. Here we investigated the potential role of MR in allergic responses to Fel d 1, a major cat allergen. Fel d 1 binding to MR was confirmed by ELISA. Using blocking, gene silencing (siRNA) experiments, and MR knock-out (MR(-/-)) cells, we have demonstrated that MR plays a major role in internalization of Fel d 1 by human and mouse antigen-presenting cells. Intriguingly, unlike other glycoallergens, recognition of Fel d 1 by MR is mediated by the cysteine-rich domain, which correlates with the presence of sulfated carbohydrates in natural Fel d 1. WT and MR(-/-) mice were used to study the role of MR in allergic sensitization to Fel d 1 in vivo. MR(-/-) mice sensitized with cat dander extract and Fel d 1 produced significantly lower levels of total IgE, Fel d 1-specific-IgE and IgG1, the hallmarks of allergic response, compared with WT mice. Our data show for the first time that Fel d 1 is a novel ligand of the cysteine-rich domain of MR and that MR is likely to play a pivotal role in allergic sensitization to airborne allergens in vivo.

Impaired allergy diagnostics among parasite-infected patients caused by IgE antibodies to the carbohydrate epitope galactose-α1,3-galactose

The carbohydrate epitope galactose-α 1,3-galactose (α-Gal) is abundantly expressed on nonprimate mammalian proteins. We have recently shown that α-Gal is responsible for the IgE binding to cat IgA, a newly identified cat allergen (Fel d 5). We sought to investigate the diagnostic relevance of IgE antibodies to Fel d 5 and α-Gal among parasite-infected patients from central Africa without cat allergy compared with patients with cat allergy from the same region. Sera from 47 parasite-infected patients and 31 patients with cat allergy were analyzed for total IgE and IgE antibodies against cat dander extract (CDE) by using the ImmunoCAP system. Inhibition assay was performed with α-Gal on solid phase-bound CDE. The presence of IgE specific for the major cat allergen Fel d 1, Fel d 5, and α-Gal was analyzed by means of ELISA. Among the 47 parasite-infected patients, 85% had IgE antibodies against α-Gal (OD; median, 0.175; range, 0.102-1.466) and 66% against Fel d 5 (OD; median, 0.13; range, 0.103-1.285). Twenty-four of the parasite-infected patients were sensitized to CDE, and 21 of them had IgE antibodies to Fel d 5 and α-Gal. There was no correlation between IgE levels to CDE and rFel d 1 among the parasite-infected patients but a strong correlation between CDE and Fel d 5 and α-Gal (P < .001). Among the group with cat allergy, only 5 patients had IgE to α-Gal, and nearly 75% (n = 23) had IgE to rFel d 1 (median, 7.07 kU(A)/L; range, 0.51-148.5 kU(A)/L). In contrast, among the patients with cat allergy, there was a correlation between IgE levels to CDE and rFel d 1 (P < .05) but no correlation between CDE and Fel d 5 and α-Gal. IgE to α-Gal causes impaired allergy diagnostics in parasite-infected patients. Screening for IgE to rFel d 1 and other allergens without carbohydrates might identify patients with true cat sensitization/allergy in parasite-infested areas.

Human allergy to environmental pet danders: a public health perspective

The prevalence of human allergy to pet danders has increased rapidly over the past six decades, as a consequence of lifestyle changes that have enhanced ambient exposure to pet allergens. This is a problem of global public health importance, as the morbidity associated with allergic diseases disproportionately affects socio-economically disadvantaged populations, particularly children. Although the public often seeks advice from veterinary health-care professionals regarding healthy pet ownership practices, including strategies for reducing residential pet dander exposure, many misconceptions persist in the public domain regarding pet dander allergy, such as the belief that certain dog and cat breeds are 'hypoallergenic' due to their hair/coat type. This review considers the epidemiology of human exposure to the major cat and dog dander allergens, Fel d 1 and Can f 1 respectively, and the sensitization mechanisms to them, including the hygiene hypothesis and the putative role of bacterial endotoxin. The literature regarding primary and secondary exposures in different ambient environments is explored including threshold effects that influence allergen sensitization and elicitation of symptoms, and environmental intervention strategies that seek to reduce allergen exposure. Pet-specific factors, including the aetiopathogenesis of the several cat and dog allergens that have been characterized to the molecular level, individual animal characteristics that influence Fel d 1 and Can f 1 shedding, and pet-directed interventions intended to reduce allergen dispersal, are discussed.

