Epitope-specific T-cell responses and allergic phenotypes: implications for T-cell peptide therapy

In the 1990s, elucidation of the primary amino acid sequence of several major allergens using molecular cloning techniques opened the door to T-cell epitope mapping studies. Such analyses underscored the complexity of the allergen-specific T-cell repertoire and the challenges to using allergen-derived peptides to identify epitope-specific differences associated with allergic and nonallergic responses. This review highlights important factors that may influence the nature of epitope-specific T-cell responses observed in vitro. These include the properties of the allergen, genetics of the host and selection of patients with defined allergic phenotypes based on serum antibody profiles and skin test reactivity. By taking these factors into account, T-cell epitope-specific differences associated with distinct allergic phenotypes can be identified. Observations at the T-cell epitope level undermine the Th1/Th2 paradigm as a model for the development of allergic versus nonallergic responses. Instead, they support the mounting data that point to a network of interactions between T helper cells and regulatory T cells, which controls the allergic response. The ability of peptides that localize to polypeptide chain 2 of the major cat allergen, Fel d 1, to preferentially induce interleukin-10 and interferon-γ is discussed. Mechanisms whereby specific allergen-derived peptides may modify the T-cell repertoire and influence the immune outcome are also outlined. Further investigation of allergen-derived T-cell epitopes is warranted in order to optimize the design of peptide vaccines for the treatment of allergic disease.     

Serum IgG and IgG4 antibodies to Fel d 1 among children exposed to 20 microg Fel d 1 at home: relevance of a nonallergic modified Th2 response

Exposure to foreign antigens is an essential element of all immune responses, including allergic sensitization. For some allergens (e.g. mite and cockroach), the prevalence of sensitization is directly correlated with exposure. However, for allergens derived from domestic animals, several studies have suggested that children with a cat in the home have a decreased risk of sensitization and asthma. We have now shown that many children exposed to greater than 20 microg of Fel d 1/g of dust at home made an IgG and IgG4 antibody response to Fel d 1 without IgE antibody. This modified Th2 response is not associated with symptoms and should be regarded as a form of immunological tolerance. The fact that the dose-response relationship between cat exposure and sensitization is bell shaped, while that for mite exposure and sensitization is linear, is highly relevant to understanding the role of allergens in the increase in allergic disease.     

Fel d 4, a cat lipocalin allergen

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals. OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels. METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed. RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog. CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.     

High prevalence of sensitization to cat allergen among Japanese children with asthma, living without cats

BACKGROUND: Cat allergy is common among children with asthma. Many cat-allergic patients in Japan and elsewhere do not keep cats, but nonetheless become sensitized through environmental exposure to cat allergen. OBJECTIVE: To assess the frequency of cat allergy and cat-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibody responses in young Japanese patients with asthma in relation to self-reported cat exposure and Fel d 1 levels in dust samples. METHODS: Cat dander-specific IgE antibody was measured in sera from asthma patients using the CAP system. IgE and IgG antibody to Fel d 1 was measured by antigen binding radioimmunoassay and by chimeric enzyme immunoassay. Fel d 1 levels in dust samples from a subset of patients' homes were measured by monoclonal antibody-based enzyme immunoassay. RESULTS: Cat-specific IgE (CAP class>/=2) was found in sera from 70% of 44 patients who kept cats and 34% of 394 patients who had never kept cats. The prevalence of sensitization increased progressively to age 6 years (40%: positive), and then increased gradually to age 16 years (approximately 60%: positive) in patients who had never kept cats. There was an excellent correlation between cat CAP values and IgE levels to Fel d 1. The absolute amount of IgE antibody to Fel d 1 ranged from 0.01 to 15.6% of total IgE. Most patients who did not keep cats were exposed to Fel d 1 levels ranging from 0.07-8 microg/g dust. CONCLUSIONS: Sensitization to cat allergen is common among young asthmatic patients in Japan, even among patients who do not keep cats. Use of CAP and the chimeric enzyme-linked immunosorbent assay allows accurate diagnosis of cat allergy and quantification of specific IgE antibody levels.     

