Major Dog Allergen Research Articles

ПРОФИЛИ МОЛЕКУЛЯРНОЙ СЕНСИБИЛИЗАЦИИ К АЛЛЕРГЕНАМ ПУШНЫХ ЖИВОТНЫХ У ПАЦИЕНТОВ РОССИЙСКОЙ ФЕДЕРАЦИИ

Molecular sensibilization patterns have not been yet described for the Russian population, which makes it difficult to develop prevention, elimination and treatment methods for patients with allergic pathology. The purpose of this research was to analyze patterns of sensibilization to components of fur animal allergens in patients examined in order to exclude the sensibilization. Materials and methods used: results of 1113 Immuno Solid-phase Allergen Chip (ISAC) in patients with suspected atopy were analyzed. In all patients, sIgE concentrations to allergenic components of fur animals were determined. Results: 468 (58.21%) of 1113 were found to have sensibilization to fur animal allergens. Of these, 396 (84.62%) were sensibilized to the cat, 277 (59.19%) to the dog, 98 (20.94%) to the horse, and 26 (5.55%) to the mouse. sIgE to Fel d 1 was determined in 90.91% of patients with cat sensibilization, and the median concentration of sIgE to Fel d 1 was higher than to Fel d 2 (p=0.011) and Fel d 4 (p<0.001) (5.4 [1,7; 15]). ISU-E, sIgE to Can f 1 was detected in 73.29%, to Can f 2 - in 18.05%, to Can f 5 - in 41.52% of patients sensibilized to dogs. Among children under 9 y/o, the frequency of detection of sensitization to Can f 5 was less frequent than in the group of children aged over 10 y/o and adults (p<0.001). Conclusion: the Russian population is characterized by a high prevalence of sensibilization to fur animal allergens. Most patients had polysensibilization to components of cat (Fel d 1) and dog (Can f 1, 2 and 5) allergens. It was revealed that Can f 2 has a higher allergenic potential, which in its turn allows it to be identified as a major dog allergen. 92.55% of patients with sensibilization to Fur-bearing animals have an increased risk of developing asthma.

TiO2-Photocatalyst-Induced Degradation of Dog and Cat Allergens under Wet and Dry Conditions Causes a Loss in Their Allergenicity.

Allergies to dogs and cats can cause enormous damage to human health and the economy. Dog and cat allergens are mainly found in dog and cat dander and are present in small particles in the air and in carpets in homes with dogs and cats. Cleaning houses and washing pets are the main methods for reducing allergens in homes; however, it is difficult to eliminate them completely. Therefore, we aimed to investigate whether a TiO2 photocatalyst could degrade dog and cat allergens. Under wet conditions, exposure to the TiO2 photocatalyst for 24 h degraded Can f1, which is a major dog allergen extracted from dog dander, by 98.3%, and Fel d1, which is a major cat allergen extracted from cat dander, by 93.6-94.4%. Furthermore, under dry conditions, the TiO2 photocatalyst degraded Can f1 and Fel d1 by 92.8% and 59.2-68.4%, respectively. The TiO2 photocatalyst abolished the binding of dog and cat allergens to human IgE by 104.6% and 108.6%, respectively. The results indicated that the TiO2 photocatalyst degraded dog and cat allergens, causing a loss in their allergenicity. Our results suggest that TiO2 photocatalysis can be useful for removing indoor pet allergens and improving the partnership between humans and pets.

Спектр сенсибілізації до молекулярних компонентів алергенів собаки у пацієнтів з респіраторною алергопатологією

