Recombinant Culicoides obsoletus complex allergens stimulate antigen-specific T cells of insect bite hypersensitive Shetland ponies in vitro.

BACKGROUND: Ponies may suffer from Insect bite hypersensitivity (IBH), an allergic IgE-mediated pruritic skin disorder, induced by allergens from biting midges of the Culicoides spp. HYPOTHESIS/OBJECTIVES: To determine whether recombinant Culicoides obsoletus allergens are able to activate T cells of ponies exposed to C. obsoletus and whether these allergen-specific responses differ between IBH-affected and healthy ponies. ANIMALS: Ten IBH-affected Shetland ponies and 10 age-matched healthy controls taken from the same stables, to ensure similar exposure to midges. METHOD: Peripheral blood mononuclear cells (PBMC) were cultured with two different pools of recombinant C. obsoletus complex allergens to expand the allergen-specific T cells. These PBMC cultures were subsequently co-cultured with mature dendritic cells (DCs) loaded with the same antigens. Induction of Th1, Th2 and regulatory T (Treg) cells in these DC/PBMC co-cultures was assessed by analysis of IFN-γ, IL-4, IL-10 and FoxP3 expression levels using quantitative RT-PCR and phenotyping by flow cytometry. RESULTS: Recombinant C. obsoletus allergens increased IFN-γ mRNA expression levels, percentages of IFN-γ expressing (Th1) cells and CD25(high) FoxP3(+) IL-10(+) Tregs compared to unstimulated DC/PBMC co-cultures. Stimulation of IL-4 expressing Th2 cells by the recombinant allergens was far less pronounced. The DC/PBMC co-cultures did not reveal significant differences between healthy and IBH-affected ponies for any of the analysed parameters, except for higher IL-4 mRNA levels in IBH affected ponies after stimulation with one of the two allergen pools. CONCLUSION AND CLINICAL IMPORTANCE: The recombinant C. obsoletus complex allergens can stimulate antigen-specific Th1 and IL10 producing Treg cells and are therefore promising candidates for the immunotherapy of IBH.

CD4+ and CD8+ skin-associated T lymphocytes in canine atopic dermatitis produce interleukin-13, interleukin-22 and interferon-γ and contain a CD25+ FoxP3+ subset.

BACKGROUND: T Cells play a major role in the immunopathogenesis of canine atopic dermatitis (cAD). However, the significance of cutaneous regulatory T cells (Tregs) and CD8(+) T cells is currently unclear. HYPOTHESIS/OBJECTIVES: The study aimed to evaluate the presence and distribution of Tregs in cAD and healthy skin and to determine the cytokine production of cutaneous CD4(+) and CD8(+) T cells. ANIMALS: Biopsies were taken from four dogs with cAD (lesional and nonlesional skin) and four healthy control dogs. METHODS: Distribution patterns of T-cell subtypes in cAD lesional, nonlesional and control skin were evaluated by immunohistochemistry. Phenotypic characterization of T cells from skin explant cultures and enzymatic digestions was performed using flow cytometry. Cytokine production of sorted CD4(+) and CD8(+) explant-derived T cells was measured by RT-qPCR. RESULTS: Regulatory T cells phenotypically characterized by CD25(+) FoxP3(+) were found in both CD4(+) and CD8(+) subsets of skin explant and digestion samples. The percentages of CD4(+) CD25(+) cells that were FoxP3(+) were similar in cAD and control skin. In atopic lesional and nonlesional explant samples, lower FoxP3(+) percentages of CD8(+) CD25(+) cells were seen compared with control skin. The presence of predominantly periadnexal CD25(+) FoxP3(+) cells was confirmed by immunohistochemistry in lesional, nonlesional and control skin. The CD4(+) /CD8(+) ratio was less than one in cAD skin with both skin explant and digestion methods. CD4(+) and CD8(+) T-cell subsets of lesional and nonlesional cAD skin were capable of producing interleukin-13, interleukin-22 and interferon-γ. CONCLUSIONS AND CLINICAL IMPORTANCE: Both CD4(+) and CD8(+) T cells are likely to contribute to the immunopathogenesis of cAD through the production of interleukin-13, interleukin-22 and interferon-γ. In both subsets, functional analysis of FoxP3(+) cells is essential to determine their role.