Human T and B cell immune responses to Fel d 1 in cat-allergic and non-cat-allergic subjects

Background In allergic individuals exposure to allergen leads to the induction of allergen-specific IgE which, upon binding to its high affinity receptors on mast cells and basophils. primes these cells for degranulation. This degranulation. a result of specific IgE allergen-interaction, initiates the debilitating symptoms of allergy and the potentially life-threatening symptoms of anaphylaxis. The lack of symptoms following antigen encounter by non-allergic individuals is probably due to the undetectable levels of allergen-specific IgE in the plasma of non-allergic individuals. Objective To compare the immune responses of allergic and non-allergic individuals. Method We compared the immune responses of 42 cat-allergic subjects with 16 nem-cat-allergic subjects to the major cat allergen. Fel d I. We have measured plasma immunoglobulin levels and the proliferative responses of fel d 1 primed T cell lines to Fel d 1 peptides. Results While these two groups have similar levels of Fel d 1 specific IgG. only subjects in the cat-allergic group have detectable Fel d 1 specific IgE. Affinity purified Fel d 1 was used to generate T cell lines from peripheral blood mononuclear cells of these same subjects. The proliferative responses of these T cell lines to intact Fel d 1 and a set of overlapping peptides covering the entire sequence of the molecule demonstrated that the pattern of epitope recognition was similar in both groups. Conclusion Our data suggest that factors other than T cell recognition of specific epitopes are responsible for the nature of allergic immune responses generated when allergen is encountered.     

Longitudinal study on the relationship between cat allergen and endotoxin exposure, sensitization, cat-specific IgG and development of asthma in childhood--report of the German Multicentre Allergy Study (MAS 90)

BACKGROUND: Controversial data have emerged regarding the question whether cat exposure in childhood favours or decreases the risk of sensitization and allergic airway disease. In a prospective birth-cohort study, we assessed the association between longitudinal cat allergen exposure, sensitization (immunoglobulin E, IgE), IgG antibody (ab) levels to cat and the development of asthma in children up to the age of 10 years. METHODS: Of 1314 newborn infants enrolled in five German cities in 1990, follow-up data at age 10 years were available for 750 children. Assessments included yearly measurements of specific serum IgE to cat and at age 6 and 18 months, 3, 4 and 10 years measurement of cat allergen Fel d 1 in house dust samples. Additionally, Fel d 1-specific IgG ab were determined in 378 serum samples of 207 children. Endotoxin exposure in mattress dust was measured in a subgroup of 153 children at age 10 years. From age 4 years on, International Study of Asthma and Allergy in Childhood (ISAAC) questionnaires were completed yearly in order to assess the prevalence of wheeze and asthma. RESULTS: Serum IgG-levels to cat showed a large variation, however, intraindividually values showed rather constant concentration over a longer time period. The IgG levels at school-age correlated with cat allergen exposure during the first 2 years of life. Specific IgE to cat was clearly associated with wheeze ever, current wheeze and bronchial hyperresponsiveness (BHR), this was also observed for children with specific IgE ab to cat (>0.35 kU/l) plus IgG levels above 125 U/ml. A large percentage of very highly exposed children showed high IgG but no IgE responses to cat, however, not all highly exposed children were found to be protected from sensitization. Children with IgG but without IgE ab to cat showed the lowest prevalence of wheeze ever and current wheeze despite high cat allergen exposure, however, this trend did not achieve significance. While homes of cat owners showed higher Fel d 1 concentrations than homes without cats, homes of cat owners were not found to have higher endotoxin levels in carpet dust samples than homes without cats. CONCLUSIONS: We could confirm that high cat allergen exposure in a cohort with lower community prevalence of cats is associated with higher serum IgG and IgE levels to cat in schoolchildren. Sensitization to cat allergen (IgE) is a risk factor for childhood asthma. While exposure to cat allergen during infancy is associated with sensitization (IgE), only in the very highly exposed children the likelihood of sensitization (IgE) is decreased and high IgG levels to cat without IgE were associated with low risk of wheeze. However, cat-specific IgG ab levels did not protect children with IgE-mediated sensitization from wheeze.     

Cat and dust mite sensitivity and tolerance in relation to wheezing among children raised with high exposure to both allergens