Treatment with a Fel d 1 hypoallergen reduces allergic responses in a mouse model for cat allergy

A hypoallergen of the major cat allergen Fel d 1, recombinant (r) Fel d 1 (DTE III), was previously shown to have retained T-cell reactivity and strongly reduced IgE-binding capacity compared to unmodified rFel d 1. Here, we evaluated the therapeutic capacity of rFel d 1 (DTE III) in a mouse model for cat allergy. Mice were subcutaneously (s.c.) sensitized with rFel d 1 and subsequently treated (s.c.) with 50 or 200 μg rFel d 1 (DTE III), or 50 μg rFel d 1, prior to intranasal challenge with cat dander extract. Airway hyperreactivity (AHR), cells and cytokines in bronchoalveolar lavage fluid, splenocyte in vitro response, and serum immunoglobulins were analyzed. Seven cat-allergic patients and ten healthy controls were tested for skin prick test (SPT) reactivity to rFel d 1 (DTE III) and rFel d 1. Mice treated with 50 and 200 μg rFel d 1 (DTE III), and 50 μg rFel d 1, produced increased serum levels of rFel d 1-specific IgG1 and IgG2a compared to sham-treated mice. IgG from all treatment groups could block binding of patients' IgE to rFel d 1. The 200 μg rFel d 1 (DTE III) treatment tended to reduce AHR. All mice tolerated treatment with rFel d 1 (DTE III), in contrast to only four of ten treated with rFel d 1. Compared to rFel d 1, the hypoallergen showed a tendency of reduced SPT reactivity. The rFel d 1 (DTE III) hypoallergen might be a promising candidate for application in immunotherapy of cat allergy with improved safety and efficacy.

Mechanisms of allergen-specific desensitization

Allergen-specific desensitization (SIT) is the most effective therapy for allergies. Although allergen-specific antibodies have an important role in the process, mechanisms of IgG-mediated inhibition of allergic reactions are not well defined. We investigated mechanisms by which SIT-induced allergen-specific IgGs inhibit allergic reactions. We generated mAbs that recognize 3 nonoverlapping epitopes of the major cat allergen Fel d 1. Each of the mAbs was produced as an IgE and different IgG isotype. IgEs against 2 nonoverlapping epitopes on Fel d 1 are necessary and sufficient to sensitize mast cells for maximal FcepsilonRI signaling and degranulation on exposure to monomeric Fel d 1. IgE antibodies of a third specificity did not further increase mast cell degranulation, indicating that formation of large FcepsilonRI clusters are not required to induce maximal activation of mast cells. A single IgG that was specific for an epitope different from those recognized by the IgEs was a potent inhibitor of Fel d 1-mediated mast cell activation in vitro and in vivo. This inhibition required Fcgamma receptor-IIB. In human beings, IgGs of a single specificity were able to block degranulation of basophils from individuals with cat allergy. The inhibitory potential of these antibodies increased when larger allergen-IgG complexes were formed. These data reconcile conflicting theories in the literature and might explain the reason IgE levels do not necessarily decrease during therapy, despite clinical efficacy. These findings have important implications for vaccine design.

Distribution of Core Fragments from the Major Cat Allergen Fel d 1 Is Maintained among the Main Anatomical Sites of Production

Background: Polymorphism of Fel d 1 has long been observed, but structural characterization of Fel d 1 variants among the various sites of production and animals is still missing. This study was aimed at elucidating the structural polymorphism of this protein as a function of the site of production and the resulting influence on the immunological behavior of the allergen. Methods: Fel d 1 was water-extracted from the 4 main sites of production, i.e. cheek zone, interdigital zone, anal sac (AS), and chest area and a panel of 10 cats. Fel d 1-like materials were immunoblotted, immunopurified and characterized by MALDI-TOF MS. Immunoreactivity was studied using ELISA dose-dependent curves at equilibrium. Results: In all areas, 4–10 isoforms ranging from 7 to 40 kDa were detected by MS. Essentially two truncated heterodimers of 13–14 and 16–17 kDa were identified together with intact Fel d 1 in all compartments and they were largely predominant in AS. These core fragments were found to contribute to a variable recognition by antibodies in a panel of ELISA. Conclusions: Our study identified truncated dimers of Fel d 1, probably resulting from proteolytic degradation of both chains and present in all cats. Core fragments are largely distributed among anatomical sites of production and especially well represented in AS. They are recognized as intact allergens by antibodies and may therefore introduce discrepancies in allergen measurement depending on the variable amount of intact and degraded Fel d 1 produced by the cat.