Can f 1 levels in hair and homes of different dog breeds: lack of evidence to describe any dog breed as hypoallergenic.

BACKGROUND: Certain dog breeds are described and marketed as being "hypoallergenic" on the basis of anecdotal reports that these dogs are better tolerated by patients allergic to dogs. OBJECTIVE: These observations were investigated by comparing Can f 1 (major dog [Canis familiaris] allergen) levels in hair and coat samples and in the home environment of various hypoallergenic (Labradoodle, Poodle, Spanish Waterdog, and Airedale terrier) and non-hypoallergenic dogs (Labrador retriever and a control group). METHODS: Hair and coat samples were obtained from dogs, and settled floor and airborne dust samples were taken from the dogs' homes. Can f 1 concentrations were measured by using ELISA, and results were analyzed by using multiple linear regression analyses. RESULTS: Significantly higher Can f 1 concentrations were found in hair and coat samples of hypoallergenic dogs (n = 196, geometric mean [GM], 2.26 µg/g, geometric standard deviation [GSD], 0.73, and GM, 27.04 µg/g, GSD, 0.57, respectively) than of non-hypoallergenic dogs (n = 160, GM, 0.77 µg/g, GSD, 0.71, and GM, 12.98 µg/g, GSD, 0.76, respectively). Differences between breeds were small, relative to the variability within a breed. Can f 1 levels in settled floor dust samples were lower for Labradoodles, but no differences were found between the other groups. No differences in airborne levels were found between breeds. CONCLUSION: So-called hypoallergenic dogs had higher Can f 1 levels in hair and coat samples than did control breeds. These differences did not lead to higher levels of environmental exposure to dog allergens. There is no evidence for the classification of certain dog breeds as being "hypoallergenic."

Enzymes involved in the conversion of arachidonic acid to eicosanoids in the skin of atopic dogs.

Canine atopic dermatitis (AD), a chronic inflammatory skin disease, shares characteristics with its human counterpart. To get insight into the role of enzymes involved in production of prostaglandin E(2) (PGE2) and leukotriene B(4) (LTB(4)), potent inflammatory mediators originating from membrane-derived arachidonic acid (AA), expression of genes encoding these enzymes and receptors was quantified by qPCR in non-lesional and lesional skin from atopic dogs and in healthy skin. Significantly higher mRNA expression of the key enzymes 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP), leukotriene A(4) hydrolase (LTA(4)H) and prostaglandin E synthase 1 (mPGES-1) and their receptors (PGE receptors 2 and 3) were observed. Being responsible for elevated levels of metabolites of the 3-series prostaglandins and the 5-series leukotrienes these enzymes may be interesting targets for therapy that should result in amelioration of clinical signs in canine atopic dermatitis.

Allergen and endotoxin exposure in a companion animal hospital.

BACKGROUND: Exposure to allergens, both in general and occupational environments, is known to result in sensitisation and exacerbation of allergic diseases, while endotoxin exposure might protect against allergic diseases. This may be important for veterinarians and co-workers. However, exposure levels are mostly unknown. OBJECTIVE: We investigated the allergen and endotoxin exposure levels of veterinary medicine students and workers in a companion animal hospital. METHODS: Airborne and surface dust was collected using various sampling methods at different locations. Allergen levels in extracts were measured with sandwich ELISAs and/or the multiplex array for indoor allergens (MARIA). Endotoxin was determined by limulus amebocyte lysate (LAL) assay. RESULTS: Fel d 1 (Felis domesticus), Can f 1 (Canus familiaris) and endotoxin were detected in all except stationary samples. The geometric mean (GM) level of personal inhalable dust samples for Fel d 1 was 0.3 ng/m(3) (range: below lower limit of detection (

Evaluation of T-cell activation in the duodenum of dogs with cutaneous food hypersensitivity.