BACKGROUND: Recent evidence has suggested that high exposure to cat allergens is associated with decreased prevalence of sensitization to cat and, in some studies, decreased asthma. OBJECTIVE: Our objective was to study antibodies to cat and mite allergens and their relationship to wheezing in a country with high exposure to both allergens. METHODS: Sera from 112 wheezing and 112 control children aged 10 to 11 years in a nested case-control study in New Zealand were assayed for specific IgE antibody, as well as IgG antibody and IgG4 antibody, to Der p 1 and Fel d 1. RESULTS: IgE antibody to both mite (99/224) and cat (41/224) were strongly associated with wheezing (odds ratios, 5.2 and 6.5, respectively). Children who had ever lived with a cat were less likely to have IgE antibody to cat (20/141 vs 21/83, P < .04); however, cat ownership had no effect on IgE antibody to mite (67/141 vs 32/83, P = .23). Among sensitized children, cat ownership was associated with a lower prevalence of IgE antibody to cat (28% vs 66%, P < .001), and this analysis remained significant after exclusion of children whose families had chosen not to own a cat. Among sensitized subjects, the mean titer of IgE antibody to cat (1.7 IU/mL) was 10-fold lower than for mite (22.1 IU/mL). A cat in the home had no significant effect on endotoxin or mite allergen in house dust, whereas cat allergen was much higher (40.8 vs 3.3 microg/g). CONCLUSION: The response to these 2 allergens was distinct on the basis of the prevalence of sensitization, the titer of IgE antibody, and the effect of cat ownership. The results suggest that induction of tolerance to cat allergen is an allergen-specific phenomenon that cannot be attributed to endotoxin or family choice. The strength of the IgE antibody response to dust mite in humid climates could contribute to the increased prevalence and severity of asthma.     

Priming with high and low respiratory allergen dose induces differential CD4+ T helper type 2 cells and IgE/IgG1 antibody responses in mice

Sensitization of allergic patients normally takes place over several years and is the result of repeated exposure to low levels of allergen. Most mouse asthma models use a high dose of allergen administered over a short period. We have investigated the role of dose in the immune response to an inhaled respiratory allergen (Blomia tropicalis). We observed the effect of priming dose on the allergic response in mice intranasally immunized with low (0·5 μg) and high (50 μg) doses of B. tropicalis extract and killed 1 day after the last challenge. For both doses of allergen, T helper type 2 (Th2) cells and Th2 cytokines were evident as well as eosinophilic inflammation accompanied by mucus hyper‐secretion. By contrast, IgE and IgG1 antibody responses were normally only detected at high‐dose priming. To investigate the mechanism for these effects, we found group 2 innate lymphoid cells (ILC2s) were increased 48 hr after challenge in the low‐dose‐treated but not the high‐dose‐treated mice. Furthermore, we determined whether repeated low‐dose exposure with different priming protocols could induce an antibody response. Repeated low‐dose exposure to 0·5 μg three times weekly for 4 weeks (cumulative 6 μg) had the same effect as a shorter high‐dose exposure (cumulative 80 μg) and increasing cumulative dose induced antibody responses. These data indicate that low doses of allergen are sufficient to prime Th2 cells and ILC2s, but insufficient to induce antibody responses. Cumulative exposure to small amounts of allergen induces both Th2 and antibody responses and may better reflect natural sensitization.     

The cat lipocalin Fel d 7 and its cross‐reactivity with the dog lipocalin Can f 1

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensitized Swedish patients and elucidated its allergenic activity and cross‐reactivity with the dog lipocalin Can f 1. Sixty‐five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross‐reactive antisera. Fel d 7 is a common allergen in a Swedish cat‐sensitized population that cross‐reacts with Can f 1, and may contribute to symptoms in cat‐ but also in dog‐allergic patients.     

Exposure and sensitization in infants and children

The complex relationship between allergen exposure, atopic sensitization and asthma in individuals and in populations has recently been a subject of controversy. A number of studies have demonstrated that allergen exposure in sensitized asthmatic individuals increases the severity of disease. A simple dose-response relationship between mite allergen exposure and specific sensitization in infants and children has been confirmed. However, the concept that there is a direct relationship between allergen exposure and the initiation of asthma has been challenged. The relationship between allergen exposure and subsequent disease development is complex, and is confounded by a number of important factors. Populations and individuals are exposed to a mixture of several allergens, irritants and pollutants, and we know very little about the impact of these mixtures and their possible synergistic effect. The dose-response relationship between allergen exposure and allergic disease may not be linear, and may be different for different allergens. For example, a protective effect of cat ownership on sensitization and allergic disease has been reported, raising the question of whether the dose-response relationship between exposure and sensitization may be different for cat compared with mite allergen. It has been suggested that many children who are exposed to a high level of cat allergen make a modified T helper type 2 response, characterized by the presence of IgG4 antibody to cat proteins without becoming allergic (i.e. no IgE response), which could be regarded as a form of tolerance. This could explain a decreased risk of asthma in children living in homes with cats, without invoking a concept of a shift in the balance of T helper types 1 and 2 responses. Cat allergen is ubiquitous, and passive exposure (e.g. home without cats and public places) may induce specific IgE responses in non-cat owners, whereas those exposed to very high levels of cat allergen may initially mount an IgE response, which may be replaced by a modified T helper type 2 response (tolerance). It is likely that the population susceptibility to allergic sensitization and also end-organ responsiveness has altered, and allergen exposure may still be important in initiating disease in an increasingly susceptible population, although the pattern may differ for different allergens.     

