Peripheral blood mononuclear cell responses to major and minor Dermatophagoides allergens in canine atopic dermatitis.

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans. Most cases involve hypersensitivity to the house dust mites (HDM) Dermatophagoides farinae and Dermatophagoides pteronyssinus. Human atopic dermatitis is associated with the HDM derived allergens Der f 1 and 2, and Der p 1 and 2. Serological data, however, suggest that a 98/104kD protein is the most important allergen in dogs with atopic dermatitis. The aim of this study was to characterise the specificity of circulating T-cells in canine atopic dermatitis for HDM derived allergens. Peripheral blood mononuclear cells (PBMCs) from dogs with atopic dermatitis that were skin test positive for D. farinae and D. pteronyssinus were cultured with crude extracts of D. farinae, D. pteronyssinus and D. microceras, a 98/104kD allergen purified from D. farinae, Der f 1 and Der f 2. There was significantly greater responsiveness of PBMCs to the D. farinae and D. pteronyssinus extracts compared to the D. microceras extract, and similarly to the purified 98/104kD allergen compared to Der f 1 and Der f 2. The close association between serological findings and PBMC proliferation implies that the 98/104kD HDM protein is a major target of immune recognition and that T-cells also participate in the pathogenesis of canine atopic dermatitis by supporting IgE production.     

IgG responses to antigens from Dermatophagoides farinae in healthy and atopic dogs

In this study, we used a semi-quantitative electrophoresis and immunoblotting technique to characterise the IgG response to antigens from Dermatophagoides farinae in 20 healthy and 20 atopic dogs. Both groups mounted an IgG response to multiple antigens from the mite. There was no significant difference in the number of bands recognised, or the molecular weights of the bands, between the two groups. The two most obvious bands in both groups were proteins with molecular weights of 98 kDa (likely to be the high molecular weight allergen Der f 15) and 44 kDa, although dogs in both groups recognised a similar pattern of other antigens. The magnitude of the IgG response was greater in the atopic group although this was not statistically significant. The results indicate that the immune system of both healthy and atopic dogs generates an IgG response to multiple antigens from D. farinae. As some of these antigens (such as the 98 and 44 kDa proteins) are also targeted by IgE in atopic dogs, immunoglobulin class switching in response to Th2 cytokines may not be as dominant a process as has been proposed.     

Characterisation of major and minor Dermatophagoides allergens in canine atopic dermatitis

Atopic dermatitis is a well-recognised chronic inflammatory skin disease of humans and dogs. Most atopic dogs are sensitised to Dermatophagoides mites. The aim of this study was to characterise allergens in different Dermatophagoides species using polyclonal and monoclonal antibodies to canine IgE. Western blots were prepared from crude extracts of D farinae, D pteronyssinus and D microceras, and purified group 1 and 2 allergens under reducing and non-reducing conditions. They were probed with sera from atopic (n = 33) and healthy (n = 27) dogs. There was no significant difference in the sensitivity or specificity between the polyclonal and monoclonal sera in detecting Dermatophagoides -specific IgE. Major allergens common to both D farinae and D pteronyssinus were detected at 97-98 kDa, 103-104 kDa and 134-139 kDa on both reducing and non-reducing blots. Major allergens at 84-85 kDa, 65-69 kDa and 44-45 kDa were only recognised on reducing blots, suggesting that these are fragments of the larger allergens. Only a few sera recognised group 1 or 2 allergens on blots of crude extracts or purified allergens. These results confirm that, in atopic dogs, high molecular weight allergens are the most important Dermatophagoides allergens, rather than the low molecular weight group 1 and 2 proteins.     

