The contribution of stem cells to epidermal and hair follicle tumours in the dog

Background –  Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog. Hypothesis –  Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours. Animals and Methods –  In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin 15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression. Results –  In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours. Conclusions and clinical importance –  Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation.     

Oxidative stress in the pathogenesis of canine zinc‐responsive dermatosis

Zinc deficiency causes skin diseases both in humans and in animals. The underlying pathogenic mechanisms remain unclear, but a growing body of evidence indicates a role for zinc in skin protection against free radical‐induced oxidative damage. The immunohistochemical expression of heat shock proteins (HSPs; Hsp27, Hsp72, Hsp73 and Hsp90), Cu/Zn superoxide dismutase (SOD), metallothionein (MT), Ki‐67 antigen and active caspase‐3 were evaluated in normal canine skin and in samples from eight dogs with zinc‐responsive dermatosis. All investigated HSPs showed intense cytoplasmic immunostaining in the affected epidermis. Focal nuclear positivity of Hsp72 was also detected in keratinocytes. Although Cu/Zn SOD expression was similar to that observed in normal skin, MT immunoreactivity occurred in both the cytoplasm and the nucleus of basal cells in normal skin but was absent from the affected epidermis. Caspase‐3 activation was also absent in the involved epidermis, which revealed a high Ki‐67 index (a 3.5‐ to 9‐fold increase compared with normal skin). These results support the hypothesis that cellular response to stress, particularly oxidative stress, is involved in the pathogenesis of skin lesions in canine zinc‐responsive dermatosis. The lack of MT immunoreactivity in the affected epidermis may be indicative of low zinc levels, thus resulting in vulnerability to oxidative damage. In contrast, high expression levels of HSPs in skin during zinc deficiency may confer protection against a variety of dangerous stimuli, contributing to inhibition of apoptosis and to cell cycle regulation of proliferating keratinocytes.     

Expression of canine Kdap in normal, hyperplastic and neoplastic epidermis

Keratinocyte differentiation-associated protein, Kdap, is a recently identified small secretory protein that may act as a soluble regulator for the cornification and/or desquamation of keratinocytes. To clarify the role of Kdap in the terminal differentiation of keratinocytes, detailed in situ localisation of Kdap was studied using canine skin with normal, hyperplastic and neoplastic epidermis. In normal canine trunk skin, Kdap was expressed by granular keratinocytes, with polarity to the apical side of the cells, suggesting that canine Kdap is present in lamellar granules, as in humans. Expression of Kdap was widespread in the spinous layers in hyperplastic epidermis, but was undetectable in squamous cell carcinomas. These findings suggest that Kdap is closely related to the delay of terminal differentiation and/or release of cells in hyperplastic epidermis.     

CD34 glycoprotein identifies putative stem cells located in the isthmic region of canine hair follicles

It is widely documented that a pool of multipotent stem cells located in humans and mice hair follicle outer root sheath (bulge region) is involved in the restoration of the whole follicular unit during each anagen phase. To the authors' knowledge, data regarding the location and characterization of hair follicle stem compartment in dogs have not been reported in the recent relevant literature. In this study, we investigated the haematopoietic stem and progenitor cell antigen CD34 as a marker of putative stem cells located in a bulge-like region of canine hair follicles. The presence of CD34 mRNA and glycoprotein was assessed on formalin-fixed, paraffin-embedded canine skin samples by in situ hybridization technique and by standard immunohistochemistry, respectively. A strong expression of CD34 mRNA and glycoprotein was observed in a well-defined area of the hair follicle isthmic region and appeared uniformly concentrated at the level of the basal layer of the outer root sheath. These findings provide compelling support to the hypothesis that in dogs, a subpopulation of basal keratinocytes located in the hair follicle isthmic region and characterized by the selective expression of CD34 is potentially associated with the stem cell compartment of this skin appendage.     

