Carsten Schou

Assay for the major dog allergen, Can f I: Investigation of house dust samples and commercial dog extracts

Monospecific rabbit antibodies were used to develop a sensitive two-site enzyme immunoassay to measure a major dog hair and dander allergen, Can f I. This Can f I assay demonstrated no reaction with 17 heterologous allergen sources, including dog albumin, cat, guinea pig, and horse. Analysis of serial dilutions of purified Can f I and the international standard for dog was parallel. The assay was considered specific for Can f I with a lower limit of detection at 0.03 micrograms/ml. Total imprecision was from 2% to 6%. Commercial dog extracts for specific immunotherapy contained from 0.7 to 290 micrograms of Can f I per milliliter. The assay was used to measure Can f I in 136 house dust samples collected from 103 homes across the United States. Concentration of the dog allergen was expressed as micrograms of Can f I per gram of dust. Prevalence of Can f I in the dust samples ranged from less than 0.3 to 10,000 micrograms/gm. Serial dilutions of samples containing Can f I were parallel to the standard. The median Can f I value for homes with a dog in residence was 120 micrograms/gm, and for homes with no dog, 3 micrograms/gm. With few exceptions, homes with no dog in residence had less than 10 micrograms/gm. This Can f I assay will provide useful information for assessing commercial extracts as well as monitoring dog-allergen exposure and allergen-control methods.

Dense mapping of chromosome 12q13.12-q23.3 and linkage to asthma and atopy

BACKGROUND Asthma is a complex disease characterized by a high prevalence of allergic diathesis and the almost ubiquitous presence of upper airway disease (eg, rhinitis). Previously, we observed linkage of asthma among Afro-Caribbean families to markers in chromosome 12q, which contains a number of genes encoding for products closely related to allergic airway inflammation and disease. OBJECTIVE To identify susceptibility loci in chromosome 12q contributing to the genetics of upper and lower airway diseases and to expand the region to include genes encoding IFN-gamma (IFNG ) and one of the signal transducers and activators of transcription (STAT6 ), we conducted further linkage studies among 33 multiplex families. METHODS We characterized 528 subjects from Barbados for asthma; 82% were characterized for allergic rhinitis. Two-point and multipoint linkage analysis of 22 microsatellite markers (spanning approximately 79 centimorgan) was performed. RESULTS Affected sib-pair analysis revealed significant evidence for linkage to asthma over approximately 30 cM (P <.05 to.002), with the best evidence for linkage at a CA repeat polymorphism in the first intron of IFNG in 12q21.1 (P =.002). Evidence of linkage to allergic rhinitis was observed in the same region (D12S313, P = 0.006, and IFNGCA, P =.01, respectively). Multipoint linkage analysis also provided evidence for linkage to asthma, with the best nonparametric linkage analysis score at D12S326 (nonparametric linkage score = 3.8, P =.0008). Modest evidence for linkage to allergic rhinitis was observed next to D12S326 at D12S1052 (P =.036). CONCLUSIONS Our findings suggest that (1) one or more loci in the chromosome 12q13. 12-q23.3 region are contributing to the expression of the clinical phenotype asthma and the strongest evidence for linkage is in a region near the gene encoding IFNG and (2) a susceptibility locus for both asthma and allergic rhinitis maps to this region.

Identification and purification of an important cross-reactive allergen from American (Periplaneta americana) and German (Blattella germanica) cockroach

