Fel d I allergen distribution in cat fur and skin.

Immunohistochemical procedures were performed to ascertain Fel d I antigen (Ag) distribution in cat fur and skin biopsy specimens and to analyze Fel d I allergen concentrations in fur. One hundred strands of fur and 24 skin biopsy specimens (6 by 4 by 3 mm) from shaved areas were collected from 11 different cats. Freshly depilated hairs were immunostained by free-floating monoclonal anti-Fel d I, avidin-biotin-peroxidase complex, and either processed for scanning electron microscopic examination or mounted on glass slides for computer-assisted densitometric analysis (SAMBA system). Skin biopsy specimens were promptly frozen and sectioned just before the immunohistochemical processing. Densitometric analysis of fur demonstrated that immunoprecipitate concentrations were tenfold higher at the root than at the tip. However, this finding may be explained by decrease of the thickness of the hair cortex that varied in similar proportions. The Ag accumulated on the strand surface but may focally penetrate into the medulla through the scale-like cortical interstices. In skin biopsy specimens, Fel d I Ag was found in epithelial squamous cells, within the epidermis and hair follicles, on the surface of the epidermis and hair follicles, and in sebaceous gland cells. These data suggest that Fel d I Ag is produced by sebaceous cells and, to a lesser extent, by basal squamous epithelial cells and that it is stored mainly on the surface of the epidermis and fur.     

Fel d I allergen: skin and or saliva?

To determine the relative importance of saliva and sebaceous glands as sources of Fel d I allergen, we compared Fel d I levels at the base and tip of the hair in areas presenting more or less sebaceous glands and areas licked more or less frequently. The amount of Fel d I was significantly higher at the base than the tip of the hair. Further, it was strongly correlated with the density of sebaceous glands. This study demonstrated that the most abundant source of Fel d I allergen is cat skin.     

Monoclonal antibodies against Fel d I and other clinically relevant cat allergens

Several mouse monoclonal antibodies (MAbs) were obtained which specifically recognized allergen molecules from cat dander extract. Two of them (C5.8 and C5.24) were specific for the main cat allergen, Fel d I. One (C5.25) recognized an important allergen with approximate molecular weight of 30000 Da and a pI between 3.9 and 4.3. A third group of MAbs comprised several hybridomas which were specific either for cat albumin or cat immunoglobulin. The immunochemical characterization and the clinical significance of the cat dander components recognized by these MAbs is presented and discussed.     

Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I

The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide-linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty-one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope-mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I.
Two monoclonal antibodies directed against reduced/alkylated chain I bound to the overlapping peptides 53–66 and 60–70 of chain 1. The monoclonal antibody directed against reduced/alkylated chain 2 bound to the overlapping peptides 36–49 and 43–56 of chain 2. Binding specificity was demonstrated by inhibition by reduced/alkylated Fel d I for all three monoclonal antibodies.
Another monoclonal antibody against reduced/alkylated Fel d I had been found to bind predominantly to reduced/alkylated chain 2 on immunoblot in previous studies (27). It bound to peptides 1–16 and 60–70 of chain 1 and peptides 1–14 and 50–63 of chain 2; it is therefore probably directed against a conformational epitope formed by these four regions. Possibly because of low affinity of this monoclonal antibody, specificity of its binding could not be verified by inhibition studies.
A panel of monoclonal antibodies directed against native Fel d I bound to peptides 1-16 and 60–70 of chain 1 and peptides 1–14 and 43–56 of chain 2. For two monoclonal antibodies, binding to each peptide was investigated and shown to be inhibitable by native Fel d I. These antibodies are therefore probably directed against a conformational epitope formed by these four regions.
These studies give us substantial information about the quaternary structure of Fel d I.     

