Canine Allergy Research Articles

Canis familiaris allergen Can f 7: Expression, purification and analysis of B cell epitopes in Chinese children with dog allergies.

Dogs are a major source of indoor allergens. However, the prevalence of dog allergies in China remains unclear, especially in children. In the present study, Can f 7, a canine allergen belonging to the Niemann pick type C2 protein family, was selected to study its sensitization rate in Chinese children with dog allergies. The Can f 7 gene was subcloned into a pET‑28a vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant Can f 7 was purified by nickel affinity chromatography, identified by SDS‑PAGE electrophoresis, and had its allergenicity assessed by western blot, ELISA and basophil activation tests. Through a series of bioinformatical approaches, B‑cell epitopes, secondary structures, and 3 dimensional (3D) homology modeling of Can f 7 were predicted. The activity of the B cell epitopes was verified by ELISA. The recombinant Can f 7 showed a distinct band with a molecular weight of 14 kDa. Six of 20 sera from dog‑allergic children reacted positively to the Can f 7. Can f 7 induced an ~4.0‑fold increase in cluster of differentiation 63 and C‑C motif chemokine receptor R3 expression in basophils sensitized with the serum of dog‑allergic children compared with those of non‑allergic controls. The secondary structure analysis showed that Can f 7 contains 6 β‑sheets. Five B cell epitopes of Can f 7 were predicted, and two of these were confirmed by ELISA. These results indicate that Can f 7 is an important canine allergen in Chinese children and provide novel data for further research concerning the use of Can f 7 in the diagnosis and treatment of Chinese children with canine allergy symptoms.

Mite allergoids coupled to nonoxidized mannan from Saccharomyces cerevisae efficiently target canine dendritic cells for novel allergy immunotherapy in veterinary medicine

We have recently reported that grass pollen allergoids conjugated with nonoxidized mannan of Saccharomyces cerevisae using glutaraldehyde results in a novel hypoallergenic mannan-allergen complex with improved properties for allergen vaccination. Using this approach, human dendritic cells show a better allergen uptake and cytokine profile production (higher IL-10/IL-4 ratio) for therapeutic purposes. Here we aim to address whether a similar approach can be extended to dogs using canine dendritic cells. Six healthy Spanish Greyhound dogs were used as blood donors to obtain canine dendritic cells (DC) derived from peripheral blood monocytes. Allergens from Dermatophagoides farinae mite were polymerized and conjugated with nonoxidized mannan. Nuclear magnetic resonance (NMR), gel electrophoresis (SDS-PAGE), immunoblotting and IgE-ELISA inhibition studies were conducted to evaluate the main characteristics of the allergoid obtained. Mannan-allergen conjugate and controls were assayed in vitro for canine DC uptake and production of IL-4 and IL-10. The results indicate that the conjugation of D. farinae allergens with nonoxidized mannan was feasible using glutaraldehyde. The resulting product was a polymerized structure showing a high molecular weight as detected by NMR and SDS-PAGE analysis. The mannan-allergen conjugate was hypoallergenic with a reduced reactivity with specific dog IgE. An increase in both allergen uptake and IL-10/IL-4 ratio was obtained when canine DCs were incubated with the mannan-allergen conjugate, as compared with the control allergen preparations (unmodified D. farinae allergens and oxidized mannan-allergen conjugate). We conclude that hypoallergenic D. farinae allergens coupled to nonoxidized mannan is a novel allergen preparation suitable for canine allergy immunotherapy targeting dendritic cells.

Analysis of feline and canine allergen components in patients sensitized to pets.