In vitro development and characterization of canine epidermis on a porcine acellular dermal matrix

The aim of this study was to develop and to characterize a canine skin epidermal model able to form a proper epidermis on a porcine acellular dermal matrix (PADM). In addition, the role of fibroblasts in skin barrier formation was studied by incorporating or omitting canine dermal fibroblasts in the PADM. Canine epidermal composites were developed by seeding keratinocytes onto the surface of PADM that were previously seeded or non-seeded with dermal fibroblasts. After 14days of culture under air-exposed conditions and in a special growth medium, skin composites were histologically processed and immunohistochemically characterized to determine the expression of cytokeratins and of vimentin and the presence of basement membrane. In all composites, keratinocytes underwent differentiation to a multilayer epidermis with 5-7 viable cell layers. The stratum basalis, stratum spinosum, stratum granulosum and stratum corneum were identified. The expression of cytokeratins was similar to that described in healthy canine epidermis. Laminin and collagen IV immunostaining revealed a homogeneous layer in the epidermal-dermal junction only when the matrix had been seeded by canine dermal fibroblasts. The model may become a simple, useful and cost-effective tool to investigate the biology and pathology of canine epidermis and could partially replace animal testing in several areas of dermatological research.     

Immunomapping of desmosomal and nondesmosomal adhesion molecules in healthy canine footpad,haired skin and buccal mucosal epithelia: comparison with canine pemphigus foliaceus serum immunoglobulin G staining patterns

Pemphigus foliaceus (PF) is the most common canine autoimmune skin disease. In contrast to human PF (hPF), desmoglein‐1 is a minor autoantigen in the canine disease. The major autoantigen(s) of canine PF (cPF) remain(s) unknown, which limits the ability to perform mechanistic studies of lesion formation and the development of novel diagnostic and therapeutic strategies for this disease. The immunofluorescence patterns of selected desmosomal (desmoglein‐1, desmoglein‐3, desmocollin‐1, desmocollin‐3, desmoplakin‐1/2, plakoglobin and plakophilin‐1) and nondesmosomal adhesion proteins (E‐cadherin, claudin‐1, zona occludens‐1 and occludin) in healthy canine footpad, haired skin and buccal mucosal epithelia were determined using hPF and pemphigus vulgaris sera and specific antibodies. The immunostaining patterns were then compared with that of indirect immunofluorescence staining with 66 cPF sera. Most cPF sera (58 of 66; 88%) exhibited positive staining along keratinocyte margins in the stratum spinosum and stratum granulosum of canine footpad. One serum contained autoantibodies binding solely to stratum granulosum keratinocytes. Concurrent intercellular fluorescence in the stratum basale was limited to seven of 66 cPF sera (11%). Only 12 of 66 cPF sera (18%) also exhibited positive IF staining of the buccal mucosa. This study confirms the immunological heterogeneity of cPF immunoglobulin G autoantibodies. Moreover, the major indirect immunofluorescence staining pattern and the inability of most cPF sera to label the buccal mucosa closely matched that of desmocollin‐1. These observations warrant further investigation of desmocollin‐1 as a potential major cPF autoantigen.     

P‐50 Clinical,histological and immunological characteristics of Neumann‐type pemphigus vegetans in a dog

Pemphigus vegetans is a very rare cutaneous autoimmune blistering acantholytic disease of humans that combines features of both pemphigus foliaceus and mucosal lesions of pemphigus vulgaris. We report here the clinical, histopathological and immunological findings in a dog whose lesions resembled those of pemphigus vegetans of humans. A 4‐year old, greater Swiss mountain dog was presented with verrucous papules and crusts on the axillae and inguinal region. Within 3 months, lesions progressed to involve the thorax and ear pinnae, and then became generalized. Ulcers were observed in the oral cavity, anus and prepuce. Microscopic examination of mucosal and cutaneous biopsy specimens revealed a mixed pattern of deep intraepidermal neutrophilic and eosinophilic pustules with isolated and clustered acantholytic keratinocytes, along with suprabasal epidermal clefts leaving rounded basal keratinocytes at the bottom of the vesicles. These dual changes were also observed within the hair follicle epithelium. Dermal inflammation was mixed and perivascular. Direct immunofluorescence revealed IgG deposited around epidermal keratinocytes. Indirect immunofluorescence performed on normal canine gingival substrate uncovered antikeratinocyte IgG autoantibodies with a serum titre of 1:2500. Immunoblotting confirmed that circulating IgG autoantibodies recognized the extracellular segment of canine desmoglein‐1 and human desmoglein‐3. Treatment with azathioprine and oral glucocorticoids resulted in long‐lasting complete remission. In this dog, clinical signs, microscopic skin lesions and immunological findings were deemed analogous to those of human Neumann‐type pemphigus vegetans. Funding: Self‐funded.     