Rational design of hypoallergens applied to the major cat allergen Fel d 1

BACKGROUND: Allergen-specific immunotherapy is the only treatment for allergic disease providing long-lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy. OBJECTIVE: The aim of this study was to provide a rational design of hypoallergen candidates for immunotherapy by using structural information and knowledge of B and T cell epitopes of an allergen. METHODS: The three-dimensional structure of the major cat allergen Fel d 1 was systematically altered by duplication of selected T cell epitopes and disruption of disulphide bonds. Seven Fel d 1 derivatives were generated and screened for allergenic reactivity in comparison with recombinant Fel d 1 in competition-ELISA. The allergenicity was further evaluated in basophil activation experiments and T cell reactivity was assessed in a lymphoproliferation assay. RESULTS: Three out of seven Fel d 1 derivatives, with two duplicated T cell epitopes and one or two disulphide bonds disrupted, were carefully evaluated. The three derivatives displayed a strong reduction in allergenicity with 400-900 times lower IgE-binding capacity than recombinant Fel d 1. In addition, they induced a lower degree of basophil activation and similar or stronger T cell proliferation than recombinant Fel d 1. CONCLUSION: By a rational approach, we have constructed three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell reactivities. This strategy may be applied to any well-characterized allergen to improve immunotherapy for allergic patients.     

High-level expression of immunoreactive recombinant cat allergen (Fel d 1): Targeting to antigen-presenting cells

BACKGROUND: Cat allergen Fel d 1 is a heterodimer encoded by 2 separate genes that has been difficult to produce as a fully immunoreactive molecule. OBJECTIVE: We sought to engineer recombinant (r) Fel d 1 with IgE and IgG antibody binding comparable with that of the natural allergen that could be targeted to antigen-presenting cells. METHODS: The rFel d 1 chains were coexpressed in baculovirus, either linked to the anti-CD64 antibody H22 (rFel d 1 H22(+)) or alone (rFel d 1 H22 (-)). Binding of expressed allergens to mouse and human antibodies was compared with that of natural (n) Fel d 1 by means of enzyme immunoassay and antigen-binding and inhibition RIAs. Binding of rFel d 1 H22 (+) to the CD64 receptor on leukocyte subpopulations and on the THP -1 cell line was analyzed by means of flow cytometry. RESULTS: The baculovirus-expressed allergens migrated with molecular weights of 49 kd (rFel d 1 H22(+)) and 22 kd (rFel d 1 H22 (-)). The rFel d 1 inhibited IgG antibody binding to nFel d 1 by greater than 95% and showed identical dose-dependent inhibition curves. There was an excellent quantitative correlation between IgE and IgG antibody binding to rFel d 1 and nFel d 1 in sera from patients with cat allergy (IgE: n = 258, r = > 0.72,P <.001). The rFel d 1 H22(+) bound to monocytes but not to lymphocytes or neutrophils, and binding of rFel d 1 H22(+) to THP-1 cells was inhibited by a soluble CD64 fusion protein. CONCLUSIONS: Recombinant Fel d 1 chains have been successfully coexpressed as mature proteins with comparable immunoreactivities to nFel d 1. The rFel d 1 can be targeted to antigen-presenting cells through CD64. These constructs will facilitate structural studies of Fel d 1 and the development of improved allergy diagnostics and therapeutics.     

Effects of various vacuum cleaners on the airborne content of major cat allergen (Fel d 1)

Background It has been shown that a vacuum cleaner (VC) can increase airborne cat allergen levels. This study aimed to compare the degree of leakage of airborne Fel d 1 levels among five different VCs, both under laboratory conditions and in an apartment with cats.
Methods Three of the VCs were marketed as antiallergic: a HEPA filter VC (VC A), a water impingement and HEPA filter VC (VC B), and a foam fabric filter VC (VC C). The other two were standard VCs: VC D and VC E. VCs were tested in a 20 m', airtight, experimental room and in a 53 m²* living room in an apartment with three cats. Air was sampled with a glass-fiber filter and an impinger at 20 1/min for 30 min before, during, and after vacuuming. Airborne Fel d 1 was measured with a two-site monoclonal ELISA assay.
Results In the experimental room, no airborne Fel d 1 level was measured before using the VCs. After introducing a dust sample containing Fel d 1 in the VCs. we found that VCs A, B, and E did not provoke any increase in airborne Fel d I. In contrast, VCs C and D significantly increased airborne Fel d 1 levels (GM: 4.9 and 5.3 ng/m, respectively). In the apartment, all VCs induced an increase in airborne Fel d 1, which was carried by particles greater than 5 nm. However, VCs C and D provoked significantly greater increases in airborne Fel d 1 than VCs A, B, and E (P=0.0001).
Conclusions Our results suggest that:

• 1)

  The two VCs with leakage in the experimental room had greater leakages in the apartment.
• 2)

  In the apartment with cats, all VCs provoked increases in airborne Fel d 1, primarily carried by large particles.
• 2)

  Given the increased marketing of "antiallergic" VCs, further studies are needed to standardize methods for testing airborne allergen leakage by VCs.