BackgroundComponent resolved allergen diagnosis allows for a precise evaluation of the sensitization profiles of patients sensitized to felines and canines. An accurate interpretation of these results allows better insight into the evolution of a given patients sensitizations, and allows for a more precise evaluation of their prognoses.Methods70 patients (42 women and 28 men, aged 18–65, with the average of 35.5) with a positive feline or canine allergy diagnosis were included in the research group. 30 patients with a negative allergy diagnosis were included in the control group. The total IgE levels of all patients with allergies as well as their allergen-specific IgE to feline and canine allergens were measured. Specific IgE levels to canine (Can f 1, Can f 2, Can f 3, Can f 5) and feline (Fel d 1, Fel d 2, Fel d 4) allergen components were also measured with the use of the ImmunoCap method.ResultsMonosensitization for only one canine or feline component was found in 30% of patients. As predicted, the main feline allergen was Fel d 1, which sensitized as many as 93.9% of patients sensitized to felines. Among 65 patients sensitized to at least one feline component, for 30 patients (46.2%) the only sensitizing feline component was Fel d 1. Only 19 patients in that group (63.3%) were not simultaneously sensitized to dogs and 11 (36.7%), the isolated sensitization to feline Fel d 1 notwithstanding, displayed concurrent sensitizations to one of the canine allergen components. Fel d 4 sensitized 49.2% of the research group.64.3% of patients sensitized to canine components had heightened levels of specific IgE to Can f 1. Monosensitization in that group occurred for 32.1% of the patients. Sensitization to Can f 5 was observed among 52.4% of the patients.ConclusionsConcurrent sensitizations to a few allergic components, not only cross-reactive but also originating in different protein families, are a significant problem for patients sensitized to animals.

Lymphocyte blastogenic response to ovalbumin in a model for canine allergy

Lymphocyte stimulation tests (LST) were performed in five dogs sensitised with ovalbumin (OVA) and seven healthy dogs. In addition, all five OVA-sensitised and two control dogs were tested after two in vivo provocations with OVA-containing eye drops. The isolated cells were suspended in culture media containing OVA and were cultured for up to 12 days. Proliferation was measured as reduction in 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity by flow cytometry on days 0, 3, 6, 9 and 12. A cell proliferation index (CPI) for each day and the area under the curve (AUC) of the CPI was calculated for each dog. All OVA-sensitised dogs demonstrated increased erythema after conjunctival OVA application. The presence of OVA-specific lymphocytes was demonstrated in 2/5 OVA-sensitised dogs before and 4/5 after in vivo provocation. Using the AUC, the difference between OVA-sensitised and control dogs was significant in all three LST before in vivo provocation ( P < 0.05) and borderline significant ( P = 0.053) in 2/3 LST after provocation. The most significant difference in CPI was observed after 9 days of culture ( P = 0.001). This pilot study indicates that the LST allows detection of rare antigen specific memory T-cells in dogs previously sensitised to, but not concurrently undergoing challenge by a specific antigen.

The ACVD task force on canine atopic dermatitis (IV): environmental allergens

Numerous environmental allergens have been incriminated in the pathogenesis of canine atopic dermatitis (AD). These include dust and storage mite antigens, house dust, pollens from grasses, trees and weeds, mould spores, epidermal antigens, insect antigens, and miscellaneous antigens such as kapok. In this paper, we review the literature concerning the allergens that have been reported to contribute to canine AD. We conclude that attempts to identify the relevant canine antigens in the past have been plagued by a lack of standardisation of extracts and techniques, and the presence of false-positive and -negative reactions in allergy tests. Until these problems are rectified, it is unlikely that we will be able to provide a list of major and minor antigens for dogs. Hence, we recommend that future studies should be aimed at determining the major patterns of reactivity and cross-reactivity to specific protein allergens within antigenic extracts using electrophoresis and immunoblotting techniques. Once this information becomes available, it may be possible to use a selection of genetically engineered, highly pure antigens for both diagnostic and therapeutic purposes in canine allergy investigations. The use of such antigens will allow standardisation of canine allergy testing and immunotherapy so that the reliability and efficacy of these procedures can be objectively assessed.

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 109 kDa (98/109 kDa). 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 98/109 kDa 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 63.2 kDa; 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.

Binding of canine anaphylactic antibodies on human basophils: application to canine allergy diagnosis.