Abstract. The aim of study: to analyze the dependence of the severity of the course of allergic rhinitis (AR) and/or bronchial asthma (BA) in patients with hypersensitivity to dog allergens from sensitization to individual dog allergenic proteins. Research materials and methods. Blood serum obtained from the venous blood of 102 patients (37 women and 65 men) with a confirmed diagnosis of persistent AR and/or BA and hypersensitivity to dog allergens was used for the study of dog allergenic proteins. Serum was analyzed for the presence of the major dog allergen proteins Can f1 and Can f5 and the minor (cross-linking) protein Can f3 using ImmunoCAP technology (Thermo Fisher Scientific, Uppsala, Sweden). Results. The vast majority of patients with a mild form of persistent AR were monosensitized to one of the main dog allergen proteins Can f1 (50 %) or Can f5 (33 %). Whereas sensitization to 2 proteins occurred in this category of patients much less frequently, as only 12 % of patients were sensitized to Can f1 and Can f5, and 5 % to Can f1 and Can f3 simultaneously. In patients with a moderate and severe form of persistent AR, the sensitization profile was dominated by hypersensitivity to several allergenic components (Can f1 and Can f5), which occurred in 40 % of the examined, and sensitization to 3 components (Can f1, Can f5 and Сan f3) of allergens dogs — in 50 % of patients. Among the examined patients with BA, the most frequent proteins in the form of mono- or co-sensitization were the main dog allergens Can f1 and Can f5. Monosensitization to the minor component of Can f3 was not detected in any patient from both groups of subjects. Conclusions. The vast majority of patients with AR and/or BA and hypersensitivity to dog allergens were sensitized to one or both of the major dog allergens Can f1 and Can f5. As a rule, sensitization to 2 or more allergenic proteins was associated with a more severe course of respiratory allergy pathology. At the same time, special attention should be paid to monosensitization to Can f5 dog urinary kallikrein, which occurred in the examined subjects, because this component is often absent in allergen extracts for skin prick tests and in allergen extracts for allergen-specific immunotherapy (AIT), which can lead to before receiving false-negative test results and ineffective AIT.

Single-cell characterization of dog allergen–specific T cells reveals TH2 heterogeneity in allergic individuals

Allergen-specific type 2 CD4+ TH2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the TH2 response has only recently been appreciated. We sought to characterize at the single-cell level the exvivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4+ T cells specific to the major dog allergens Can f 1, Can f 4, and Can f 5 in subjects with and without dog allergy. Dog allergen-specific memory CD4+ T cells were detected exvivo by flow cytometry using a CD154-based enrichment assay and single-cell sorted for targeted gene expression analysis and TCR sequencing. Dog allergen-specific T-cell responses in allergic subjects were dominantly of TH2 type. TH2 cells could be phenotypically further divided into 3 subsets, which consisted of TH2-like (CCR6-CXCR3-CRTH2-), TH2 (CCR6-CXCR3-CRTH2+CD161-), and TH2A (CCR6-CXCR3-CRTH2+CD161+CD27-) cells. All these subsets were nonexistent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the TH2-biased signature in allergen-specific T cells from allergic subjects and revealed a TH1/TH17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen-specific T cells were diverse and allergic subjects demonstrated less clonality compared to nonallergic donors. Finally, TCR and transcriptomic analyses revealed a close relationship between TH2-like, TH2, and TH2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype. Our study demonstrates heterogeneity within allergen-specific TH2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches.

Structure-based prediction of the IgE epitopes of the major dog allergen Can f 1.

Allergy to dogs has become increasingly prominent worldwide. Seven dog allergens have been identified, including Canis familiaris allergen 1-7 (Can f 1-7). Although Can f 1 is a major dog allergen sensitized to 50-75% of dog-allergic subjects, its IgE epitopes have not been identified. The structural analysis of an allergen is important to identify conformational epitopes. In this study, we generated a recombinant Can f 1 protein and determined its crystal structure using X-ray crystallography. Can f 1 had a typical lipocalin fold, which is composed of an eight-stranded β-barrel and α-helix, and has high similarity to Can f 2, Can f 4, and Can f 6 in overall structure. However, the localizations of surface charges on these proteins were quite different. Based on sequence alignment and tertiary structure, we predicted five critical residues (His86, Glu98, Arg111, Glu138, and Arg152) for the IgE epitopes. The relevance of these residues to IgE reactivity was assessed by generating Can f 1 mutants with these residues substituted for alanine. Although the effects of the mutation on IgE binding depended on the sera of dog-allergic patients, H86A and R152A mutants showed reduced IgE reactivity compared with wild-type Can f 1. These results suggest that Can f 1 residues His86 and Arg152 are candidates for the IgE conformational epitope.