OBJECTIVE: To determine whether skin-related clinical signs in cutaneous food hypersensitivity (CFH) coincide with immune reactivity in the intestine in dogs. ANIMALS: 11 dogs with CFH without intestinal clinical signs and 8 healthy control dogs. PROCEDURES: After a provocation and elimination diet, the duodenal gene expression levels of Th1-, Th2- and Treg-related cytokines and transcription factors were investigated by means of quantitative PCR assay. The presence of CD3(+), CD8(+), CD4(+), CD1c(+), gammadelta T-cell receptor(+), and major histocompatibility complex II(+) cells in duodenal epithelium and lamina propria were determined. RESULTS: The expression of Th1-, Th2-, and Treg-related genes in dogs with CFH and healthy control dogs was similar. Although clinical signs disappeared, there was no effect of the elimination diet on cytokines, transcription factors, or cellular phenotypes. CONCLUSIONS AND CLINICAL RELEVANCE: No change in T-cell phenotypes or a distinct Th1, Th2, or Treg profile was detected in the duodenum of dogs with only cutaneous clinical signs of food hypersensitivity. This suggested that the intestinal mucosa is not the primary site of T-cell activation that eventually leads to cutaneous food hypersensitivity.

A GeNorm algorithm-based selection of reference genes for quantitative real-time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis.

Quantitative real time PCR (Q-PCR) is the method of choice to study mRNA expression levels. Since Q-PCR is very sensitive, normalization of the data with stably expressed reference genes if of utmost importance. The stability of reference genes depends on the tissue and the species of interest. Therefore, evaluation of the stability of reference genes must be performed for each new tissue and species under study. The stability of B2M, GAPDH, HPRT, SRPR, hnRNPH, GUSB, RPL8, RPS5, and RPS19 was analyzed with the GeNorm software in snap frozen canine skin biopsies. Healthy dogs (n=7) and dogs with confirmed atopic dermatitis (n=28) were included. Lesional and non-lesional skin was analyzed. The study indicated that the most appropriate reference genes in canine skin are the ribosomal gene products RPL8, RPS5 and RPS19 besides GUSB and HPRT. As little as three reference genes will reveal highly reliable Q-PCR calculations.

The efficacy of cyclosporine A in cats with presumed atopic dermatitis: a double blind, randomised prednisolone-controlled study.

The objective of this study was to compare the efficacy of cyclosporine A (CsA) and prednisolone in feline atopic dermatitis (AD) in a randomised, controlled double blind study. Twenty-nine cats with feline AD were randomly allocated to two groups. Eleven cats were treated orally with prednisolone (1mg/kg SID) and 18 were treated with CsA (5mg/kg/day) for 4 weeks. At day 0 (D0) and D28, skin lesions were graded by means of the canine atopic dermatitis extent and severity index (CADESI). Skin biopsies and intradermal allergy tests were performed at D0 and blood samples for haematology and serum biochemistry were collected at D0 and D28. During the trial the cat owners were asked to evaluate the intensity of the pruritus once weekly on a linear analog scale and to record side effects. Based on the CADESI there was no significant difference between the two groups in the amount of remission (P=0.0562) or in the number of cats that improved by >25% (P=0.0571). The effect of CsA and prednisolone on pruritus as evaluated by the owners was not significantly different (P=0.41) between the two groups. No serious side effects were observed. The conclusion was that CsA is an effective alternative to prednisolone therapy in cats with presumed atopic dermatitis.

Intradermal injection of Hsp60 induces cytokine responses in canine atopic and healthy skin.

The purpose of this study was to investigate the immunoregulatory potential of Hsp60 in the skin of dogs with atopic dermatitis. Three dogs with chronic atopic dermatitis and four healthy dogs were injected intradermally with Hsp60 and phosphate-buffered saline. Biopsies were taken before testing from non-injected control skin, lesional and non-lesional atopic skin, and 48 and 72 h after injection. Analysis of cytokine messenger RNA was performed using quantitative real-time polymerase chain reaction. Forty-eight hours after Hsp60 injection, a rise in interleukin (IL)-10 was found (P = 0.034) with the highest expression levels in non-lesional atopic and control skin. A rise of transforming growth factor beta (P = 0.015) and IL-12p40 (P = 0.017) was noticed 72 h after Hsp60 injection in control skin. No significant differences were observed for the expression of IL-4, IL-12p35, and interferon gamma. The results indicate that Hsp60 is able to induce cytokines of a regulatory and Th1 phenotype in the skin. Furthermore, this study seems to provide a first indication of deficient Hsp60 response in atopic dermatitis affected skin.

Effects of cetirizine in dogs with chronic atopic dermatitis: a randomized, double blind, placebo-controlled trial.