Abstract Using the technique of human basophil passive sensitization, as employed for human allergy díagnosis, we checked the ability of canine anaphylactic antibodies to sensitize human basophils. Therefore, by sensitizing human basophils with sera taken from dogs allergic to house dust mite, we demonstrated basophil activation as measured by alcian blue staining. Basophil activation was inhibited by heating dog sera at 56 °C for 6 h and by a human myeloma IgE. Basophil activation was also shown by histamine and leukotriene (LTC4) release. These results indicate canine anaphylactic antibodies bind to human basophil IgE receptors and also that they are IgE. The three methods described here for measuring basophil activation may lead to díagnostic methods applicable to canine allergy díagnosis. Resumen Mediante el método de la sensibilización pasiva de basófilos humanos como se utiliza para el díagnóstico de la alergia humana, evaluamos la capacidad de los anticuerpos anafilácticos caninos de sensibilizar basófilos humanos. Asi, sensibilizando basófilos humanos con suero extraido de perros con alergia al ácaro del polvo, demostramos la activación de basófilos medíante la tinción de Azul de Alcián. Se inhibió calentando suero canino a 56 °C durante 6 h. y por IgE de mieloma humano. La activación de los basófilos se mostró también por la liberación de histamina y leucotrieno (LTC4). Estos resultados indican que los anticuerpos caninos anafilácticos se unen a los receptores de IgE en basófilos humanos y también que son IgE. Los tres métodos descritos aqui para medir la activación de basófilos pueden llevar a métodos de díagnóstico aplicables al díagnóstico de la alergia canina. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Union de anticuerpos anafilácticos caninos a basófilos humanos: aplicacion al díagnóstico de alergia canina). Veterinary Dermatology 1996; 7: 185-91.] Résumé Utilisant une technique de sensibilisation passive de basophiles humains, employee pour le díagnostic allergologique chez l'homme, nous avons testé la capacité des anticorps anaphylactiques canins à sensibiliser des basophiles humains. Ainsi, par sensibilisation de basophiles humains avec des sérums provenant de chiens allergiques aux acariens de la poussière de maison, nous avons démontré l'activation des basophiles mesurée par coloration au bleu alcian. Celle-ci est inhibée par des sérums canins chauffés à 56 °C pendant 6 heures et par un myélome IgE humain. L'activation des basophiles a été aussi démontrée par libération d'histamine et de leucotriénes (LTC4). Ces résultats prouvent la présence d'anticorps anaphylactiques canins fixés à des récepteurs IgE de basophiles humains et que ceux-ci sont des IgE. Les trois méthodes décrites ici pour mesurer l'activation des basophiles peuvent être utilisées pour le díagnostic allergologique chez le chien. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Fixation d'anticorps anaphylactiques canins sur des basophiles humains). Veterinary Dermatology 1996; 7: 185-91.] Zusammenfassung Mit der Technik der passiven Basophilensensibilisierung beim Menschen, wie man sie für die Allergiedíagnose beim Menschen anwendet, untersuchten wir die Möglichkeit, menschliche Basophile durch kanine anaphylaktische Antikörper zu sensibilisieren. Dazu wurden humane Basophile mit Sera von Hunden sensibilisiert, die allergisch auf Hausstaubmilben reagierten. Dabei demonstrierten wir eine Basophilenaktivierung, die durch Elsässerblau-Färbung gemessen werden konnte. Der Vorgang wurde verhindert durch Erhitzen der Hundesera auf 56 °C für 6 Stunden und durch humanes Myelom-IgE. Basophilenaktivierung wurde auch durch Histamin-und Leukotrien(LTC4)-Ausschüttung gezeigt. Diese Ergebnisse zeigen, daß kanine anaphylaktische Antikörper sich an humane basophile IgE-Rezeptoren binden und auch IgEs darstellen. Die drei hier beschriebenen Methoden zur Messung der Basophilenaktivierung können zu einer díagnostischen Methode führen, die für die Diagnostik kaniner Allergie anwendbar ist. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Die Bindung von anaphylaktischen Antikörpern des Hundes an Basophile Zellen des Menschen: Anwendung für die Allergiedíagnose beim Hund). Veterinary Dermatology 1996; 7: 185-91.].

Canine Allergy: A Model of a Spontaneous Hypersensitivity Disease.

Excerpt Spontaneous pollen allergy occurs in animals and provides a model of this type of hypersensitivity useful for laboratory investigation. A colony of four dogs with spontaneous pollen allergy...