Comparing the concentration levels of allergens and endotoxins in employees\u2019 homes and offices

ObjectiveThe aim of the study was to find out whether allergen and endotoxin concentrations in offices differ from those measured at the homes of employees, and identify the parameters that influence exposure.MethodsElectrostatic dust collectors (EDCs) were placed in five office buildings (68 rooms, 436 EDCs), as well as the homes of the office workers (145 rooms, 405 EDCs) for 14 days, four times a year. In addition, surface samples were collected from the offices four times a year by vacuuming the carpeted floors. Domestic mite (DM), and the major cat and dog allergens (Fel d 1 and Can f 1) were quantified in all samples using fluorescence enzyme immunoassays. Endotoxin was measured in the EDC samples, using the Limulus amoebocyte lysate assay. The allergen and endotoxin concentrations were log transformed and analysed with multilevel models.ResultsEndotoxin concentrations were significantly higher in personal homes compared to levels measured in the offices, and depended on the number of persons living in each household, as well as the presence of a dog. DM allergens were significantly higher in households than in offices, and were significantly higher in bedrooms compared to living rooms. Offices occupied by cat owners had significantly higher Fel d 1 concentrations than offices or homes without. Additionally, Can f 1 concentrations were significantly higher in offices occupied by dog owners compared to those without.ConclusionsPet owners appear to transfer cat and dog allergens to their offices. Therefore, in case of allergy complaints at the office, employers and physicians might consider possible contamination by cat and dog allergens.

Molecular Allergy Diagnostics Refine Characterization of Children Sensitized to Dog Dander

To analyze patterns of immunoglobulin E (IgE) reactivity to dog allergen components in sensitized children and investigate the association between those patterns and clinical symptoms by nasal provocation tests (NPT).Children 10 to 18 years (n = 60) with known sensitization to dog identified by a skin prick test wheal >3 mm or serum-specific IgE >10 kU/L were included.NPTs were performed with a commercial dog extract by using a standardized protocol. The extract was analyzed by radioallergosorbent test inhibition and contained all 6 dog allergens (Can f 1, 2, 3, 4, 5, and 6) used in subsequent analyses. The allergens were present in a wide range of concentrations. Serum-specific IgE levels to dog extract and each of the allergenic components were measured.All but 2 children had allergic rhinitis or conjunctivitis, and 41 out of 60 (68%) reported symptoms with dog exposure. The mean serum-specific IgE level was 11.5 kU/L (interquartile range: 3.15–43.0 kU/L). Sensitization to Can f 1 was most common, followed in frequency by Can f 5, 4, 6, 2, and 3. There was a wide range of IgE levels to the components, with the highest levels to Can f 1, 2, and 5. History of dog-related rhinitis was most common among those sensitized to Can f 3 (94% vs 59% of those not sensitized, P = .012) and Can f 6 (86% vs 63%, P = .007). Similar associations were found for histories of dog-induced asthma. Most patients were sensitized to several components, with great overlap in their sensitization to the proteins belonging to different groups (lipocalins: Can f 1, 2, 4, and 6; serum albumin: Can f 3; and prostatic kallikrein: Can f 5). No single protein group was associated with history of dog-induced airway symptoms, but sensitization to more proteins increased the odds ratio (OR) of dog-induced asthma or rhinitis. Those sensitized to all proteins from all 3 groups were at higher risk of dog-induced rhinitis (OR: 8.36; 95% confidence interval: 1.0–69.5). Forty-two percent (25 out of 60) of patients had positive NPT results, 14 out of 60 (23%) had inconclusive NPTs, and 21 out of 60 (35%) had negative challenges. Those with positive NPT results were significantly more likely to report dog-induced rhinitis and asthma as compared with those with negative NPT results. After adjusting for the effects of different protein groups, only sensitization to the lipocalins (Can f 1, 2, 4, and 6) was associated with positive NPT results. Those sensitized to all 3 groups had the highest OR for a positive NPT result, but as with reported symptoms above, sensitization to increasing numbers of proteins was associated with increasing risk for a positive NPT result.Sensitization to lipocalins and to an increasing number of allergenic dog proteins is associated with dog-induced airway symptoms (dog allergy).“I am not allergic to my dog, but other dogs bother me.” My dog is “hypoallergenic.” How many times have we heard comments like these from patients? Every study that has tried to identify allergenic differences between breeds of dogs has failed to find that elusive hypoallergenic dog. In fact, there is great homology of Can f 1 (the major dog allergen) among breeds. Furthermore, all breeds have comparable amounts of airborne Can f 1 in the homes of dog owners. So, what is going on here? The answer might lie in component-resolved diagnostics (CRD). Commercially available CRDs have already helped our understanding of some food allergies by enhancing our ability to make accurate diagnoses of allergy in sensitized patients. Options for follow-up studies to this one are obvious. Do different breeds of dog produce varying amounts of individual or total lipocalins? Are some patients more highly sensitized to particular proteins or more types of proteins, placing them at differential risk of allergy to certain breeds? Or, are some less highly sensitized, making them more tolerant of different breeds? When those questions are answered and when CRDs for dog allergens are commercially available, we still might not identify a consistently less-allergenic dog. But, we might be able to identify the breeds most likely to be tolerated by a child sensitized to dog.