Skin lipid profiling in normal and seborrhoeic shih tzu dogs

Background – Seborrhoea is a clinical condition resulting in excessive lipid and/or scale on the skin and is a common and important skin disease of dogs. However, there is little information on the skin surface lipid composition of dogs with seborrhoea. Hypothesis/Objectives – To compare skin surface lipid profiles in normal and seborrhoeic shih tzu dogs. Methods – Fourteen client‐owned dogs (seven seborrhoeic and seven normal) were investigated. Lipids in sebaceous glands (SGs) were extracted from homogenized tissues of SG hyperplasia. Surface lipid was collected by tape stripping [stratum corneum (SC)‐enriched fraction] and acetone‐wetted cotton swab (acetone‐extracted fraction). Lipids in SGs, SC‐enriched fractions and acetone‐extracted fractions were evaluated by high‐performance thin‐layer chromatography. Results – Lipids in SGs mainly consisted of cholesterol esters, wax esters and triglycerides, whereas lipids in the SC‐enriched fraction mainly consisted of ceramides. The acetone‐extracted fraction contained a mixture of lipid classes recognized in SG‐ and SC‐enriched fractions. In seborrhoeic dogs, concentrations of wax esters and triglycerides in the acetone‐extracted fraction were significantly higher than in control dogs (P = 0.0285). Amounts of total ceramides (in micrograms) per milligram of SC were not significantly different between the two groups (P = 0.5204). Interestingly, two unknown ceramide fractions, which accounted for 20% of the total ceramides, were recognized exclusively in seborrhoeic dogs. Conclusions and clinical importance – These results provide evidence that the skin surface lipid profiles are altered in shih tzu dogs with seborrhoea.     

Identification and cornification-related gene expression of canine keratinocyte differentiation-associated protein, Kdap

The outermost layer of skin, the epidermis, is cornified epithelial tissue composed of keratinocytes. To maintain the structure and function of the epidermis, the regulation of proliferation, differentiation, and cornification of keratinocytes is crucial, and various soluble factors secreted by keratinocytes are involved in these regulations. Previously, work has shown that keratinocytes secreted the protein Kdap (keratinocyte differentiation-associated protein) associated with the formation of cornified cell envelopes, a specialized protective barrier structure on the periphery of terminally differentiating keratinocytes. In the present report, the canine counterpart of human Kdap is identified and an attempt has been made to define its physiological role in canine keratinization. Canine Kdap (cKdap) showed structural features commonly observed in other counterparts and is secreted from transfected cells. The expression profile of cKdap mRNA, which was restrictively expressed in cornified epithelial tissues besides skin has also been determined. These findings indicate that there is a strong association between cKdap expression and cornification, which supports previous observations that Kdap is involved in the synthesis and/or degradation of cornified cell envelopes in humans and mice.     

