Molecular cloning of canine thymus and activation-regulated chemokine (TARC) gene and its expression in various tissues

Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.     

Expression of RANTES mRNA in skin lesions of feline eosinophilic plaque

Abstract One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine–cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT–PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.     

Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion

CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane receptor that is selectively expressed on Th2 cells and plays an important role in the trafficking of Th2 cells into inflammatory sites. In this study, a full-length canine CCR4 cDNA was cloned and characterized in order to examine the potential role of CCR4 in allergic responses that produce skin lesions in canine atopic dermatitis (AD). The canine CCR4 cDNA reported in this study contained an open reading frame of 1083 nucleotides encoding 360 amino acids. The predicted amino acid sequence of canine CCR4 showed 91.9, 85.3 and 84.5% similarity with those of the human, mouse and guinea pig counterparts, respectively. Expression of CCR4 mRNA was detected in various tissues including thymus, spleen, heart, small intestine and lymph node. Furthermore, it was found that CCR4 mRNA was preferentially expressed in lesional skin of dogs with AD, together with the mRNA of thymus and activation-regulated chemokine (TARC), which is a ligand for CCR4. The present study demonstrates that CCR4 contributes strongly to the immunopathogenesis of canine AD.     

Prospective open pilot study on the use of ciclosporin for feline allergic skin disease

OBJECTIVES: To evaluate the efficacy of ciclosporin in cats with allergic skin disease. METHODS: Ten cats with signs of allergic skin disease were administered ciclosporin daily at a dose of 3.6 to 8.3 mg/kg for one month. None of these cats had previously responded to a hypoallergenic diet trial, and all animals had previously been treated with endectoparasiticidal drugs, with no improvement two weeks before entering the trial. On days 0 and 30, owners assessed pruritus with a visual analogue scale, and veterinarians evaluated cutaneous lesions. RESULTS: All the cats had pruritus and erythema, five had alopecia, two had an eosinophilic plaque, one had miliary dermatitis and two had both alopecia and an eosinophilic plaque. Good or excellent improvement was observed in 40 per cent of cats for pruritus, 57 per cent of cats for alopecia and 60 per cent of cats for erythema. A significant decrease in mean scores was observed for pruritus only, while for erythema and alopecia, it was close to being significant (P < 0.052). CLINICAL SIGNIFICANCE: Ciclosporin may be helpful in symptomatically treating signs of feline allergic skin disease. However, it is important to remember that ciclosporin is not licensed for use in cats.     

Production of a monoclonal antibody to canine thymus and activation-regulated chemokine (TARC) and detection of TARC in lesional skin from dogs with atopic dermatitis

A monoclonal antibody to canine thymus and activation-regulated chemokine (TARC/CCL17) was developed to examine the association of TARC with the immunopathogenesis of canine atopic dermatitis (AD). Recombinant canine TARC was prepared using an E. coli expression system. Results of transwell chemotaxis assay demonstrated that the recombinant canine TARC showed chemotactic activity for canine lymphoid cells expressing CC chemokine receptor 4 (CCR4). Mice were then immunized with the recombinant canine TARC to obtain monoclonal antibodies. Among the monoclonal antibodies thereby obtained, one monoclonal antibody (CTA-1) was found to react with both recombinant and authentic canine TARC in ELISA and flowcytometric assays, respectively. Immunohistochemical analysis using the monoclonal antibody CTA-1 demonstrated that keratinocytes were major TARC producing cells in lesional skin of dogs with AD.     

Measurement of serum immunoglobulin E (IgE) specific for house dust mite antigens in normal cats and cats with allergic skin disease