Aqueous whole body extracts from two major domiciliary cockroaches, the American, Periplaneta americana, and the German, Blattella germanica, were analyzed in crossed immunoelectrophoresis and immunoblotting. Forty-five antigens were found in P. americana and 29 in B. germanica. IgE-binding antigens were identified by crossed radioimmunoelectrophoresis with sera from 30 cockroach-allergic patients. Seven and three precipitates from P. americana and B. germanica bound significant amounts of IgE. A cross-reactive, apparently homologous allergen, from P. americana and B. germanica bound IgE from 100% and 70%, respectively, of the patients. These important allergens were tentatively named Per a I and Bla g I. The allergens were purified by sequential ion exchange, gel filtration, and isoelectric focusing. Both allergens had a molecular size of 33 to 37 kd in Sephadex G-75 gel filtration, and 28 kd in high-performance liquid chromatography gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a minor band at approximately 25 kd, and most of the protein at 6 kd. The isoelectric point of both allergens was found be to 3.5. In amino acid analysis, the allergens were highly similar. Skin test revealed the allergens to be important in vivo sensitizing agents. The allergens may be used for environmental assays for cockroach exposure in the homes of allergic subjects.

Association between quantitative traits underlying asthma and the HLA-DRB1 locus in a family-based population sample

The region of human chromosome 6 containing the MHC has been identified as influencing asthma and atopy (allergy) by several genome-wide searches. The MHC contains many genes with potential effects on innate and specific immunity. As a first step in dissecting MHC influences on asthma and its underlying quantitative phenotypes, we have examined the HLA-DRB1 locus in a population sample consisting of 1004 individuals from 230 families from the rural Australian town of Busselton. The locus was strongly associated with the (loge) total serum IgE concentration, accounting for 4.0% of the σ2 (variance) in that trait (multi-allelic test, P=0.00001). The locus also influenced specific IgE titres to common allergens (multi-allelic tests, 2.8% σ2 for the house dust mite allergen Der p I, P=0.0013; 3.0% of σ2 for Der p II, P=0.0007; and 2.1% of σ2 for the cat allergen Fel d I, P=0.014). No associations were found to the categorical phenotype of asthma, or to the quantitative traits of peripheral blood eosinophil counts and bronchial hyper-responsiveness. Transmission disequilibrium tests excluded genetic admixture as a cause of false-positive findings. The results indicate that HLA-DRB1 alleles modulate the total serum IgE concentration and IgE responses to allergens, but do not account for the previous observations of linkage of asthma to the MHC.

Genetic influences of chromosomes 5q31-q33 and 11q13 on specific IgE responsiveness to common inhaled allergens among African American families

BACKGROUND We have recently conducted a genome-wide screening for genes influencing Dermatophagoides pteronyssinus-specific IgE responsiveness as a part of the Collaborative Study on the Genetics of Asthma (CSGA), which showed evidence for linkage in some regions, including chromosomes 5131-q33 and 11q13 in African American families. OBJECTIVES To clarify relative contributions of these regions to atopy in the same African American population, we have conducted further genetic linkage studies of specific IgE responses toward common inhaled allergens. METHODS We studied 328 individuals in 58 African American families participating in the CSGA. Specific IgE responses toward Dermatophagoides farinae, cat, dog, American cockroach, rye grass, and Bermuda grass, as measured by skin tests, were used for multipoint linkage analysis with polymorphic markers on chromosomes 5q31-q33 and 11q13. RESULTS Specific IgE response toward American cockroach showed evidence for linkage to chromosomes 5q31-q33 (P = .0050) and 11q13 (P = .017). Specific IgE response toward dog showed evidence for linkage with chromosome 5q31-q33 (P = .0043). Evidence for linkage with chromosome 11q13 was obtained for specific IgE responses toward Dermatophagoides farinae (P = .012), cat (P = .035), and Bermuda grass (P = .017). The presence of a positive ST response for at least 1 of 30 common allergens showed evidence for linkage to chromosomes 5q31-q33 (P = .017) and 11q13 (P = .00058). CONCLUSIONS These data support that genes on both chromosomes 5q31-q33 and 11q13 confer susceptibility to upregulated IgE-mediated immune responses in this African American population. The putative genes on chromosomes 5q31-q33 and 11q13, however, showed contrasting effects on atopy, which may result from strong gene-environmental interactions.