This study was conducted to evaluate the effects of cetirizine in dogs with atopic dermatitis (AD) while fulfilling Favrot's diagnostic clinical criteria. Dogs received either 3 mg/kg cetirizine (n = 27), or a placebo (n = 23) orally once daily for 14 days in a randomized, double blind, placebo-controlled study, without concomitant medication. The effects were evaluated using a pruritus visual analog scale at the start (day 0) and at day 14. After 14 days, cetirizine clearly had no effect on the pruritus in dogs with chronic AD, and there was no significant difference between groups. These findings indicated that cetirizine (and likely H1 histamine receptor antagonists in general) should not be recommended for the control of pruritus in dogs with long term allergies.

The effect of long-term feeding of skin barrier-fortified diets on the owner-assessed incidence of atopic dermatitis symptoms in Labrador retrievers.

We investigated the effect of feeding a skin barrier function-augmenting diet early in dogs' lives on the appearance of clinical signs associated with canine atopic dermatitis. Pregnant bitches (starting 5 weeks after mating) and their subsequent litters (up to 1 year of age) were fed either supplemented or unsupplemented diets. Nutrients supplemented were nicotinamide, pantothenate, histidine, inositol and choline. Circulating IgE levels to dust mute allergens Der f and Der p were measured when the puppies were 6 and 12 months old. Two owner questionnaires were used to assess the occurrence of typical signs associated with atopic dermatitis when dogs were between the ages of 22 and 36, and 34 and 48 months. Using linear mixed models we observed higher levels of circulating anti-Der f (P = 0·021) and -Der p IgE (P = 0·01) during the first year in the dogs fed the unsupplemented than in those fed the supplemented diet. The owner-assessed incidence of atopic dermatitis signs amongst the dogs was significantly greater in the unsupplemented group at the time of the second follow-up questionnaire (10/33 dogs v. 2/24 dogs). These outcomes suggest that a nutritionally derived improvement to barrier function early in life may reduce the frequency of signs associated with atopic dermatitis. The effect is possibly the result of making the epidermis, now thought to be a major route of environmental allergen exposure, more resistant to penetration.

Allergen-Specific Cytokine Polarization Protects Shetland Ponies against Culicoides obsoletus-Induced Insect Bite Hypersensitivity.

The immunological mechanisms explaining development of an allergy in some individuals and not in others remain incompletely understood. Insect bite hypersensitivity (IBH) is a common, seasonal, IgE-mediated, pruritic skin disorder that affects considerable proportions of horses of different breeds, which is caused by bites of the insect Culicoides obsoletus (C. obsoletus). We investigated the allergen-specific immune status of individual horses that had either been diagnosed to be healthy or to suffer of IBH. Following intradermal allergen injection, skin biopsies were taken of IBH-affected and healthy ponies and cytokine expression was determined by RT-PCR. In addition, allergen-specific antibody titers were measured and cytokine expression of in vitro stimulated, allergen-specific CD4 T-cells was determined. 24 hrs after allergen injection, a significant increase in mRNA expression of the type-2 cytokine IL-4 was observed in the skin of IBH-affected Shetland ponies. In the skin of healthy ponies, however, an increase in IFNγ mRNA expression was found. Analysis of allergen-specific antibody titers revealed that all animals produced allergen-specific antibodies, and allergen-specific stimulation of CD4 T-cells revealed a significant higher percentage of IFNγ-expressing CD4 T-cells in healthy ponies compared to IBH-affected ponies. These data indicate that horses not affected by IBH, in contrast to the so far established dogma, are not immunologically ignorant but have a Th1-skewed allergen-specific immune response that appears to protect against IBH-associated symptoms. To our knowledge this is the first demonstration of a natural situation, in which an allergen-specific immune skewing is protective in an allergic disorder.

Basophil-derived amphiregulin is essential for UVB irradiation-induced immune suppression.