The purpose of this study was to determine whether cats with allergic skin disease have significant concentrations of serum Immunoglobulin E (IgE) specific for antigens derived from the house dust mites (HDM) Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus (DP). Enzyme-linked immunosorbent assays (ELISA) were developed for this purpose. Binding of serum allergen-specific IgE was detected via the use of biotinylated Fc-epsilon receptor alpha chain protein (FcvarepsilonRIalpha). Following optimisation of the assay, serum samples from 59 cats with allergic skin disease and 54 clinically normal cats were screened. Results were expressed as ELISA units per ml (EU/ml) compared to a standard curve. Serological findings were correlated with the clinical presentation of affected cats. Cats with symptoms of feline allergic skin disease were grouped as follows: self-induced alopecia without lesions (group 1), papulocrusting dermatitis (group 2), eosinophilic granuloma complex (group 3), papular/ulcerative dermatitis of head and neck/facial dermatitis (group 4), and a combination of symptoms (group 5). Control normal cats comprised the final group (group 6). The Kruskal-Wallis test was used for statistical analysis. There was no significant difference between groups for DF- and DP-specific IgE concentrations with a p-value of 0.875 and 0.705, respectively. Although the FcvarepsilonRIalpha-based ELISA was able to detect house dust mite-specific feline IgE, the presence of this allergen-specific IgE correlates poorly with the presence of clinical manifestations of allergic skin disease. The results of this study question the clinical relevance of house dust mite-specific IgE in feline allergic skin disease.     

Quantitative analysis of tryptase- and chymase-containing mast cells in eosinophilic conditions of cats

The presence and density of tryptase-positive/chymase-positive mast cells (MCs) (MC(TC)), chymase-positive/tryptase-negative MCs (MCC), and tryptase-positive/chymase-negative MCs (MC(T)) in lesional skin from cats with eosinophilic conditions were investigated. Skin biopsy specimens from eight cats with eosinophilic plaque (three cats), eosinophilic granuloma (two cats), and eosinophilic dermatitis (three cats) were studied. Toluidine blue staining and a double-enzyme-immunohistochemical staining technique were performed to determine MC density and MC subtypes, respectively. MC density varied from 170.3 to 503 cells/mm2 (mean value of 314.9 cells/mm2). In the superficial dermis, 5.9% of the MC belonged to the MC(T), 12.8% to the MC(C), and 81.2% to the MC(TC) subtype. In the deep dermis, 12.8% belonged to the MC(T), 12.8% to the MC(C), and 73.8% to the MC(TC) subtype. It is the first time that MC(C) have been identified. The double-labeling procedure proved to be a reliable tool for identifying simultaneously the presence of MC subtypes in feline skin.     

Responsiveness and validity of the SCORFAD, an extent and severity scale for feline hypersensitivity dermatitis

Background – Hypersensitivity (allergic) dermatitis (HD) is commonly seen in cats, causing pruritus and various patterns of skin lesions, including at least one of the following: head and neck excoriations, self‐induced alopecia, eosinophilic plaques and miliary dermatitis. Few studies have evaluated the efficacy of therapeutic interventions for feline HD, and although various scales have been considered, none has been formally validated for the assessment of disease severity and its response to therapy. Objective – To design and validate a novel scale (SCORing Feline Allergic Dermatitis; SCORFAD) to assess the value of different criteria used as outcome measures for the treatment of feline HD and to set minimal thresholds for defining the clinical success of tested interventions. Animals – One hundred client‐owned cats. Methods – The SCORFAD scale was designed to include the four most frequently identified lesion types in feline HD (eosinophilic plaque, head and neck excoriations, self‐induced alopecia and miliary dermatitis) across 10 body regions. The extent and severity of each lesion type were graded prior to inclusion and after 3 and 6 weeks in a clinical study to compare the efficacy of two doses of ciclosporin with placebo. Results – The SCORFAD scale was found to exhibit satisfactory content, construct, criterion and sensitivity to change. The percentage reduction in SCORFAD from baseline was determined to be the most valid assessment of clinical response. Inter‐ and intra‐observer reliability was not assessed. Conclusions and clinical importance – The SCORFAD scale is proposed for use as a validated tool for the assessment of disease severity and response to therapeutic interventions in clinical trials for feline HD.     

Lack of autologous tissue transmission of eosinophilic plaques in cats

Autologous tissue transmission of spontaneously developing feline eosinophilic plaques was attempted in 5 cats. Macerated tissue from the plaque was vigorously rubbed onto 2 scarified skin sites in each cat. The inoculated areas were observed daily for 30 days. During that time, no clinical or histologic evidence of transmission was found.     

