Catherine McCall

CHARACTERIZATION AND CLONING OF A MAJOR HIGH MOLECULAR WEIGHT HOUSE DUST MITE ALLERGEN (DER F 15) FOR DOGS

Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109kDa (98/109kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15.

Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.

Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

Recombinant canine IL-13 receptor α2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.

Late-phase reactions to intradermal testing with Dermatophagoides farinae in healthy dogs and dogs with house dust mite-induced atopic dermatitis.

OBJECTIVE To determine the prevalence of late-phase reactions to intradermal testing with Dermatophagoides farinae in healthy dogs and dogs with atopic dermatitis and an immediate reaction to D farinae. ANIMALS 6 healthy dogs and 20 dogs with atopic dermatitis and immediate reactions to D farinae. PROCEDURE ntradermal tests were performed with D farinae at 1:1,000 wt/vol and 1:50,000 wt/vol concentrations, and skin reactivity was evaluated after 0.25, 6, and 24 hours. Serum D farinae-specific IgE antibodies were assayed. Extent of lesions (atopy index) and pruritus (visual analogue scale) were evaluated in dogs with atopic dermatitis. RESULTS Late-phase reactions were observed in healthy dogs at 6 hours (n = 2 dogs) and 24 hours (1) with the 1:1,000 wt/vol concentration, and at 6 hours (1) and 24 hours (1) with the 1:50,000 wt/vol concentration of allergen. Late-phase reactions in healthy dogs were only observed in dogs with an immediate reaction to D farinae. Late-phase reactions were observed in 11 of 20 dogs with atopic dermatitis at 6 and 24 hours with the 1:1,000 wt/vol concentration and in 10 of 20 at 6 and 24 hours with the 1:50,000 wt/vol concentration of allergen. There was no difference in mean atopy index, mean visual analogue scale of pruritus, or mean serum D farinae-specific IgE concentration of dogs with a late-phase reaction, compared to dogs without a late-phase reaction. CONCLUSIONS AND CLINICAL RELEVANCE Late-phase reactions may be observed after an immediate reaction to intradermal skin testing in healthy and allergic dogs but are more commonly observed in dogs with atopic dermatitis.

Canine interleukin-5: molecular characterization of the gene and expression of biologically active recombinant protein.

Interleukin-5 (IL-5), which is produced primarily by type 2 T helper lymphocytes (Th2), is an eosinophil differentiation and activation factor. Increased numbers of eosinophils in peripherial blood or tissues (eosinophilia) are observed in asthmatic human patients, in animals with helminth infections, and in dogs with allergic diseases. Antagonism of IL-5 activity is being explored as a potential treatment of a number of disease conditions associated with eosinophils in animal models. In order to study the expression and function of this cytokine in the dog, we have isolated and characterized the canine IL-5 gene. The canine IL-5 polypeptide deduced from the cDNA is composed of 134 amino acids that share varying degrees of homology with IL-5 isolated from several mammals. The genomic structure of the canine IL-5 gene consists of four exons and three introns in the coding region, similar to that of the previously characterized human and mouse IL-5 genes. Recombinant canine IL-5 protein, expressed in Pichia pastoris, is biologically active in a cell proliferation assay. Canine IL-5 gene sequences and the biologically active protein described in this study will be useful reagents for future studies of this cytokine in physiologic processes and in pathologic conditions of the dog.