     

Structure of the major cat allergen Fel d I in different allergen sources: an immunoblotting analysis with monoclonal antibodies against denatured Fel d I and human IgE.

In this paper we show the reactivity of monoclonal antibodies (mAbs) and human IgE with Fel d I from different allergen sources in reduced SDS-PAGE immunoblots. By SDS-PAGE analysis of affinity-purified 125I-Fel d I, a 14- to 20-kD band was found, which dissociated under reducing conditions into a 4- to 5-kD chain (chain 1) and a 11- to 15-kD chain (chain 2). In initial immunoblotting experiments with mAbs against Fel d I however, only chain 1 was detected, while the mAbs lost activity upon reduction of Fel d I. Therefore mAbs were raised against reduced and alkylated Fel d I. Two of the four mAbs to 'denatured' Fel d I that were obtained did react with chain 2 on an immunoblot under reducing conditions; the other two reacted with chain 1. The mAbs did not react with native Fel d I. With these mAbs and human IgE, differences between allergen source materials in blot patterns of Fel d I were detected. A variable molecular weight for the protein stained with mAb antichain 2 was found, and occasionally the presence of a 12-kD band stained with mAb antichain 1. Human IgE strongly bound to chain 1 of Fel d I, while only 2 out of 6 sera gave a strong reaction with chain 2. The additional 12-kD band was also recognized by human IgE. In a competitive radioimmunoassay with mAb antichain 1, differences in levels of 'denatured' Fel d I between commercial extracts were quantitated. In vitro 'denatured' Fel d I was generated under high pH conditions. The reactivity of human IgE with this 'denatured' Fel d I was demonstrated in indirect RAST experiments with mAb antichain 1. We conclude that mAb antichain 1 recognizes a form of Fel d I that is not detected by mAb antinative Fel d I, but does react with human IgE.     

Health impacts of second-hand exposure to cat allergen Fel d 1 in infants

BACKGROUND: Recent cross-sectional studies suggested that highest sensitization prevalences occur with moderate cat allergen exposures. We aimed to assess the impact of moderate levels of second-hand cat allergen exposure on the incidence of specific sensitization and wheezing in the framework of a birth cohort study. Therefore we restricted our analysis to infants without a cat at home since birth. METHODS: At infant's age 3 months, cat allergen levels were measured in the mattress dust of 1840 families without cats. At age 2 years, serum IgE specific to Fel d 1 was analyzed. Incidence of wheezing apart from respiratory infection was assessed by questionnaire. Logistic regression models were used to calculate adjusted odds ratios (OR) for the association between second-hand cat allergen exposure and health outcomes. RESULTS: Until age 2 years, 13 of 1301 infants (1%) were sensitized to cat allergen and 56 of 1492 infants (4%) had ever-wheezing without infection. Early exposure to second-hand cat allergen levels >or= 1 microg/g dust increased substantially the risk for specific sensitization to Fel d 1 (OR 10.9, 95% CI 3.4-35.0) and ever-wheeze without infection (OR 2.0, 95% CI 1.1-3.9) at age 2 years. CONCLUSIONS: Second-hand exposure to cat allergen in homes without cats is detrimental in terms of allergy development in infants.     

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited. OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1. METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies. RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds. CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.     

The effect of reducing levels of cat allergen (Fel d 1) on clinical symptoms in patients with cat allergy.