Canis familiaris allergen Can f 6: expression, purification and analysis of B-cell epitopes in Chinese dog allergic children.

Dog allergy is common worldwide. However, the allergenicity of dog allergy is still unclear in China as well as in special group, such as children. In this study, we chose Can f 6, a major dog allergen which belongs to the lipocalin to study its allergenicity in Chinese dog allergic children. Can f 6 gene was subcloned into pET-28a vector and transformed into E. coli BL21 (DE3) cells for expression. The recombinant Can f 6 was purified by nickel affinity chromatography, identified by SDS-PAGE, and tested for its allergenicity by Western blot with sera and basophil activation test. Secondary structures, B cell epitopes and homology modeling of Can f 6 were predicted by using a series of bioinformatical approaches. And the verification of B cell epitopes was detected by ELISA. The recombinant allergen showed an explicit band with the molecular weight of 20 kDa by SDS-PAGE. Sera from 56.3 % (18/32) of dog-allergic children patients reacted with Can f 6. The induction of the expression of CD63 and CCR3 of dog allergic children in passively sensitized basophils was up to approximately 5.0 times higher than healthy subjects. The secondary structure of Can f 6 contains 3 α-helices, 9 β-sheets and random coils. Five B cell epitopes of Can f 6 were predicted and were confirmed successfully by ELISA. The results showed Can f 6 is a major allergen in Chinese children, which provides a basis for further study of Can f 6 in diagnosis and treatment of symptoms in children in China. The structural information of Can f 6 will help to form a foundation for the future design of vaccines and therapies for Can f 6 related allergies.

Clustering of conformational IgE epitopes on the major dog allergen Can f 1

Immunoglobulin E (IgE)-associated allergy affects more than 25% of the population. Can f 1 is the major dog allergen associated with respiratory symptoms but the epitopes recognized by allergic patients IgE on Can f 1 are unknown. To characterize IgE epitopes of Can f 1 recognized by dog allergic patients, six overlapping peptides spanning the Can f 1 sequence were synthesized. In direct IgE epitope mapping experiments peptides were analyzed for IgE reactivity by dot blot and Enzyme-linked immunosorbent assay (ELISA) with sera from dog allergic patients. For indirect epitope-mapping, rabbits were immunized with the peptides to generate specific IgG antibodies which were used to inhibit allergic patients’ IgE binding to Can f 1. IgE binding sites were visualized on a model of the Can f 1 three-dimensional structure. We found that Can f 1 does not contain any relevant sequential IgE epitopes. However, IgE inhibition experiments with anti-peptide specific IgGs showed that Can f 1 N- and C-terminal portion assembled a major conformational binding site. In conclusion, our study is the first to identify the major IgE epitope-containing area of the dog allergen Can f 1. This finding is important for the development of allergen-specific treatment strategies.

Exposure to Indoor Allergens in Different Residential Settings and Its Influence on IgE Sensitization in a Geographically Confined Austrian Cohort.