Phenotypic analysis for a cell line of canine epidermal keratinocytes

Epidermal keratinocytes have the potential to produce inflammatory mediators that are considered to play an important role in skin diseases such as atopic dermatitis (AD). Thus, cell lines of canine epidermal keratinocytes are useful for studying the biological reactivity of keratinocytes in vitro. However, there has been no report on properly analyzing the phenotype of canine keratinocyte cell lines. In this work, we performed phenotypic analysis of CPEK, which was derived from the epidermis of an adult dog in order to examine the phenotypic similarity with epidermal keratinocytes. The present findings indicated that CPEK cells expressed markers for epidermal keratinocytes including cytokeratin 14, alpha6 integrin and PCNA. Our findings demonstrated that CPEK could be a useful cell line for investigating the central role of epidermal keratinocytes in the pathogenesis of AD in vitro.     

Expression of thymic stromal lymphopoietin in canine atopic dermatitis

Background – In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). Hypothesis/Objectives – Our aim was to characterize canine TSLP and to assess its expression in CAD. Methods – Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT‐PCR. Real‐time quantitative RT‐PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. Results – Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full‐length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll‐like receptor ligands lipopolysaccharide and poly I:C. Conclusions and clinical importance – Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.     

Heat shock proteins expression in canine intracutaneous cornifying epithelioma and squamous cell carcinoma

Heat shock proteins (HSPs) are strongly implicated in the control of cell growth, differentiation and biological behaviour of many human cutaneous neoplasms. To our knowledge, no data have been published in the veterinary literature concerning either normal or neoplastic skin. In this study, the immunohistochemical expression of Hsp27, Hsp72 and Hsp73 was evaluated in normal canine skin, 14 intracutaneous cornifying epitheliomas (ICE), 10 well-differentiated and 5 moderately differentiated squamous cell carcinomas (SCC). Expression was correlated with the histological degree of keratinocyte differentiation and proliferation, and investigated as to its usefulness in the differential diagnosis of these canine tumours. In normal epidermis, Hsp27 exhibited cytoplasmic labelling in the spinous and granular layers, whereas in neoplastic tissues it was detected particularly in those areas showing squamous differentiation. Hsp72 immunoreactivity was more intense in ICE and well-differentiated SCC than in normal skin; however, reduced immunolabelling was observed in moderately differentiated SCC. Unlike Hsp72, Hsp73 showed less intense labelling in ICE and well-differentiated SCC than in normal epithelium and an increased positivity in moderately differentiated SCC. These results indicate that HSP immunoreactivity differs between normal and neoplastic canine skin. Hsp27 expression seems to correlate directly with cellular differentiation; by contrast, the involvement of Hsp72/73 in proliferation and differentiation of tumour cells remains controversial. The pattern and intensity of immunolabelling of each investigated HSP did not show, however, significant differences between ICE and SCC; therefore, they do not seem to be useful in the differential diagnosis of these two canine tumours.     

A case of apparent canine erysipeloid associated with Erysipelothrix rhusiopathiae bacteraemia

Background – Erysipelothrix rhusiopathiae is a Gram‐positive facultative anaerobe found worldwide and is most commonly associated with skin disease in swine, while anecdotal reports of cases in dogs have been associated with endocarditis. Hypothesis/Objectives – Clinicians should consider systemic infectious diseases as a potential cause of erythematous skin lesions. Animals – A 5‐year‐old female spayed Labrador retriever presented with lethargy, anorexia and erythematous skin lesions while receiving immunosuppressive therapy for immune‐mediated haemolytic anaemia. Four days prior to presentation, the dog had chewed on a raw turkey carcase. Methods – Complete blood count, serum chemistry profile, urinalysis and blood cultures. Results – Blood cultures yielded a pure growth of E. rhusiopathiae serotype 1b. Amoxicillin 22 mg/kg orally twice daily for 2 weeks and discontinuation of azathioprine resulted in remission of fever and skin lesions. Conclusions and clinical importance – This report is the first documentation, to the best of the authors’ knowledge, of Erysipelothrix infection, a known zoonosis, in an immunosuppressed dog, highlighting the need for infectious disease monitoring in patients receiving such therapy. This information may also help educate veterinarians to include Erysipelothrix infection as a differential diagnosis in dogs with fever and skin lesions, as well as the role of blood cultures in diagnosing this disease.     