UVB irradiation (290-320 nm) is used to treat skin diseases like psoriasis and atopic dermatitis, and is known to suppress contact hypersensitivity (CHS) reactions in mouse models. Regulatory T cells (Treg cells) have been shown to be responsible for this UVB-induced suppression of CHS. The epidermal growth factor (EGF)-like growth factor amphiregulin (AREG) engages EGFR on Treg cells and, in different disease models, it was shown that mast cell-derived AREG is essential for optimal Treg cell function in vivo. Here we determined whether AREG has a role in UVB-induced, Treg cell-mediated suppression of CHS reactions in the skin. Our data show that AREG is essential for UVB-induced CHS suppression. In contrast to the general assumption, however, mast cells were dispensable for UVB-induced immune suppression, whereas basophil-derived AREG was essential. These data reveal, to our knowledge, a previously unreported function for basophils in the homeostasis of immune responses in the skin. Basophils thus fulfill a dual function: they contribute to the initiation of effective type 2 immune responses and, by enhancing the suppressive capacity of local Treg cell populations, also to local immune regulation in the skin.

Evaluation of skin test reactivity to environmental allergens in healthy cats and cats with atopic dermatitis.

OBJECTIVE: To evaluate skin test reactivity to environmental allergens in healthy cats and in cats with atopic dermatitis (AD). ANIMAL: 10 healthy cats and 10 cats with AD. PROCEDURE: 10 allergens in serial dilutions were injected ID on the lateral aspect of the thorax of sedated cats. Histamine (0.01% solution) and buffer solutions were used as positive and negative controls, respectively. Immediately after the last injection, 10% fluorescein solution was administered IV. Skin test results were evaluated with ultraviolet light after 15 to 30 minutes and at 4 and 6 hours by 2 independent observers. In the control group, skin tests were repeated after 6 weeks. Skin test reactivity and the nature of the immunoglobulin involved were investigated by use of the Prausnitz-Küstner test with untreated and heat-treated cat sera. RESULTS: Intertest and interobserver agreement were high when measurement of the diameter of the fluorescent wheal was used to evaluate skin test responses, compared with assessment of its intensity. In both groups of cats, immediate skin test reactivity was observed as an IgE-mediated reaction, as an IgG-mediated reaction, and as a result of nonspecific mast cell degranulation. There was no correlation between allergen concentration and the type of reaction observed. CONCLUSIONS AND CLINICAL RELEVANCE: Skin test reactivity in cats should be evaluated after IV administration of 10% fluorescein solution by means of a Prausnitz-Küstner test to differentiate among IgE-mediated, IgG-mediated, and nonspecific reactions.

Evaluation of Felis domesticus allergen I as a possible autoallergen in cats with eosinophilic granuloma complex.

OBJECTIVE: To investigate the role of Felis domesticus allergen I (Feld I) in the pathogenesis of eosinophilic granuloma complex (EGC) in cats. ANIMALS: 7 healthy cats and 6 cats with EGC. PROCEDURE: Epidermis was removed from 4 areas. Rubber stoppers filled with Feld I, saline (0.9% NaCl) solution, and PBS solution were glued to the skin lesions and removed 48 hours later. Fluid within each stopper was collected. Biopsy specimens were obtained at each site, snap frozen, and stored at -70 C. Total and differential numbers of cells in fluid were counted. Biopsy specimens were stained by use of monoclonal antibodies against feline CD4, CD8 and CD3. Data were analyzed by use of multivariate repeated-measures analysis. RESULTS: Healthy cats had a significant increase in number of CD3+ cells, compared with number of CD4+ and CD8+ cells, and Feld I caused a significant increase in number of CD3+ cells, compared with PBS or saline solutions. Cats with EGC had a significant increase in number of CD3+ cells, compared with number of CD4+ and CD8+ cells, and Feld I caused a significant increase in number of CD3+ and CD4+ cells, compared with PBS or saline solutions. Cats with EGC had an increased CD4+ response, a significantly decreased CD8+ response, and a significantly increased CD4-to-CD8 ratio compared with healthy cats. CONCLUSIONS AND CLINICAL RELEVANCE: The increased CD4+ response, significantly decreased CD8+ response, and significantly increased CD4-to-CD8 ratio are comparable to results in atopic people and allergic cats. Therefore, Feld I could be an autoallergen responsible for chronic inflammatory reactions in cats with EGC.

Familial footpad hyperkeratosis and inheritance of keratin 2, keratin 9, and desmoglein 1 in two pedigrees of Irish Terriers.