Clinical and histological evaluation of an analogue of palmitoylethanolamide, PLR 120 (comicronized Palmidrol INN) in cats with eosinophilic granuloma and eosinophilic plaque: a pilot study

Fifteen cats with eosinophilic granuloma or eosinophilic plaque were given PLR 120 at the dosage of 10 mg kg⁻¹ twice daily for one month. PLR-120 down-modulates mast cell degranulation via a receptor-mediated mechanism. No other drugs were permitted and cats were kept free of parasites throughout the study. A clinical evaluation and skin biopsies were performed before and after the treatment. Clinical improvement was assessed at 15 and 30 days. Mast cell numbers were counted and their granular content was assessed by densitometric analysis on toluidine blue-stained sections before and after the treatment. Ten of 15 (67%) cats showed clinical improvement of signs and lesions. There was no significant difference between mast cell numbers in skin biopsies taken before and after the trial, whereas the number of granules was significantly increased ( P < 0.009). This pilot study suggests that PLR-120 might be a useful drug for the treatment of eosinophilic granuloma and eosinophilic plaque.     

Serum allergen‐specific immunoglobulin E in atopic and healthy cats: comparison of a rapid screening immunoassay and complete‐panel analysis

Feline and canine atopic dermatitis are thought to have a similar immunopathogenesis. As with dogs, detection of allergen‐specific IgE in cat serum merely supports a diagnosis of feline atopy based on compatible history, clinical signs and elimination of other pruritic dermatoses. In this study, a rapid screening immunoassay (Allercept^® E‐Screen 2nd Generation; Heska AG, Fribourg, Switzerland; ES2G) was compared with a complete‐panel serum allergen‐specific IgE assay (Allercept^®; Heska AG; CP) in healthy cats with no history of skin disease and in atopic cats. The latter had no diagnosis of external parasitism, infection, food hypersensitivity or other skin disease explaining their pruritus, and expressed cutaneous reaction patterns typically associated with feline allergic skin disease (head, neck or pinnal pruritus, miliary dermatitis, self‐induced alopecia, eosinophilic granuloma complex). The proportion of cats positive on either the ES2G or the CP assays was not significantly different between the atopic and healthy cat groups. There was, however, strong agreement between the results of the ES2G and CP assay; overall, the two tests were in agreement for 43 of 49 (88%) serum samples. There was also strong agreement when individual allergen groups were evaluated (agreement noted: indoor, 41 of 49 samples; grasses/weeds, 37 of 49 samples; and trees, 41 of 49 samples). These results indicate that although neither test is diagnostic for feline atopic dermatitis, the screening assay is beneficial for predicting the results of a complete‐panel serum allergen‐specific IgE assay in cats.     

The use of oral cyclosporin to treat feline dermatoses: a retrospective analysis of 23 cases

Limited information is available regarding the use of cyclosporin A (CsA) for the treatment of feline dermatoses. The aim of this retrospective study was therefore to describe the efficacy of CsA for the therapy of eosinophilic granuloma (EG), eosinophilic plaque, indolent ulcer, linear granulomas, idiopathic pruritus and stomatitis. A computer search for feline dermatological cases treated with CsA between 1999 and 2004 was performed. Based on history, clinical signs and laboratory diagnostic tests, it was then possible to divide cases into three groups and to select 23 cats. Seven cats had one or more of the following conditions: EG, eosinophilic plaque, indolent ulcer and/or linear granuloma (group A); eight cats had idiopathic pruritus (group B) and eight cats had plasmacytic stomatitis (group C). Doses ranged from 5.8 to 13.3 mg kg(-1) oral CsA. All cats were monitored, with complete serum blood analysis and physical examination, monthly for a minimum of 6 months. Response to therapy was scored (severity of lesions and pruritus) with a 0-10 visual analogue scale at each visit (day 0, day, 30, day 60, day 90). All cats in groups A and B were cured and were maintained on alternate day therapy. In group C, 4/8 patients went into remission, while remaining cats have a fair to good improvement. Routine haematological and biochemical examination failed to reveal abnormalities related to CsA administration.     