Decrease of nuclear reactivity to growth-regulatory galectin-1 in senescent human keratinocytes and detection of non-uniform staining profile alterations upon prolonged culture for galectin-1 and -3

Summary Multipotent stem cells (source for interfollicular epidermis, hairs and sebaceous glands) are localized in the bulge region of the outer root sheath of hair follicles, while stem cells giving rise to interfollicular epidermis reside in its basal. Using the multifunctional lectin galectin-1 as a marker to localize accessible binding sites in situ as a step to figure out galectin functionality in stem cells, we studied hair follicle-derived keratinocytes. Specific nuclear binding of galectin-1 associated with expression of DeltaNp63alpha, a potential marker of epidermal stem cells, was detected. Binding of chimera-type galectin-3 to a nuclear site was not found in parallel assays. During the process of ageing in culture when cells acquire properties of senescence, disappearance of the nuclear signal for galectin-1 binding was accompanied by a similar decrease of nuclear DeltaNp63alpha expression and increased binding of galectin-3 to the cell membrane, namely in regions of intercellular contacts. Expression of cytokeratin 10, a marker of the terminal differentiation was seen only in a small fraction of the cell population. These data extend the evidence for nuclear sites with galectin-1 reactivity in squamous epithelial cells, the expression of which is modulated upon senescence. Moreover, the results document the divergence of galectin-1 and -3 on the level of ligand selection in this cell type, underscoring the importance of the technical aspect to employ tissue lectins as probe and to perform a fingerprinting with several markers of the galectin family in parallel.     

Staphylococcus pseudintermedius exfoliative toxin EXI selectively digests canine desmoglein 1 and causes subcorneal clefts in canine epidermis

Staphylococcal exfoliative toxins are known to digest desmoglein (Dsg) 1, a desmosomal cell–cell adhesion molecule, thus causing intraepidermal splitting in human bullous impetigo, staphylococcal scalded skin syndrome and swine exudative epidermitis. Recently, a novel exfoliative toxin gene (exi), whose sequence shares significant homology with previously identified exfoliative toxins, was isolated from Staphylococcus pseudintermedius. Little is known about the pathogenic involvement of this toxin in canine pustular diseases such as impetigo. The aim of this study was to determine whether EXI, the product of the exi gene, digests canine Dsg1 and causes intraepidermal splitting in canine skin. An exi gene was isolated from chromosomal DNA of an S. pseudintermedius strain obtained from a pustule of a dog with impetigo, and was used to produce a recombinant EXI by Escherichia coli expression. When purified recombinant EXI was injected intradermally into normal dogs, it caused the development of vesicles or erosions with superficial epidermal splitting. In addition, the EXI abolished immunofluorescence for Dsg1, but not for Dsg3, at the injection sites. Moreover, the EXI directly degraded baculovirus‐secreted recombinant extracellular domains of canine Dsg1, but not that of canine Dsg3, in vitro. The EXI also degraded mouse Dsg1α and swine Dsg1, but not human Dsg1, mouse Dsg1β and Dsg1γ. Conversely, recombinant SIET, previously designated as S. intermedius exfoliative toxin, did not cause intraepidermal splitting or degradation of any Dsgs. These findings indicate that EXI has a proteolytic activity that digests canine Dsg1, and this characteristic might be involved in the pathogenesis of intraepidermal splitting in canine impetigo.     

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.     

Altered immunohistochemical staining for desmoglein in skin biopsies in canine pemphigus foliaceus.

In this study, 50 cases of canine pemphigus foliaceus and 49 cases of canine superficial pyoderma were examined by immunohistochemical staining for patterns of desmoglein expression. In 31/50 (62%) of pemphigus foliaceus cases, there was an altered staining pattern for desmoglein consisting of distinct clumped deposits at the periphery of keratinocytes and/or dark cytoplasmic staining of acantholytic cells (consistent with internalization of desmoglein). In contrast, desmoglein staining in biopsies from cases of superficial pyoderma was diffusely pale without evidence for clumping or distinct internalization. This study demonstrates that epidermal desmoglein expression is altered in some cases of pemphigus foliaceus in dogs and suggests that immunohistochemical staining for this protein may be useful in diagnosis.     