BACKGROUND: Treatment of cat allergy normally entails removal of the cat from the household, but cat owners are often unwilling to part with their pets, despite clinically relevant allergies. OBJECTIVE: To determine whether levels of Fel d 1 can be reduced without removal of the cat and whether this will affect symptoms of cat allergy. METHODS: Cat-allergic patients underwent randomization to either a group instructed in environmental control (EC) and a group with unchanged environment (UE). Dust samples were obtained and settled Fel d 1 measured by enzyme-linked immunosorbent assay. Patients recorded daily nasal inspiratory flow rates. At baseline, 3 months, and 8 months, patients underwent symptom evaluation. RESULTS: Eighteen patients were randomized to the EC group and 22 to the UE group; the final number completing the study was 31, 15 in the EC group, and 16 in the UE group. At 8 months, home Fel d 1 levels had diminished to 6.8% of baseline levels in the EC group, whereas no reduction in levels was noted in the UE group. In the EC group, significant improvements were found in nasal inspiratory flow rate and symptoms compared with the UE group. Patients did not have difficulties adhering to EC measures. CONCLUSION: A decrease in the allergen load was found in the EC group, which had a significant effect on symptoms of nasal allergy.     

猫主要过敏原Fel d 1的T细胞抗原表位分析及三维结构模拟

目的:预测猫主要过敏原Fel d 1与MHC Ⅰ、MHCⅡ类分子的结合力获得T细胞优势抗原表位,并模拟Fel d 1的三维结构,为猫主要过敏原的改造及其临床研究等提供依据.方法:从Uniprot数据库中得到猫主要过敏原Fel d 1的氨基酸序列,通过在线软件NetMHCⅡ2.2对Fel d 1氨基酸序列进行MHCⅡ抗原表位分析,使用软件SYFPEITHI分析MHC Ⅰ的抗原表位,再通过在线软件Swiss-Model预测Feld1的三维结构,以Ramachandran图评估三维结构的稳定性.结果:运用生物学软件分析得到Fel d 1上两个高值MHCⅡ抗原表位区域分别为22～43、80～95,MHC Ⅰ抗原表位优势区为17～30、56～84、91 ～103.Ramachandran图显示Feld 1的空间构象稳定.结论:本研究有助于确定Fel d 1的T细胞优势抗原表位及其三维结构模型,为Fel d 1抗原性改造提供理论依据,及将来的临床研究奠定基础.     

Dog allergen (Can f 1) and cat allergen (Fel d 1) in US homes: results from the National Survey of Lead and Allergens in Housing

BACKGROUND: Exposures to dog and cat allergens are believed to play important roles in the etiology of asthma; however, the levels of these allergens have never been assessed in a representative sample of US homes. OBJECTIVE: The objective of this study was to estimate and characterize exposures to Can f 1 (dog allergen) and Fel d 1 (cat allergen) in US homes. METHODS: Data were obtained from the National Survey of Lead and Allergens in Housing, a nationally representative survey of 831 US homes. Vacuumed-collected dust samples from the bed, bedroom floor, living room floor, and living room sofa were analyzed for concentrations of Can f 1 and Fel d 1 (micrograms of allergen per gram of dust). RESULTS: Although a dog or cat had lived in only 49.1% of homes in the previous 6 months, Can f 1 and Fel d 1 were detected in 100% and 99.9% of homes, respectively. Averaged over the sampled sites, geometric mean concentrations (microg/g) were 4.69 for Can f 1 and 4.73 for Fel d 1. Among homes with an indoor dog and cat, respectively, geometric mean concentrations were 69 for Can f 1 and 200 for Fel d 1. Among homes without the indoor pet, geometric mean concentrations were above 1.0. The independent predictors of elevated concentrations in homes without pets were all demographic variables that were also linked to a higher prevalence of pet ownership. CONCLUSIONS: Can f 1 and Fel d 1 are universally present in US homes. Levels that have been associated with an increased risk of allergic sensitization were found even in homes without pets. Because of the transportability of these allergens on clothing, elevated levels in homes without pets, particularly among demographic groups in which pet ownership is more prevalent, implicate the community as an important source of these pet allergens.     