BackgroundExposure to indoor allergens is crucial for IgE sensitization and development of allergic symptoms. Residential settings influence the allergen amount in house dust and hence allergic sensitization. Within this study, we investigated allergen exposure and molecule-based IgE levels in a geographically confined region and evaluated the impact of housing, pets and cleaning.Methods501 adolescents from Salzburg, Austria participated in this cross-sectional study. House dust samples were examined regarding major mite, cat, dog, and mold allergens using a multiplex assay. Serum samples of participants were analyzed for specific IgE to Der p 1, Der p 2, Fel d 1, Can f 1 and Alt a 1 using the multiplex array ImmunoCAP ISAC. Information on allergies, living areas, dwelling form (house, flat, farm), pets, and household cleanliness were obtained by a questionnaire.ResultsIn investigated house dust samples, the concentration of cat allergen was highest while the prevalence of mold allergens was very low. Participants showed IgE sensitization to Der p 1 (13.2%), Der p 2 (18.2%), Fel d 1 (14.4%), Can f 1 (2.4%) and Alt a 1 (2.0%). In alpine regions, lower mite allergen concentrations were detected which correlated with reduced IgE levels. A trend for increased sensitization prevalence from rural to alpine to urban regions was noted. Living on farms resulted in lower sensitization prevalence to mite and cat allergens, even though exposure to mites was significantly elevated. The presence of cats was associated with a lower sensitization rate and IgE levels to cat and mite allergens, and less frequent allergic diseases. Cleaning did not impact allergen concentrations, while IgE reactivity to mites and allergic diseases were more pronounced when living in cleaner homes.ConclusionAllergen exposure to indoor allergens was influenced by setting of homes. Living in a farm environment and having a cat at home showed a protective effect for IgE sensitization and allergies. This cross-sectional study in combination with hereditary and lifestyle factors enables development of risk schemes for a more efficient management and potential prevention of allergic diseases.

Human memory CD4+ T cell response to the major dog allergen Can f 5, prostatic kallikrein.

Human CD4+ T cell responses to important animal allergens are still insufficiently understood. To comprehensively characterize invitro and exvivo the peripheral blood memory CD4+ T cell responses of subjects with and without allergy to the major dog allergen Can f 5, the only known animal allergen in the kallikrein family of proteins. Can f 5-specific memory CD4+ T cell lines (TCLs) were established from the peripheral blood of 12 subjects with and 12 subjects without allergy to Can f 5 and characterized for their functional and phenotypic properties. The results were evaluated with those obtained exvivo with a novel CD154 enrichment method. The epitopes recognized by the Can f 5-specific TCLs were determined with 72 overlapping 16-mer peptides covering the sequence of the allergen. Can f 5-specific TCLs were obtained at about tenfold higher frequency from allergic than from non-allergic subjects. Functionally, the TCLs of allergic subjects displayed a Th2-biased cytokine phenotype and increased T cell receptor avidity, whereas the TCLs of non-allergic subjects displayed a Th1-/Th0-biased cytokine phenotype and lower TCR avidity. The higher frequency and the Th2 phenotype of Can f 5-specific memory CD4+ T cells in allergic subjects were confirmed by the CD154 enrichment method exvivo. Six distinct T cell epitope regions of Can f 5 were predominantly recognized by the TCLs from allergic subjects. Can f 5-specific memory CD4+ T cell responses differ considerably between subjects with and without allergy, as assessed by both invitro and exvivo approaches. Peptides containing the dominant T cell epitopes of Can f 5 can be employed for developing peptide-based immunotherapy for dog allergy.

The NPC2 protein: A novel dog allergen

BackgroundDogs are an important source of indoor allergens that cause rhinoconjunctivitis, urticaria, and asthma in sensitized individuals. Can f 1 is reported as a major dog allergen, but other allergens have also been identified. Identification of immunologically important allergens is important for both the diagnosis and treatment of dog allergy. ObjectiveTo identify and characterize the canine NPC2 protein, a novel dog allergen. MethodsWe screened commercial and laboratory-generated aqueous dog extracts by 2-dimensional polyacrylamide gel electrophoresis with IgE immunoblotting using human serum samples from 71 dog-allergic individuals. A target of interest was excised from the gel and sequenced. Canine NPC2 sequence was generated, and recombinant proteins expressed in yeast and bacteria were used to determine allergenicity. An IgE enzyme-linked immunosorbent assay was used for screening 71 dog-positive and 30 dog-negative serum samples. ResultsA 16-kDa protein (pK = 8.5) in dog allergen extracts was recognized by specific IgE. The protein was identified by sequencing as a CE1 protein or NPC2 protein. Human IgE bound to recombinant protein was expressed in both yeast and bacteria. Ten (14%) of 71 individuals had specific IgE to NPC2 protein from bacteria, and 12 (17%) had IgE to NPC2 protein from yeast. Binding of pooled dog-allergic serum IgE to the dust mite protein Der p 2 was partially inhibited by recombinant NPC2 protein. ConclusionNPC2 protein, a member of the MD-2–related lipid recognition family, is identified as a dog allergen (Can f 7), with an apparent seroprevalence of 10% to 20%.