Mechanisms of peripheral tolerance to allergens

The immune system is regulated to protect the host from exaggerated stimulatory signals establishing a state of tolerance in healthy individuals. The disequilibrium in immune regulatory vs effector mechanisms results in allergic or autoimmune disorders in genetically predisposed subjects under certain environmental conditions. As demonstrated in allergen‐specific immunotherapy and in the healthy immune response to high‐dose allergen exposure models in humans, T regulatory cells are essential in the suppression of Th2‐mediated inflammation, maintenance of immune tolerance, induction of the two suppressive cytokines interleukin‐10 and transforming growth factor‐β, inhibition of allergen‐specific IgE, and enhancement of IgG4 and IgA. Also, suppression of dendritic cells, mast cells, and eosinophils contributes to the construction of peripheral tolerance to allergens. This review focuses on mechanisms of peripheral tolerance to allergens with special emphasis on recent developments in the area of immune regulation.     

Mechanisms of tolerance to inhalant allergens: the relevance of a modified Th2 response to allergens from domestic animals

Subjects can be non-allergic because (1) they are not exposed, (2) they fail to make an immune response, or (3) they make an immune response that does not include IgE antibodies (Ab). The recent observation that children raised in a house with a cat are less likely to become allergic to cat allergen than those who only get indirect exposure provides a model to investigate the factors controlling allergic responses. Many of these highly exposed children have made an IgG and IgG4 Ab response to Fel d 1 without IgE Ab, i.e., a modified Th2 response. In countries where cats are a major cause of asthma, the presence of a cat may decrease the risk of asthma. By contrast, in countries with high exposure to dust mites, cats can induce specific tolerance to Fel d 1 without influencing asthma or the IgE Ab response to dust mites. Using overlapping peptides to investigate T cell responses to Fel d 1 suggests that the structure of the molecule plays a special role in inducing the T cell responses that can control the immune response to cat allergens. This T cell response is characterized by high levels of IL-10 production, but this is not restricted to those who have made a modified Th2 response. The results suggest that there are major differences in the immune response to different allergens that profoundly affect their role in allergic disease. Dust mite and cockroach differ from cat (and rat) allergens not only in the quantity inhaled and the particles sizes but also in the biochemistry of the molecule.     

The relevance of maternal immune responses to inhalant allergens to maternal symptoms,passive transfer to the infant,and development of antibodies in the first 2 years of life

BACKGROUND: Asthma and other atopic diseases are strongly hereditary. Although the mother might play a special role, the mechanisms for such an effect are not clear. OBJECTIVE: We sought to investigate the influence of maternal immune responses to cat and mite allergens on (1) maternal symptoms, (2) the development of immune responses in the infant, and (3) the development of allergic disease during the first 3 years of life. METHODS: In sera from 465 mothers and 424 infants (cord blood), as well as in sera from 230 of the children at age 2 to 3 years, total IgE and IgE antibodies were measured by using CAP testing; IgG and IgG4 antibodies for the cat allergen Fel d 1 were measured by means of radioimmunoprecipitation. RESULTS: In both mothers and children, approximately 15% of sera contained IgG antibodies to Fel d 1 without IgE antibodies to cat. The strongest predictor of the maternal IgG antibody response was exposure to greater than 8 microg of Fel d 1/g of dust. Thus approximately 70% of children living in a house with a cat had received IgG antibodies from their mothers. In many cases the infant received IgG and IgG4 antibodies to Fel d 1 from a nonallergic mother. Maternal IgE antibodies were consistently associated with asthma; by contrast, the IgG antibody was not independently related to asthma but was related to rhinitis in the mothers (odds ratio, 2.6; 95% CI, 1.1-6.2) and to eczema in children. At age 3 years, 13 of 230 sera contained IgE antibodies to mite, but only 5 had IgE antibodies to cat. CONCLUSIONS: A significant proportion (approximately 15%) of mothers and children exposed to high concentrations of cat (but not mite) allergens have serum IgG antibodies without IgE antibodies. This IgG antibody is freely transferred to the infant and might influence IgG antibody production in the child. The results indicate the importance of understanding the mechanisms of tolerance to cats and raise questions about the independent role of the mother in the inheritance of allergy.     