Evaluation of IgG subclass responses against Dermatophagoides farinae allergens in healthy and atopic dogs

A semiquantitative chemiluminescent Western blot analysis system was developed and validated to evaluate antigen-specific IgG subclass responses to electrophoretically separated proteins of Dermatophagoides farinae in healthy and atopic dogs. Both groups mounted similar D. farinae-specific IgG1 and IgG4 responses to multiple antigens, but IgG2 and IgG3 responses were difficult to detect. The most commonly recognized bands in both groups were 18 and 98 kDa antigens for IgG1 and 18, 45, 66, 98, 130 and 180 kDa for IgG4. The number of bands recognized per dog did not differ significantly, but significantly more atopic dogs had an IgG1 response to a 180 kDa protein. The overall D. farinae-specific IgG1 and IgG4 responses were slightly higher, but not significantly different, in the healthy group. The results suggest that some antigens produced by D. farinae can induce different subclass responses. However, as most of these responses are seen in both healthy and atopic dogs, they are likely to merely represent recognition of foreign proteins presented to the immune system, rather than involvement in the pathogenesis of atopic dermatitis. The role of the 180 kDa antigen warrants further study.     

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.     

Dermatophagoides farinae house dust mite allergen challenges reduce stratum corneum ceramides in an experimental dog model of acute atopic dermatitis

Background – Ceramides are essential stratum corneum (SC) lipids and they play a pivotal role in maintaining effective cutaneous barrier function. Objectives – The present study aimed at determining the effect of a Dermatophagoides farinae house dust mite (Df‐HDM) allergen challenge on SC ceramides of atopic dogs experimentally sensitized to these allergens. Animals – Six Df‐HDM‐sensitized atopic Maltese–beagle dogs were used. Methods – Prechallenge SC was obtained by cyanoacrylate stripping. One week later, the dogs were challenged topically with Df‐HDM allergens, which resulted in mild to moderate inflammation 24 h later. Two weeks after challenge, SC of lesional and nonlesional skin was obtained. Finally, SC was collected from challenge sites 2 months after lesion resolution. The different SC lipids were quantified blindly by thin‐layer chromatography. Results – Significantly lower amounts of ceramides [AH], [AP], [AS], [NP], [EOP], [NS] and [EOS] were observed in lesional SC compared with prechallenge samples, while no significant effect was found on the amount of other lipids, including cholesterol and free fatty acids. The ceramide profile of nonlesional skin generally showed the same postchallenge reduction pattern. Ceramide amounts returned to normal within 2 months after lesion remission. Conclusion and clinical importance – These findings suggest that the allergic reactions caused by Df‐HDM allergens lead to a selective reduction of SC ceramides, not only at sites of inflammation but also at sites away from those of allergen application. There is normalization of ceramide amounts after inflammation subsides. These observations suggest that the deficiency of ceramides observed in canine atopic skin occurs, at least in part, secondary to inflammation.     

Breed-associated phenotypes in canine atopic dermatitis

Canine atopic dermatitis is a multifaceted disease, whose clinical presentation may be affected by numerous factors, including the genetic background of the animal, the environment, the offending allergens and flare factors. In particular, breed-associated differences have often been mentioned but never defined precisely. Using a large data set of atopic dogs, we document in this study the clinical presentation of nine often-affected breeds and demonstrate the existence of substantial differences between the clinical phenotype of each breed and the whole population. Some of the differences may be due to genetic differences while others are most likely to be associated with variations in environmental factors.     

Serum antibodies to Malassezia yeasts in canine atopic dermatitis

Significant numbers of humans with atopic dermatitis develop Malassezia-specific IgE. Immediate skin-test reactivity to Malassezia has been demonstrated in atopic dogs. The aim of this study was to compare the serum IgG and IgE response to Malassezia in atopic dogs with and without clinical evidence of Malassezia dermatitis and/or otitis, nonatopic dogs with clinical evidence of Malassezia dermatitis and/or otitis and healthy dogs. Cytology was used to diagnose clinically significant Malassezia dermatitis and otitis. Contact plate cultures confirmed the validity of this technique. Reproducible enzyme-linked immunosorbent assays for Malassezia-specific IgG and IgE in canine serum were established. Atopic dogs had significantly higher serum IgG and IgE levels than either healthy dogs or nonatopic dogs with clinical evidence of Malassezia dermatitis and/or otitis. There was no significant difference in IgG and IgE levels between atopic dogs with and without clinical evidence of Malassezia dermatitis and/or otitis. The implications of these findings in the pathogenesis and management of canine atopic dermatitis are discussed.     