Cat sensitization identified by recombinant Fel d 1 several years before symptoms - results from the bamse cohort

Exposure to cat is one of the most important causes of allergic disease. The objective of this study was to investigate IgE reactivity to the recombinant major cat allergen, rFel d 1, as an early marker of cat sensitization. Based on questionnaires, 144 children with allergic symptoms due to cat, or where such symptoms were suspected, were selected from the birth cohort BAMSE and allocated into three study groups. Blood samples taken at age 4 and 8 yrs were analysed for IgE to rFel d 1 and cat dander extract (CDE) by quantitative ELISA (cut-off limit 0.037 kU(A)/l) and the ImmunoCAP System (cut-off limit 0.35 kU(A)/l), respectively. At 4 yrs, 25/33 children with certain allergic symptoms to cat had IgE to both rFel d 1 and CDE, while 14/42 of those suspecting symptoms at 4 had IgE to rFel d 1 and 9/42 to CDE. In a group developing symptoms after 4 yrs, 60/69 had IgE to rFel d 1 and 57/69 to CDE at 8, while 33/69 had IgE to rFel d 1 already at 4 and 26/69 to CDE. This was the only one of the three study groups where a significant increase in the IgE levels to rFel d 1 was found from 4 to 8 yrs (p < 0.001), even when only children with IgE to rFel d 1 already at 4 were included (p < 0.001). We show that the single major cat allergen rFel d 1 is at least as good as CDE in the diagnosis of cat allergy in childhood. With a sensitive rFel d 1 assay cat sensitization can be detected several years before symptoms to cat are reported.

Higher Immunoglobulin E Antibody Levels to Recombinant Fel D 1 in Cat-Allergic Children With Asthma Compared With Rhinoconjunctivitis

Grönlund H, Adédoyin J, Reininger R, et al. Clin Exp Allergy. 2008;38(8):1275–1281PURPOSE OF THE STUDY. To measure immunoglobulin E (IgE) and IgG4 antibodies to an engineered recombinant major cat allergen, rFel d 1, among sera from cat-allergic children and adults.STUDY POPULATION. One hundred forty cat-allergic children and adults with rhinoconjunctivitis and/or asthma were selected; all had positive skin-prick test results to cat dander extract (CDE). Seventy-five healthy, age-matched, CDE-skin-test–negative children and adults were selected as control subjects.METHODS. Sera from the 140 patients were tested for IgE and IgG4 antibodies to CDE and rFel d 1 by ImmunoCAP (Phadia, AB Uppsala, Sweden) and for IgE to rFel d 1 by enzyme-linked immunosorbent assay.RESULTS. Ninety-eight percent of patients (all but 1) and none of the control subjects had evidence of specific IgE to rFel d 1. Specific IgE results to rFel d 1 and CDE correlated strongly (rs = 0.85; P < .001) among the 140 patients; however, results to rFel d 1 were, on average, 30% higher (P < .0001). IgE responses to rFel d 1 among children with asthma were higher (median: 19.4 kU/L), compared with children with rhinoconjunctivitis only (median: 6.6 kU/L; P < .05) and adults with asthma (median: 3.0 kU/L; P < .01). There was a threefold increased risk of asthma for one half of the children with the highest IgE levels (odds ratio: 3.23 [95% confidence interval: 1.19–8.79]) by the enzyme-linked immunosorbent assay. Children with asthma also displayed significantly higher IgG4 levels than did adults with asthma.CONCLUSIONS. Recombinant major cat allergen (rFel d 1) seems to be at least equally sensitive for in vitro diagnosis of cat allergy, compared with the current extract-based test. Elevated specific IgE antibody levels to rFel d 1 are suggested to be a risk factor for asthma in cat-allergic children.REVIEWERS COMMENTS. In vitro diagnosis of allergic sensitization is currently confirmed by using allergen extracts derived from natural source materials. The authors previously described creation of this recombinant major cat allergen, rFel d 1. Investigation with such purified recombinant proteins is a current area of intense interest within the field of allergy and immunology, because these engineered proteins may offer improved diagnostic specificity and theoretically may offer superior therapy for type 1 hypersensitivity. Previous research showed that allergen-specific IgE levels are predictive for the likelihood of allergic disease including asthma; however, some allergen-avoidance studies failed to show significant evidence of improvement or protection. This study gives further support to cat allergy causing asthma.