IgE-AB to animals molecular allergens in patients with respiratory allergy

Background. The purpose of this study was to identify IgE-antibodies to the major and minor allergenic components of pets in the blood serum of patients with bronchial asthma and/or allergic rhinitis (BA/AR). Methods. The study included 327 patients from Moscow and St. Petersburg. All of them were detected for specific IgE-antibodies (IgE-AB) to the animal allergens by ImmunoCAP® (ThermoFisherScientific, Sweden). 105 patients wanted to detect IgE-AB to the main animal allergens molecules ImmunoCAP ISAC® (ThermoFisherScientific, Sweden). Results. In the sera of 327 patients IgE-AB to cat’s (62%) allergen dominated. IgE-antibodies to dog’s, house dust mites (D.pteronyssinus, D. farinae), mold fungi, horses and rodents allergens were found in 2,6-31 times less. IgE-AB to uteroglobin (rFel d 1), major cat allergen, was detected in 80% of patients (84/105). The detection rate of IgE-AB to other cat allergens was 4,2-8 times lower (rFel d 2 - 10%, nFel d 4 - 19%). IgE-AB to major dog allergen rCan f 1 was detected in 39% of patients (41/105). Positive level of IgE-AB to other dog allergens was detected in 2,2-4,9 times lower (rCan f 2 - 8%, nCan f 3 - 9%, rCan f 5 - 18%). Positive level of IgE-AB to lipocalin was assayed in 6% of patients, to serum albumin - in 7%. Conclusion. Prevalence of IgE-AB to cat and dog allergens was almost 3 times higher than the frequency of detection of IgE-AB to house dust mite allergens in citizens of Moscow and S. Petersburg. The frequency of IgE-AB to rFel d 1 was dominated and detected 2 and 4-11 times often than to rCan f 1 and other animal allergens consequently.

Allergenic Can f 1 and its human homologue Lcn-1 direct dendritic cells to induce divergent immune responses.

Why and when the immune system skews to Th2 mediated allergic immune responses is still poorly characterized. With two homologous lipocalins, the major respiratory dog allergen Can f 1 and the human endogenous, non-allergenic Lipocalin-1, we investigated their impact on human monocyte-derived dendritic cells (DC). The two lipocalins had differential effects on DC according to their allergenic potential. Compared to Lipocalin-1, Can f 1 persistently induced lower levels of the Th1 skewing maturation marker expression, tryptophan breakdown and interleukin (IL)-12 production in DC. As a consequence, T cells stimulated by DC treated with Can f 1 produced more of the Th2 signature cytokine IL-13 and lower levels of the Th1 signature cytokine interferon-γ than T cells stimulated by Lipocalin-1 treated DC. These data were partially verified by a second pair of homologous lipocalins, the cat allergen Fel d 4 and its putative human homologue major urinary protein. Our data indicate that the crosstalk of DC with lipocalins alone has the potential to direct the type of immune response to these particular antigens. A global gene expression analysis further supported these results and indicated significant differences in intracellular trafficking, sorting and antigen presentation pathways when comparing Can f 1 and Lipocalin-1 stimulated DC. With this study we contribute to a better understanding of the induction phase of a Th2 immune response.

Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients.

BackgroundDog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality.ObjectiveTo assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog.MethodsA linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production.ResultsThe linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses.ConclusionWe provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.

Human CD4+ T cell responses to the dog major allergen Can f 1 and its human homologue tear lipocalin resemble each other.