OBJECTIVE: To investigate the possibility that variants in the acidic or basic keratin genes or in desmoglein 1 may cause the clinical manifestation of familial footpad hyperkeratosis in Irish Terriers. ANIMALS: 11 dogs belonging to 2 related affected pedigrees of Irish Terriers. PROCEDURE: Genomic DNA was extracted from blood samples obtained from each dog. The DNA markers linked to the genes keratin 2, keratin 9, and desmoglein 1 were amplified by use of a polymerase chain reaction technique, and length of the products was determined by use of an automatic DNA analyzer. RESULTS: All tested markers yielded information. None of the markers (genotype) cosegregated with the clinical status of the dogs (phenotype) in the 2 pedigrees. CONCLUSIONS AND CLINICAL RELEVANCE: Mutations in the genes encoding keratin 2 and 9 as well as desmoglein 1 are highly unlikely to be the primary cause of familial footpad hyperkeratosis in Irish Terriers.

The histology of normal llama skin.

Skin biopsy specimens of normal llama skin were examined histologically. Adnexal structures similar to those of most other domestic mammals included epitrichial sweat glands, sebaceous glands and arrector pili muscles. Unique features of normal llama skin included a very thick dermis with marked differences between superficial and deep dermis, prominent cutaneous vascular plexuses, unidentified cells with eosinophilic granules within the adventitia of the vascular plexuses, both simple and compound hair follicles, 'metatarsal glands', 'interdigital glands', footpad glands and the absence of eyelid tarsal glands.

The immunostimulatory effect of CpG oligodeoxynucleotides on peripheral blood mononuclear cells of healthy dogs and dogs with atopic dermatitis.

Synthetic oligodeoxynucleotides containing cytosine phosphatidyl guanine-rich DNA sequences (CpG ODN) can promote T-helper type 1 (Th1) responses, reduce T-helper type 2 (Th2) responses and/or favour regulatory T cell (Treg) responses in vitro and in vivo in humans and animals, by acting via Toll-like receptor 9 (TLR9). Since CpG ODN can be used as immune-modulators for canine atopic dermatitis (AD), the aim of the current study was to investigate their immunostimulatory potential on peripheral blood mononuclear cells (PBMC) and their subsets, from AD and healthy dogs. Expression of TLR9 and cytokine mRNA in CpG ODN-stimulated and unstimulated cells was assessed by real-time quantitative PCR. Stimulation of PBMC with CpG class C ODN upregulated mRNA expression of interleukin (IL)-6, interferon (IFN)-γ and IL-12p40 in AD dogs (P<0.05). It also stimulated IFN-γ protein secretion by PBMC of atopic and healthy dogs as measured by ELISA. In healthy dogs only, CpG class C ODN stimulated IFN-α mRNA production by CD21(+) cells, and IL-10, IL-13 and IFN-γ mRNA production by CD3(+) cells. Increased expression of TLR9 mRNA was only observed in CD3(+) cells from AD dogs. No significantly increased gene expression was found in the CD11c(+) subset upon stimulation, for those genes evaluated. The results indicate that PBMC of healthy and atopic dogs are sensitive to stimulation with CpG ODN class C, with a resulting Th1 cytokine response in AD dogs and a mixed Th1/Th2/Treg cytokine response in healthy dogs. From this study, little evidence was found to support the use of CpG ODN class C for therapeutic purposes in dogs affected with AD.

Two loci on chromosome 5 are associated with serum IgE levels in Labrador retrievers.

Crosslinking of immunoglobulin E antibodies (IgE) bound at the surface of mast cells and subsequent mediator release is considered the most important trigger for allergic reactions. Therefore, the genetic control of IgE levels is studied in the context of allergic diseases, such as asthma, atopic rhinitis, or atopic dermatitis (AD). We performed genome-wide association studies in 161 Labrador Retrievers with regard to total and allergen-specific immunoglobulin E (IgE) levels. We identified a genome-wide significant association on CFA 5 with the antigen-specific IgE responsiveness to Acarus siro. We detected a second genome-wide significant association with respect to the antigen-specific IgE responsiveness to Tyrophagus putrescentiae at a different locus on chromosome 5. A. siro and T. putrescentiae both belong to the family Acaridae and represent so-called storage or forage mites. These forage mites are discussed as major allergen sources in canine AD. No obvious candidate gene for the regulation of IgE levels is located under the two association signals. Therefore our studies offer a chance of identifying a novel mechanism controlling the host's IgE response.