Displaying Fel d1 on virus-like particles prevents reactogenicity despite greatly enhanced immunogenicity: a novel therapy for cat allergy

Allergen-specific desensitization is the only disease-modifying therapy currently available for the treatment of allergies. These therapies require application of allergen over several years and some may induce life-threatening anaphylactic reactions. An ideal vaccine for desensitization should be highly immunogenic and should alleviate allergic symptoms upon few injections while being nonreactogenic. We describe such a vaccine for the treatment of cat allergy, consisting of the major cat allergen Fel d1 coupled to bacteriophage Qβ-derived virus-like particles (Qβ–Fel d1). Qβ–Fel d1 was highly immunogenic, and a single vaccination was sufficient to induce protection against type I allergic reactions. Allergen-specific immunoglobulin G antibodies were shown to be the critical effector molecules and alleviated symptoms by two distinct mechanisms. Although allergen-induced systemic basophil degranulation was inhibited in an FcγRIIb-dependent manner, inhibition of local mast cell degranulation in tissues occurred independently of FcγRIIb. In addition, treatment with Qβ–Fel d1 abolished IgE memory responses upon antigen recall. Despite high immunogenicity, the vaccine was essentially nonreactogenic and vaccination induced neither local nor systemic anaphylactic reactions in sensitized mice. Moreover, Qβ–Fel d1 did not induce degranulation of basophils derived from human volunteers with cat allergies. These data suggest that vaccination with Qβ–Fel d1 may be a safe and effective treatment for cat allergy.

Peptide immunotherapy in allergic asthma generates IL-10–dependent immunological tolerance associated with linked epitope suppression

Treatment of patients with allergic asthma using low doses of peptides containing T cell epitopes from Fel d 1, the major cat allergen, reduces allergic sensitization and improves surrogate markers of disease. Here, we demonstrate a key immunological mechanism, linked epitope suppression, associated with this therapeutic effect. Treatment with selected epitopes from a single allergen resulted in suppression of responses to other (“linked”) epitopes within the same molecule. This phenomenon was induced after peptide immunotherapy in human asthmatic subjects and in a novel HLA-DR1 transgenic mouse model of asthma. Tracking of allergen-specific T cells using DR1 tetramers determined that suppression was associated with the induction of interleukin (IL)-10+ T cells that were more abundant than T cells specific for the single-treatment peptide and was reversed by anti–IL-10 receptor administration. Resolution of airway pathophysiology in this model was associated with reduced recruitment, proliferation, and effector function of allergen-specific Th2 cells. Our results provide, for the first time, in vivo evidence of linked epitope suppression and IL-10 induction in both human allergic disease and a mouse model designed to closely mimic peptide therapy in humans.

Prolonged antigen‐exposure with carbohydrate particle based vaccination prevents allergic immune responses in sensitized mice