Lipocalin allergens form a notable group of proteins, as they contain most of the significant respiratory allergens from mammals. The basis for the allergenic capacity of allergens in the lipocalin family, that is, the development of T-helper type 2 immunity against them, is still unresolved. As immunogenicity has been proposed to be a decisive feature of allergens, the purpose of this work was to examine human CD4+ T cell responses to the major dog allergen Can f 1 and to compare them with those to its human homologue, tear lipocalin (TL). For this, specific T cell lines were induced in vitro from the peripheral blood mononuclear cells of Can f 1-allergic and healthy dog dust-exposed subjects with peptides containing the immunodominant T cell epitopes of Can f 1 and the corresponding TL peptides. We found that the frequency of Can f 1 and TL-specific T cells in both subject groups was low and close to each other, the difference being about two-fold. Importantly, we found that the proliferative responses of both Can f 1 and TL-specific T cell lines from allergic subjects were stronger than those from healthy subjects, but that the strength of the responses within the subject groups did not differ between these two antigens. Moreover, the phenotype of the Can f 1 and TL-specific T cell lines, determined by cytokine production and expression of cell surface markers, resembled each other. The HLA system appeared to have a minimal role in explaining the allergenicity of Can f 1, as the allergic and healthy subjects' HLA background did not differ, and HLA binding was very similar between Can f 1 and TL peptides. Along with existing data on lipocalin allergens, we conclude that strong antigenicity is not decisive for the allergenicity of Can f 1.

The homologous lipocalins Can f 1 and Lcn-1 induce different immune responses

Background Why and when the immune system provokes TH2 mediated allergic immune responses is still not well understood. Dendritic cells are one of the master regulators during the induction of allergic airway inflammation providing stimuli for TH2 cell differentiation. Most of the major as well as minor mammalian allergens causing respiratory sensitization belong to the lipocalin family. We investigated two homologous lipocalins, the endogenous non-allergenic human Lcn-1 and the major respiratory dog allergen Can f 1 to study their effects on human monocyte derived dendritic cells in order to initiate allergic immune responses.

Can f 1 Levels in Hair and Homes of Different Dog Breeds: Lack of Evidence to Describe Any Dog Breed as Hypoallergenic

DW Vregegoor, T Willemse, MD Chapman. J Allergy Clin Immunol. 2012;130(4):904–909 To compare levels of, and determine the relationships between, the major dog allergen (Can f 1) from the hair and coat of various breeds of dogs and in the homes in which the dogs live. A total of 356 dogs (Labradoodles, Labrador retrievers, poodles, Spanish waterdogs, airedales, and a heterogeneous control group) were recruited from breeders, breeder associations, and a veterinary hospital in the Netherlands. In addition, 168 homes with 1 dog had floor and airborne dust samples collected and a survey of their 502 owners was conducted. Dog …

Retagging Identifies Dendritic Cell-specific Intercellular Adhesion Molecule-3 (ICAM3)-grabbing Non-integrin (DC-SIGN) Protein as a Novel Receptor for a Major Allergen from House Dust Mite

Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens.

Efficacy of Occupant-Collected Dust Samples in the Evaluation of Residential Allergen and Fungal Levels

This study evaluated the ability of a resident to evaluate their home for allergens and mold using a settled dust test kit compared with evaluation and collection of settled dust by an industrial hygienist. Forty-three home residents were provided with a kit containing written instructions and a vacuum cleaner attachment for collecting a settled dust sample. Within 2 weeks of receiving the occupant-collected sample, an industrial hygienist evaluated these homes, including a visual inspection, collection of settled dust, and collection of spore trap samples. Settled dust samples were analyzed for major dog, cat, dust mite, and cockroach allergens using immunoassay methods, and for mold spore equivalents using quantitative polymerase chain reaction methods for the 13 mold species or species groups comprising the American Relative Moldiness Index (ARMI). Allergen concentrations and ARMIs were compared between the resident- and industrial hygienist-collected samples. Linear regression between the two sets of samples showed strong correlations for dog allergen (r2 = 0.92) and cat allergen (r2 = 0.90). Correlations for dust mite (r2 = 0.57) and cockroach allergens (r2 = 0.22) were lower, likely due to most samples being near the limit of detection. ARMIs were highly correlated (r2 = 0.68) and were in categorical (high, medium, or low) agreement for 76% of residences. These results show that residents can reliably follow directions and collect settled dust samples, providing an efficient method to remotely screen homes for elevated allergen levels and to identify homes with a potential mold or moisture problem that may need further evaluation.