Oxidative stress markers in canine atopic dermatitis

There are no data in the veterinary literature relating to oxidative stress in canine atopic dermatitis (CAD). The study aimed to determine levels of oxidative stress markers, plasma malondialdehyde (MDA), total antioxidant capacity (TAC), whole blood glutathione peroxidase (GPX) and erythrocyte superoxide dismutase (SOD), in 15 CAD patients and 17 healthy dogs. A correlation between CADESI (Canine Atopic Dermatitis Extent and Severity Index) score and MDA was also determined. Significantly higher plasma MDA levels were found in patients than in healthy dogs. The significant, highly positive correlation determined between CADESI score and MDA in the patient group indicates an association between the severity of CAD and the extent of oxidative damage to membrane lipids. There were no significant differences in TAC, GPX and SOD between patients and healthy dogs. Our findings suggest that oxidative stress with increased lipid peroxidation could be involved in the pathogenesis of atopic dermatitis in dogs.     

Adherence by Staphylococcus intermedius to canine keratinocytes in atopic dermatitis

The adherence of Staphylococcus intermedius to canine keratinocytes in normal dogs was compared to that in dogs suffering from atopic dermatitis, primary seborrhoea and bacterial pyoderma. Statistically significant greater adherence by S. intermedius to keratinocytes occurred in atopic dogs and dogs suffering from pyoderma when compared with the normal group (P < 0.01) and dogs suffering from primary seborrhoea (P < 0.05). This is similar to the results of a study of human atopic dermatitis by Cole and Silverberg (1986) who demonstrated increased adherence by S. aureus to keratinocytes from atopic dermatitis patients when compared with adherence to keratinocytes in a variety of non-atopic dermatoses. This increased adherence by pathogenic staphylococci to keratinocytes may in part explain the high incidence of staphylococcal pyoderma seen in both canine and human patients suffering from atopic dermatitis.     

Advances in our understanding of canine atopic dermatitis

Canine atopic dermatitis (cAD) is a genetically inherited clinical syndrome that encompasses a diversity of mechanisms and can have a variety of triggers. Development of clinical disease is the result of genetic factors and environmental conditions, which shape the resulting immunological response. Clinical disease becomes evident once a threshold of inflammatory response is achieved. Skin barrier impairment plays a role in promoting cutaneous dysbiosis and increased allergen penetration. Keratinocytes shape the response of dendritic cells and subsequent lymphocytic response. Thymic stromal lymphopoietin is one of the links between the damaged skin barrier and the modulation of a T-helper (Th)2 response. It is still unclear whether mutations in skin barrier genes exist in atopic dogs, as they do in humans, or whether the observed alterations are purely secondary to inflammation. A dysregulated immune response with increased Th2, Th17 and CD4+ CD25+ regulatory T cells has been reported. A variety of cytokines [interleukin(IL)-31, IL-34, Macrophage migration inhibitory factor] are proposed as potential biomarkers and treatment targets because they are increased in the serum of atopic dogs when compared to controls, although a correlation between serum levels of these factors and severity of disease is not always present. The main issue with many published studies is that atopic dogs are always only compared to normal controls. Thus, it is unclear whether the changes that we find are truly a signature of cAD or merely a manifestation of nonspecific broad inflammatory responses. Studies considering comparison with other inflammatory diseases different from cAD are urgently needed to correctly identify what is specific to this complicated syndrome.     

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.     

Calcineurin inhibitors: a novel approach to canine atopic dermatitis

The introduction of calcineurin inhibitors represents a major addition to the armamentarium of drugs available to veterinary clinicians for the management of allergic skin diseases. Both cyclosporine and tacrolimus have been proven to be well tolerated and effective for the treatment of atopic dermatitis in dogs. Although broad spectrum in their mechanism of action, they lack the major adverse effects of glucocorticoids and provide an appealing alternative to traditional therapies. The purposes of this article are to review clinically relevant information regarding these agents and to provide tips for maximizing the benefit obtained from these therapies.