Defined particles carrying tightly bound allergens at high density have been suggested as alternatives in allergy vaccination. Carbohydrate based particles (CBP), sized 2 microm, provide a platform for covalent coupling of allergens. To investigate the mechanisms of antigen presentation by CBP, as well as cellular and humoral responses after vaccination with the major cat allergen Fel d 1, covalently coupled to CBP. Mice (n = 10/group) were subcutaneously vaccinated with CBP-rFel d 1, CBP or phosphate buffer saline (PBS) before sensitization with rFel d 1 and challenged with cat dander extract. Fluorescent and (75)Se-radiolabeled tracking of allergens and particles were performed with flow cytometry and whole-body autoradiography. Humoral, cellular and regulatory immune responses were analyzed by ELISA and flow cytometry. Cytokines were measured in bronchoalveolar lavage fluid and splenocyte cultures. CBP-rFel d 1 prevented induction of airway inflammation and induced allergen-specific T-cell anergy. CBP-rFel d 1 also induced rapid IgM and IgG1-responses compared with soluble rFel d 1. Particles were phagocytosed by antigen-presenting cells and transported to draining lymph nodes and spleen. Moreover, antigen coupled to CBP remained longer at the injection site compared with alum. Covalent coupling of rFel d 1 to CBP induces rapid antibody production, prevents induction of allergic immune responses and systemic allergen spreading. Thus, CBP comprise several attractive adjuvant features for use in allergy vaccination. Prolonged allergen exposure through covalent coupling to particles suitable for phagocytosis, provides an adjuvant for safer and efficient allergy vaccination.

Targeting the MHC class II pathway of antigen presentation enhances immunogenicity and safety of allergen immunotherapy

Current s.c. allergen-specific immunotherapy (SIT) leads to amelioration of IgE-mediated allergy, but it requires numerous allergen injections over several years and is frequently associated with severe side-effects. The aim of this study was to test whether modified recombinant allergens can improve therapeutic efficacy in SIT while reducing allergic side-effects. The major cat allergen Fel d 1 was fused to a TAT-derived protein translocation domain and to a truncated invariant chain for targeting the MHC class II pathway (MAT-Fel d 1). The immunogenicity was evaluated in mice, while potential safety issues were assessed by cellular antigen stimulation test (CAST) using basophils from cat-dander-allergic patients. MAT-Fel d 1 enhanced induction of Fel d 1-specific IgG2a antibody responses as well as the secretion of IFN-gamma and IL-2 from T cells. Subcutaneous allergen-specific immunotherapy of mice using the modified Fel d 1 provided stronger protection against anaphylaxis than SIT with unmodified Fel d 1, and MAT-Fel d 1 caused less degranulation of human basophils than native Fel d 1. MAT-Fel d 1 allergen enhanced protective antibody and Th1 responses in mice, while reducing human basophil degranulation. Immunotherapy using MAT-Fel d 1 allergen therefore has the potential to enhance SIT efficacy and safety, thus, shortening SIT. This should increase patient compliance and lower treatment costs.

Targeting allergen to FcγRI: a strategy to treat allergic disease?

Targeting allergens to surface receptors on antigen presenting cells may provide a therapeutic strategy for allergic disease. This article discusses the immunomodulatory capacity of a molecule (H22-Fel d 1), which targets the major cat allergen, Fel d 1, to the high affinity IgG receptor, Fc gammaRI, on human dendritic cells. The fusion protein, H22-Fel d 1, induced a semi-mature phenotype in dendritic cells characterized by production of inflammatory cytokines with no change in surface markers, suggesting tolerogenic capacity. At the T-cell level, H22-Fel d 1 stimulated increased proliferation coupled with amplification of T cells expressing IL-5 and IL-10. Further analysis revealed induction of diverse T cell subtypes characteristic of Th0, regulatory Th1 and regulatory Th2 cells. Notably, this effect was restricted to T cells isolated from cat-allergic patients. Despite the increase in IL-5-expressing T cells, responses induced by H22-Fel d 1 appeared to be regulated by IL-10. Comparison with nonreceptor-targeted allergens from cat and house dust mite confirmed that qualitative T-cell changes induced by H22-Fel d 1 were unique. H22-Fel d 1 induces a novel variation of the Th2 response, which incorporates elements of a protective T-cell response. Exploiting Fc gammaRI-mediated pathways for allergen delivery may offer a new approach for treatment.

Katzenallergie: aktualisierte Datenlage zu Epidemiologie und Therapie und Neues aus der Molekularbiologie von Fel d 1

Cat allergy belongs to the most common animal allergies and is not just of high scientific and clinical interest but plays also an increasing socioeconomic role. This is underlined by studies revealing the tetrameric structure of the major cat allergen Fel d 1. Recent epidemiologic studies concerning the sensitization to cat and resulting allergic diseases still provide conflicting results. However there is increasing evidence that childhood sensitization presents a risk factor for later wheeze and asthma. Furthermore new scientific approaches for specific immunotherapy may result in a better effectiveness and tolerance. In this review we summarize the most recent data on the molecular biology ofFel d 1, on epidemiology and therapy of cat allergy.