Correlation of histologic and immunologic findings in cats with miliary dermatitis

Eighteen cats with miliary dermatitis were evaluated, using skin testing and histology. Sixteen cats had allergic skin disease (14 cats had positive skin-test reactions to flea antigen [two of which were also atopic], one was atopic only, and one was allergic to beef). In the two remaining cats, the cause of miliary dermatitis was not identified. Histologically, 17 of the cats had superficial eosinophilic dermatitis and epidermal spongiosis, crusting, and ulceration, which were compatible with an allergic cause. Four of these cats had concurrent eosinophilic plaques, which histologically resembled miliary lesions. This overlap of plaques with miliary lesions indicated that when plaques and miliary dermatitis are found concurrently, both lesions may be caused by the same allergens.     

Ulcerative facial and nasal dermatitis and stomatitis in cats associated with feline herpesvirus 1

Ulcerative dermatitis of the nasal planum or haired skin of the face, associated with intranuclear inclusion bodies compatible with herpesvirus, was identified in nine cats. Clinically, lesions were ulcerative and crusted, and often persistent. A tenth cat had focal proliferative ulcerative stomatitis, also associated with intranuclear inclusion bodies. Microscopically, there was necrosis and ulceration associated with prominent eosinophilic inflammation. Intranuclear inclusion bodies were noted in all cases, within the surface or adnexal epithelium. Ultrastructural examination of skin from two cats revealed virions morphologically compatible with a herpesvirus. Polymerase chain reaction (PCR) specific for feline herpesvirus 1 on DNA extracted from fresh-frozen or formalin-fixed paraffin-embedded biopsy samples and/or consensus primer PCR with DNA sequencing performed on DNA extracted from formalin-fixed paraffin-embedded biopsy samples from seven cats revealed that the virus was indistinguishable from feline herpesvirus 1. PCR was negative in one of eight cats tested.     

Allergen-specific IgG antibodies in cats with allergic skin disease

An enzyme-linked immunosorbent assay was developed for quantifying feline serum allergen-specific IgG directed against selected house dust, pollen and flea allergens. The assay was used to compare allergen-specific IgG concentrations in sera from healthy cats, cats with non-dermatologic illness, confirmed allergic cats and undiagnosed pruritic cats. Our results demonstrate that cats with confirmed allergic skin disease have significantly more IgG directed against house dust, flea and ryegrass allergens than other cat groups examined. These results support the theory that cats with allergic skin disease have an underlying T_H2 lymphocyte response that directs production of both allergen-specific IgG and IgE.     

Malassezia spp. overgrowth in allergic cats

A series of 18 allergic cats with multifocal Malassezia spp. overgrowth is reported: atopic dermatitis was diagnosed in 16, an adverse food reaction in another and one was euthanized 2 months after diagnosis of Malassezia overgrowth. All the cats were otherwise healthy and those tested (16 out of 18) for feline leukaemia or feline immunodeficiency virus infections were all negative. At dermatological examination, multifocal alopecia, erythema, crusting and greasy adherent brownish scales were variably distributed on all cats. Cytological examination revealed Malassezia spp. overgrowth with/without bacterial infection in facial skin (n = 11), ventral neck (n = 6), abdomen (n = 6), ear canal (n = 4), chin (n = 2), ear pinnae (n = 2), interdigital (n = 1) and claw folds skin (n = 1). Moreover, in two cats Malassezia pachydermatis was isolated in fungal cultures from lesional skin. Azoles therapy alone was prescribed in seven, azoles and antibacterial therapy in eight and azoles with both antibacterial and anti-inflammatory therapy in three of the cats. After 3-4 weeks of treatment, substantial reduction of pruritus and skin lesions was observed in all 11 cats treated with a combined therapy and in five of seven treated solely with azoles. Malassezia spp. overgrowth may represent a secondary cutaneous problem in allergic cats particularly in those presented for dermatological examination displaying greasy adherent brownish scales. The favourable response to treatment with antifungal treatments alone suggests that, as in dogs, Malassezia spp. may be partly responsible for both pruritus and cutaneous lesions in allergic cats.