Demonstration of a partially cryptic epitope of the major cat allergen Fel d 1: consequences for mAb-based standardization of cat extracts

BACKGROUND: Antiallergen mAbs that do not recognize clinically important isoforms have been described, raising the question of the selection of mAbs for quantifying major allergens in order to standardize allergenic extracts. This question is even more critical if mAbs can discriminate between different forms of allergen molecules with the same amino acid sequence. OBJECTIVE: We sought to demonstrate that an anti-Fel d 1 mAb was able to discriminate between two forms of the major cat allergen independently of its amino acid sequence and to determine the relative importance and stability of both forms in various cat extracts. METHODS: Anti-Fel d 1 mAbs were raised in mice and characterized. By using two of these mAbs, a two-site ELISA was developed to quantify Fel d 1 in mass units. RESULTS: One of the anti-Fel d 1 mAbs developed was shown to specifically recognize a particular form of Fel d 1. A two-site ELISA with this mAb to capture Fel d 1 was able to quantify the allergen specifically in this form. It was then shown that (1) the quantitative importance of this form of Fel d 1 could vary from one cat extract to another, (2) Fel d 1 was converted into this form under certain conditions, and (3) both converted and unconverted forms of Fel d 1 may bear IgE epitopes that are specific. CONCLUSION: Although the present study emphasizes the issue of selecting mAbs that are not too specific to standardize allergenic extracts, it also demonstrates that very specific mAbs can be of interest, especially to verify the stability of allergens in extracts, since this stability might have clinical implications.     

The 18-kDa form of cat allergen Felis domesticus 1 (Fel d 1) is associated with gelatin- and fibronectin-degrading activity

BACKGROUND: Fel d 1, an important allergen from domestic cats, is a significant cause of asthma. In addition to directly promoting IgE synthesis, other biological activities of allergens may contribute to either allergic sensitization or the magnitude of allergic effector responses. For example, allergens that degrade proteins have been suggested to facilitate allergen presentation by increasing parallelular permeability of airways epithelium. However, little information exists to indicate whether Fel d 1 has other activities relevant to allergic responses. OBJECTIVE: To study whether Fel d 1 is associated with enzyme activity. METHODS: Fel d 1 was obtained by a rigorous purification strategy and its identity confirmed by laser desorption mass spectrometry, cleavage and sequencing. The ability of Fel d 1 to degrade gelatin, fibronectin and the artificial substrate N-benzoyl-FVR-p-nitroanilide was studied. The effect of Fel d 1 on the morphology of tight junctions in epithelial cell monolayers was also investigated. RESULTS: The 18-kDa form of Fel d 1 caused degradation of denatured collagens (gelatin) and cleaved a 20-kDa fragment from the A chain of plasma fibronectin. Catalytic activity was not altered by inhibitors of cysteine peptidases, matrix metallopeptidases or by removal of divalent cations. In contrast, aprotinin and TLCK were inhibitors of Fel d 1. The absence of a serine peptidase catalytic triad in Fel d 1, together with the stoichiometry of the inhibition of TLCK and aprotinin, suggest that their inhibitory action may be due to noncatalytic site interactions. Alternatively, highly purified Fel d 1 may be associated with an active contaminant, although none were found. CONCLUSION: These results suggest that Fel d 1 is another example of a domestic allergen which is associated with enzyme activity. It remains to be established whether the activity resides in Fel d 1 itself or in an unresolved, and possibly related, protein.     

Focus on cat allergen (Fel d 1): immunological and aerodynamic characteristics, modality of airway sensitization and avoidance strategies

The increasing frequency of pet ownership (especially cats) in many industrialized countries has raised the level of exposure to the allergens produced by these animals. Moreover, it is likely that modern energy-saving systems and the wide use of upholstered furniture has resulted in closer contact between cats (and their allergens) and humans. Many different methods have been developed to quantify the main cat allergen (Fel d 1) in settled dust and in ambient air. The threshold levels of cat allergen inducing sensitization or triggering respiratory symptoms in sensitized patients have been calculated in settled dust, but airborne amounts of Fel d 1 probably represent a more reliable index of allergen exposure. Noticeably, the amount of Fel d 1 may be relatively high also in confined environments where cats have never been kept. It has been demonstrated that clothes of cat owners are the main source for dispersal of allergens in cat-free environments. This fact may be of relevance, because recent studies have shown that allergic sensitization to cats is more likely to develop in children exposed to moderate levels of this allergen than in children exposed to high amounts of Fel d 1. The ubiquity of cat allergen may justify the common observation that allergen avoidance is often insufficient to reduce the risk of developing allergic sensitization and/or symptom exacerbation in highly susceptible patients. Further efforts are needed to improve the efficacy of Fel d 1 avoidance strategies to try to reduce the risk of allergic sensitization to this allergen.