Cat sensitization according to cat window of exposure in adult asthmatics

Background In adults, there is limited information on tolerance to cat, which may be reflected by high IgG₄ without IgE sensitization. Early exposure to cat may play a critical role.
Objective The aim was to assess among adults the association of Fel d 1 IgG₄, Fel d 1 IgE, skin prick test (SPT) response to cat and pet-related symptoms in relation to exposure to cat considering the period of exposure.
Methods SPT response to cat, specific IgE and IgG₄ to Fel d 1 were assessed in 167 asthmatics recruited in chest clinics (40 years of age in average) from the French Epidemiological study on the Genetics and Environment of Asthma (EGEA). Childhood and/or current exposure to cat were studied retrospectively.
Results IgG₄ was higher in relation to current cat exposure (0.53 vs. 0.09 ng/mL; P <0.001) and higher in women than in men. The period of cat exposure was significantly related to Fel d 1 IgE, the IgE/IgG₄ pattern and cat weal size. The lowest values of Fel d 1 IgE, cat weal size, pet-related nasal or respiratory symptoms were observed in those with both childhood and current exposure as well as the highest proportion of the IgE⁻/IgG₄⁺ pattern observed in 1.4%, 4.0%, 38.1% and 12.5% of those with −/−, +/−, +/+, −/+ childhood/current exposure, respectively.
Conclusions Adult asthmatics exposed to cats since childhood present an immunologic pattern with high IgG₄ and low IgE. Continuous exposure may maintain a state of immunological tolerance to cat.     

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.     

Allergens and their role in the allergic immune response

Allergens are recognized as the proteins that induce immunoglobulin E (IgE) responses in humans. The proteins come from a range of sources and, not surprisingly, have many different biological functions. However, the delivery of allergens to the nose is exclusively on particles, which carry a range of molecules in addition to the protein allergens. These molecules include pathogen-associated molecular patterns (PAMPs) that can alter the response. Although the response to allergens is characterized by IgE antibodies, it also includes other isotypes (IgG, IgA, and IgG4), as well as T cells. The challenge is to identify the characteristics of these exposures that favor the production of this form of response. The primary features of the exposure appear to be the delivery in particles, such as pollen grains or mite feces, containing both proteins and PAMPs, but with overall low dose. Within this model, there is a simple direct relationship between the dose of exposure to mite or grass pollen and the prevalence of IgE responses. By contrast, the highest levels of exposure to cat allergen are associated with a lower prevalence of IgE responses. Although the detailed mechanisms for this phenomenon are not clear, it appears that enhanced production of interleukin-10 in response to specific Fel d 1 peptides could influence the response. However, it is striking that the animal sources that are most clearly associated with decreased responses at high allergen dose are derived from animals from which humans evolved more recently (～65 million years ago). Although the nose is still recognized as the primary route for sensitization to inhalant allergens, there is increasing evidence that the skin is also an important site for the generation of IgE antibody responses. By contrast, it is now evident that delivery of foreign proteins by the oral route or sublingually will favor the generation of tolerance.     

The role of indoor allergen exposure in the development of sensitization and asthma

The role of indoor allergen exposure in the development of sensitization and asthma remains a subject of controversy. From a number of cross-sectional and longitudinal studies we can conclude that there is a very close association between allergen exposure and the sensitization of an individual. The dose-response relationships seem to differ between allergens; house dust mite and cockroach allergens appear to have a positive linear relationship, whereas cat allergens appear to act quite differently, with maximum sensitization developing at moderate exposure levels. Very low levels of cat allergen exposure are likely to induce no response and very high levels are likely to develop a form of tolerance, with a modified T helper cell type 2 response and the production of IgG4 antibodies and but not IgE. The relationship between indoor allergen exposure and asthma is, however, less clear. The proposed mechanism for the development of disease is that allergen exposure causes sensitization, and continued exposure leads to airway responsiveness and inflammation. As yet, the evidence for allergen exposure being a primary cause of asthma remains weak, and the results of ongoing prospective, randomized allergen avoidance trials are awaited to clarify this issue.     

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.     

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

BACKGROUND: Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. OBJECTIVE: To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. METHODS: Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. RESULTS: The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). CONCLUSION: The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.     

Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy

BACKGROUND: Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.