Purification and characterization of the major dog allergen, Can f I

An important dog‐hair and dander‐specific allergen Ag13 has been purified by means of immunoaffinity chromatography utilizing rabbit antibody specific for Ag13. Purity was judged to be very high as detected by crossed immunoelectrophoresis and SDS‐PAGE. The purified allergen was subjected to amino acid analyses. Molecular weight was about 22 kD in HPLC‐gel filtration and 25 kD in SDS PAGE with an additional band at 18 kD. In vitro IgE binding of the allergen was investigated by luminescence immunoassay (LIA) inhibition. Removal of Ag13 from dog hair and dander extract (DHD) removed 50 ± 1.5% of the IgE binding capacity. The purified allergen inhibited up to 56.5% of the IgE activity to DHD as measured with a pool of serum from dog‐allergic patients. Out of 26 dog‐allergic patients, 24 had a positive skin‐prick test to the allergen. Out of 23 dog‐allergic patients, 16 reacted with the allergen in IgE immunoblotting. We suggest that Ag13 be termed Can f I. The allergen will be a marker allergen for environmental dog hair and dander exposure.

Germline TCR-A restriction of immunoglobulin E responses to allergen.

Abstract Immunoglobulin E responses to known environmental antigens (allergens) may serve as a general model to investigate germline genetic restriction of the immune response. We have previously shown genetic linkage between IgE responses to major allergens and the T-cell receptor (TCR) A/D locus, but not to TCR-B, implying that elements in TCR A/D restrict the ability to react to specific antigens. We now show, in two sets of subjects from the same population, a strong allelic association between a VA8.1 polymorphism (VA8.1*2) and reactivity to Der p II, a major antigenic component of the house dust mite Dermatophagoides pteronyssinus. Association was also seen between Der p II IgE titres and HLA-DRB1*1501 alleles. Reactivity to Der p II was confined to subjects who were positive for VA8.1*2 and HLA-DRB1*1501, demonstrating germline HLA-DR and TCR-A interaction in restricting the response to exogenous antigen.

Crossreactivity and T‐cell epitope specificity of Bet v 1‐specific T cells suggest the involvement of multiple isoallergens in sensitization to birch pollen

Background Allergen‐specific T lymphocytes play an important role in the pathophysiology of atopic disease. Detailed studies of their epitope‐specificity and crossreactivity are required for the development of novel approaches for specific immunotherapy.

Environmental assay for cockroach allergens

A sandwich ELISA was developed to measure the concentration of cockroach allergen in the environment. The assay was based on a monospecific rabbit antibody preparation reactive with determinants shared by the important allergens, Per a I and Bla g I, from American and German cockroaches. The sensitivity was 0.2 ng Lowry protein of Per a I equivalents per milliliter, corresponding to 1 ng of Per a I equivalents per gram of dust (Per a I eq/gm). The assay did not react with noncockroach-allergen sources. Dust samples from 73 households in a cockroach-infested area were assayed. The concentration in these samples varied from below detection to 200,000 ng of Per a eq/gm of dust. Three commercial cockroach-allergen extracts all contained the allergen. The assay will be valuable for studies of the clinically relevant cockroach-allergen exposure levels and for assessment of efficacy of allergen-avoidance measures. Furthermore, the assay could be used for sanitary documentation in bakeries, restaurants, etc.

Genetic Basis of IgE Responsiveness: Relevance to the Atopic Diseases

Genetic analysis of 170 subjects in 11 extended Amish families revealed evidence for linkage of five markers in chromosome 5q31.1 with a gene controlling total serum IgE levels. No linkage was found between these markers and specific IgE antibody levels. Analysis of total IgE within a subset of 128 IgE-antibody-negative sib pairs confirmed evidence for linkage to 5q31.1, especially IL4 (p = 4 x 10(-6)). These and other data suggest that IL4 or a nearby gene regulates IgE production in a non-antigen-specific (noncognate) fashion and provide evidence for a possible link between asthma and the IL4 gene.