Histologic morphology and involucrin, filaggrin, and keratin expression in normal canine skin from dogs of different breeds and coat types

The purpose of this study was to measure the thickness of canine epidermis at various anatomical sites according to localization of cornified envelopes (involucrin and filaggrin), keratins (keratin 10, 5), and their mRNA expression. This was done in the skin of five breeds of dogs including seven poodles, six golden retrievers, six Shih Tzus, four pugs, and four Labrador retrievers. Epidermal thickness of the stratum corneum and nucleated epidermal layer was significantly different. The greatest thickness was observed in the digital web area and the thinnest epidermis was in the axilla. Epidermal thickness was also significantly different between the breeds (p < 0.05). Immunohistochemical staining scores revealed significant decreases of involucrin, filaggrin, and keratin 10 in the ventral and weight-bearing sites, and a relative increase of keratin 5 (p < 0.05). q-PCR analysis showed that their the levels of mRNA were positively correlated with expression of the corresponding proteins in skin samples (p < 0.05). The present study is the first to report the relationship between epidermal gene expression and histologic morphology of the skin in normal dogs. Further studies will be essential to fully understand the pathogenesis of skin barrier dysfunctions in canines.     

Cytokeratins in the canine epidermis

The purpose of this study was to characterize the cytokeratins (CKs) present in the clinically normal skin of dogs. Skin samples from five German shepherds, five Boxers, five Cocker spaniels, five Yorkshire terriers and five mongrels were examined biochemically (using gel electrophoresis and western blotting) and immunohistochemically (using a alkaline phosphatase anti-alkaline phosphatase technique). Results indicated that the canine epidermis expressed the cytokeratins 1, 5, 6, 10/11, 14 and 16. There were no consistent differences in CK expression between the examined breeds with the exception of an individual polymorphism in CK1 and CK10/11. Immunohistochemical studies showed CK 14 labelling of the basal cell layer whereas CK10/11 staining was seen in the suprabasal cell layer of epidermis. Surprisingly, expression of CK6, known as 'stress' cytokeratin, was demonstrated in all epidermal samples. These results indicate that there is a striking consistency of cytokeratin expression in different breeds which should be useful in the investigation and characterization of canine skin diseases.     

Characterization of canine filaggrin: gene structure and protein expression in dog skin

Background – Filaggrin (FLG) is a key protein for skin barrier formation and hydration of the stratum corneum. In humans, a strong association between FLG gene mutations and atopic dermatitis has been reported. Although similar pathogenesis and clinical manifestation have been argued in canine atopic dermatitis, our understanding of canine FLG is limited. Hypothesis/Objectives – The aim of this study was to determine the structure of the canine FLG gene and to raise anti‐dog FLG antibodies, which will be useful to detect FLG protein in dog skin. Methods – The structure of the canine FLG gene was determined by analysing the publicly available canine genome DNA sequence. Polyclonal anti‐dog FLG antibodies were raised based on the canine FLG sequence analysis and used for defining the FLG expression pattern in dog skin by western blotting and immunohistochemistry. Results – Genomic DNA sequence analysis revealed that canine FLG contained four units of repeated sequences corresponding to FLG monomer protein. Western blots probed with anti‐dog FLG monomer detected two bands at 59 and 54 kDa, which were estimated sizes. The results of immunohistochemistry showed that canine FLG was expressed in the stratum granulosum of the epidermis as a granular staining pattern in the cytoplasmic region. Conclusions and clinical importance – This study revealed the unique gene structure of canine FLG that results in production of FLG monomers larger than those of humans or mice. The anti‐dog FLG antibodies raised in this study identified FLG in dog skin. These antibodies will enable us to screen FLG‐deficient dogs with canine atopic dermatitis or ichthyosis.