Higher immunoglobulin E antibody levels to recombinant Fel d 1 in cat‐allergic children with asthma compared with rhinoconjunctivitis

Current diagnosis of allergy and asthma to cat is confirmed using cat dander extract (CDE). We have previously engineered a recombinant major cat allergen, rFel d 1, with properties identical to the natural molecule. The aim of the study was to evaluate IgE and IgG4 antibodies to rFel d 1 among sera from cat-allergic children and adults suffering from asthma and/or rhinoconjunctivitis (RC) in populations from Sweden and Austria. Cat-allergic children and adults from Sweden (n=27 and 31, respectively) and Austria (n=41 and 41) with RC and/or asthma were selected. Sera were tested for IgE and IgG4 antibodies to CDE and rFel d 1 by CAP, and IgE to rFel d 1 by ELISA. Healthy subjects and non-cat-allergic patients (n=75) were included as controls. There was a high correlation between IgE responses to rFel d 1 and CDE among the 140 patients (r(s)=0.85, P<0.001); however, measured levels to rFel d 1 were on average 30% higher (P<0.0001). Ninety-eight percent of patients and none of the controls showed IgE to rFel d 1 and there was a threefold increased risk of asthma for half of the children with the highest IgE levels [odds ratio 3.23; 95% confidence interval (CI), 1.19-8.79] by ELISA. IgE responses to rFel d 1 among children with asthma were higher (median 19.4 kU/L) compared with children with RC (median 6.6 kU/L, P<0.05) and adults with asthma (median 3.0 kU/L, P<0.01). Furthermore, children with asthma displayed higher IgG4 levels than the asthmatic adults. A single recombinant molecule, rFel d 1, is at least as sensitive for in vitro diagnostics of cat allergy as the current extract-based test. Elevated IgE antibody levels to Fel d 1 are suggested to be a risk factor for asthma in cat-allergic children.

Low Levels of Endotoxin Enhance Allergen-Stimulated Proliferation and Reduce the Threshold for Activation in Human Peripheral Blood Cells

Background: Endotoxins, comprised of bacterial cell wall lipopolysaccharides (LPS), have been reported to have both protective and exacerbating effects on the development and maintenance of allergic disease in humans and on markers of allergic inflammation in animal models of allergy. In this study, we investigated the effect of low concentrations of LPS on human peripheral blood mononuclear cells (PBMC) stimulated with the major cat allergen Fel d 1. Methods: Extensive purification of recombinant (r) Fel d 1 yielded essentially endotoxin-free rFel d 1 (0.2 ng LPS /mg protein). PBMCs prepared from 15 subjects having IgE to cat (>0.7 kU_A/l) and 8 subjects IgE negative to cat were stimulated with 2, 10 or 25 µg/ml of rFel d 1 in the presence or absence of 50 pg/ml LPS. Proliferation was measured after 7 days of culture and supernatants were analyzed for IFNγ, IL-5 and IL-10. Results: LPS (50 pg/ml) increased rFel d 1-stimulated proliferation of PBMCs both from subjects IgE-positive and subjects negative to cat allergens. PBMCs from 13 of the subjects did not proliferate in response to stimulation with 2 and 10 µg/ml rFel d 1 alone but did so in the presence of LPS. Moreover, LPS increased the levels of rFel d 1-stimulated IFNγ in cultures from cat-negative subjects, IL-5 from cat-positive subjects and IL-10 from both groups. Conclusion: Very low doses of LPS enhance proliferation and decrease the apparent threshold level for cell activation, prompting careful evaluation of allergen stimulated T cell activation in vitro.

Structural Characterization of the Tetrameric form of the Major Cat Allergen Fel d 1

Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1 + 2)) and chain 2 to chain 1 (construct Fel d 1 (2 + 1)). Although the crystal structure of Fel d 1 (2 + 1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1 + 2) tetramer at 1.6 Å resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca 2+-binding sites correspond to a putative Ca 2+-binding site previously suggested for uteroglobin. The second Ca 2+-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca 2+-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin.