Apoptosis as a Mechanism for Keratinocyte Death in Canine Toxic Epidermal Necrolysis.

In humans and dogs, toxic epidermal necrolysis (TEN) is a life-threatening dermatosis characterized by sudden epidermal death resulting in extensive skin detachment. There is little information on the pathogenesis of keratinocyte cell death in canine TEN. We studied the occurrence of apoptosis in skin lesions of dogs with TEN to determine if apoptosis contributes to the pathogenesis of this disease. Immunostaining with antibodies to activated caspase-3 and the terminal deoxynucleotidyl-transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling technique revealed positive apoptotic keratinocytes in basal and suprabasal epidermal compartments in 17 biopsy specimens collected from 3 dogs with TEN and 16 from 3 dogs with erythema multiforme (EM). There was no significant difference in the number of positively stained epidermal cells between TEN and EM. These results suggest that apoptosis of epidermal keratinocytes and lymphocytic satellitosis represent one of the early steps in the pathogenesis of canine TEN, as in the human disease counterpart.

Usefulness of collagen IV immunostaining for diagnosis of canine epidermolysis bullosa acquisita.

In dogs, autoimmune subepidermal blistering diseases (AISBDs) encompass several distinct entities that exhibit varying clinical signs, microscopic characteristics, prognosis, and response to treatment. The identification of targeted autoantigens is usually required to make the diagnosis, but immunological tests to determine these antigens are not commercially available. Epidermolysis bullosa acquisita (EBA) is an AISBD characterized by the production of autoantibodies against collagen VII in sublamina densa anchoring fibrils. This article reports on the usefulness of collagen IV immunostaining on paraffin-embedded skin biopsies as an aid to diagnose EBA in dogs. In this disease, collagen IV, which forms the fibrous 2-dimensional network of lamina densa, is detected more commonly above subepidermal vesicles than below. In other canine AISBDs, this is rarely the case. Collagen IV immunostaining therefore offers an inexpensive means to help making a suggestive diagnosis of EBA in the absence of serological determination of the targeted autoantigen.

Epicutaneous sensitization with Dermatophagoides farinae induces generalized allergic dermatitis and elevated mite-specific immunoglobulin E levels in a canine model of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a cutaneous hypersensitivity associated with elevated levels of antigen-specific IgE, commonly to house dust mites (HDMs). It remains controversial as to whether sensitization and clinical disease are induced by cutaneous exposure to HDM. OBJECTIVES: The objectives of this study were to determine whether repeated applications of Dermatophagoides farinae slurry to intact skin of Maltese-Beagle atopic (MAB) dogs would result in the development of clinical signs or lesions resembling spontaneous canine AD, to determine whether repeated slurry applications would induce elevations in mite-specific IgE and/or IgG, and to determine whether mite antigens could be demonstrated within the dermis of application sites. METHODS: Dogs received weekly slurry applications to the axilla and groin, and were patch tested at 120 days, or were patch tested at days 1, 60 and 120, but did not receive further slurry applications. Skin biopsies and serum samples were obtained on days 1, 60 and 120. RESULTS: Pruritic dermatitis was seen in all dogs by day 60. D. farinae-specific IgE was elevated by day 60. Histologic examination of early application sites revealed mild, mononuclear perivascular dermatitis. Later application sites were characterized by a dense inflammatory infiltrate and oedema in both the dermis and the epidermis. Immunofluorescent staining confirmed the presence of D. farinae antigens in the dermis. CONCLUSIONS: This study demonstrated that epicutaneous application of HDM slurry to MAB dogs results in elevations of HDM-specific IgE, localized and generalized pruritic dermatitis resembling spontaneous canine AD, and histologic changes typical of IgE-driven inflammation. We feel that these results suggest that epicutaneous exposure to allergen may play an important role during both the sensitization and the perpetuation of AD, and provide support for the use of a canine model in the investigation of the pathogenesis of AD.

A natural canine homologue of alopecia areata in humans.

BACKGROUND: Alopecia areata (AA) is suspected to be an autoimmune disease directed preferentially against hair follicles (HF) affecting both humans and various mammalian species. Recently, two rodent models of AA were described, namely the ageing C3H/HeJ mouse and the DEBR rat. Despite several case reports of canine AA in the literature, there has been no systematic assessment of the disease in these companion animals, and it is also not known whether dogs with AA could be useful as an outbred homologue of this disease in humans. OBJECTIVES: To evaluate the clinical, histopathological and immunopathological features of 25 dogs with AA and compare these data with those found in the human disease. PATIENTS/METHODS: Twenty-five client-owned dogs exhibiting macroscopic alopecia with peri- or intrabulbar lymphocytic infiltrates were selected for study. Biopsies and sera were obtained and assessed by histopathology, direct immunofluorescence of immunoreactant deposition, immunohistochemistry for lymphocyte markers, indirect immunofluorescence and immunoblotting analysis of circulating serum IgG, selective immunoprecipitation of HF proteins by serum IgG, and passive transfer of purified canine IgG into naïve C57BL/10 mice. RESULTS: Clinical signs including alopecia, skin hyperpigmentation and leucotrichia usually developed during adulthood and were first seen on the face, followed by the forehead, ears and legs. Spontaneous remission of alopecia occurred in 60% of dogs and regrowing hair shafts were often non-pigmented. Histological examination of skin biopsy specimens revealed peri- and intrabulbar mononuclear cell infiltrates affecting almost exclusively anagen HF. Direct immunofluorescence analysis detected HF-specific IgG in 73% of dogs, while indirect immunofluorescence revealed circulating IgG autoantibodies to the HF inner and outer root sheaths, matrix and precortex. Immunoblotting analysis revealed IgG reactivity to proteins in the 45-60 kDa molecular weight range and with a 200-220 kDa doublet. The latter was identified as trichohyalin by selective immunoprecipitation. Purified HF-reactive IgG, pooled from AA-affected dogs, was injected intradermally to the anagen skin of naïve mice where it was associated with the local retention of HFs in an extended telogen phase in AA-treated skin compared with that seen in controls. CONCLUSIONS: These findings are very similar to those reported for human AA patients; therefore, they support the consideration of dogs with AA as a useful homologue for the study of the pathogenesis of this common autoimmune disease of humans.

Effect of substrate selection on indirect immunofluorescence testing of canine autoimmune subepidermal blistering diseases.

The detection by indirect immunofluorescence (IIF) of circulating antibodies in the serum of dogs with autoimmune subepidermal blistering diseases (AISBD) was regarded for a long time as an unrewarding tool. It was, however, demonstrated in humans that the sensitivity of IIF assays depended on the selection of the substrates used. The effects of substrate selection on IIF tests was thus studied by examining sera from 12 dogs with AISBD tested against 8 different substrates from 3 different normal dogs. Patients with AISBD suffered from bullous pemphigoid (n = 4 sera), mucous membrane pemphigoid (n = 4 sera), and epidermolysis bullosa acquisita (n = 4 sera). Substrates included canine tongue, canine lip, canine dorsal haired skin, and ventral haired skin. The same 4 substrates were also split with salt splitting technique (using 1 M sodium chloride), in order to cleave the basement membrane within the lamina lucida and to expose the targeted antigens. The strength of the specific fluorescence of each slide was scored after processing for IIF testing with anti-canine IgG polyclonal antibody. Other criteria, such as background fluorescence, easiness of the interpretation, and variations within a same substrate, were also assessed. Intact canine lip and canine salt-split lip demonstrated consistently stronger intensity of fluorescence and a better ease of interpretation. We concluded that the performance of IIF tests with such substrates was a reliable tool for the detection of circulating IgG autoantibodies of canine patients with AISBD.

Electron microscopic observations of stratum corneum intercellular lipids in normal and atopic dogs.

The barrier function of mammalian skin is maintained by intercellular stratum corneum lipids. In human patients with atopic dermatitis, an abnormal lipid barrier results in dry skin and increased transepidermal water loss. At this time, it is not known if a defective lipid barrier is present in atopic dogs. Normal and atopic canine skin were postfixed in ruthenium tetroxide and studied using transmission electron microscopy to determine structural differences within stratum corneum lipids. Intercellular lipid lamellae were graded on a semiquantitative scale. The deposition of stratum corneum lipid lamellae in atopic canine skin appeared markedly heterogeneous compared with that seen in normal canine skin. When present, the lamellae often exhibited an abnormal structure. The continuity and thickness of the intercellular lipid lamellae were significantly less in nonlesional atopic than in normal canine skin. These preliminary observations suggest that the epidermal lipid barrier is defective in atopic canine skin. Additional studies are needed to further characterize the biochemical defect and to possibly correct it with nutritional and/or pharmacologic intervention.

A spontaneous canine model of mucous membrane (cicatricial) pemphigoid, an autoimmune blistering disease affecting mucosae and mucocutaneous junctions.

Mucous membrane pemphigoid (MMP) is a rare autoimmune blistering dermatosis of humans that was previously known as cicatricial pemphigoid. It is characterized by vesicles, ulcers and scarring that affect predominantly mucosae and mucocutaneous junctions. Circulating autoantibodies recognize epitopes on basement membrane proteins such as collagen XVII or laminin-5/6. Herein, we describe the clinico-pathological and immunological characteristics of 17 dogs afflicted with a dermatosis homologous to MMP of humans. Patients exhibited vesicles and erosions predominantly on mucous membranes or mucocutaneous junctions of the mouth, nose, eyes, genitalia or anus. Histopathology revealed subepithelial vesicles with variable dermal inflammation. Direct immunofluorescence demonstrated IgG or complement at the dermoepithelial junction. Indirect immunofluorescence using salt-split epithelia permitted the detection of circulating basement membrane-specific IgG autoantibodies in 15 cases. In 11 patients, autoantibodies recognized the NC16A segment of collagen XVII, as determined by salt-split indirect immunofluorescence, immunoblotting using canine keratinocytes and ELISA with synthetic canine peptides. In one dog, autoantiodies bound to the dermal side of salt-split epithelia and recognized epitopes within the 30 kDa carboxy-terminal segment of human collagen XVII. Canine MMP, like its human counterpart, exhibits distinctive clinical signs and histopathological lesions, yet circulating autoantibodies target different antigenic epitopes. This spontaneous canine model of MMP could prove useful for studies on the pathogenesis or therapy of this human disease.

Characterization of the inflammatory infiltrate during IgE-mediated late phase reactions in the skin of normal and atopic dogs.

In canine and human atopic patients, the intracutaneous injection of offending allergens is followed by the development of both immediate and late-phase reactions. The present study was performed to expand on the characterization and dynamics of inflammatory cell subsets during IgE-mediated late-phase reactions in canine skin. Three normal dogs and three Dermatophagoides farinae-allergic dogs were selected for this experiment. All dogs were challenged intradermally with mite allergen, purified anticanine IgE antibodies (positive control) or phosphate-buffered saline (negative control). Skin biopsies were obtained before and 6, 12 and 24 h post-injection. Sections were stained with metachromatic and eosinophil-specific histological stains. Additionally, we used an immunohistochemical method with antibodies specific for canine leukocyte antigens. This study confirmed the occurrence of a late-phase reaction in atopic skin following allergen challenge, and in normal and atopic canine skin after intradermal injection of IgE-specific antibodies. Whereas early emigrating dermal cells were composed chiefly of neutrophil and activated eosinophil granulocytes, there was an influx of alpha beta T-lymphocytes and dermal dendritic cells in later stages of the late-phase reactions. Because IgE-mediated late-phase reactions resemble spontaneous atopic canine skin lesions, both at macroscopic and microscopic levels, we propose the use of similar challenges to study the anti-inflammatory effects of anti-allergic drugs in a pre-clinical setting.

Parvovirus infection of keratinocytes as a cause of canine erythema multiforme.

Erythema multiforme major was diagnosed in a dog with necrotizing parvoviral enteritis. Skin lesions consisted of ulceration of the footpads, pressure points, mouth, and vaginal mucosa; vesicles in the oral cavity; and erythematous patches on the abdomen and perivulvar skin. Microscopic examination of mucosal and haired skin specimens revealed lymphocyte-associated keratinocyte apoptosis at various levels of the epidermis. Basophilic cytoplasmic inclusions were seen in basal and suprabasal keratinocytes. Immunohistochemical staining, performed with canine parvovirus-2-specific monoclonal antibodies, confirmed the parvovirus nature of the inclusions in the nucleus and cytoplasm of oral and skin epithelial cells. This is the first case of canine erythema multiforme reported to be caused by a viral infection of keratinocytes. This case study indicates that the search for epitheliotropic viruses should be attempted in cases of erythema multiforme in which a drug cause cannot be identified.

Autoantibodies against the processed ectodomain of collagen XVII (BPAG2, BP180) define a canine homologue of linear IgA disease of humans.

Linear IgA disease (LAD) is an acquired autoimmune subepidermal blistering dermatosis that affects human children and adults. In contrast to bullous pemphigoid, in which autoantibodies recognize transmembrane type XVII collagen (BP180, BPAG2), LAD is associated with skin-fixed and circulating IgA autoantibodies that target LAD-1, the processed extracellular form of type XVII collagen. An immunologic homologue of LAD in humans was identified in two dogs according to the following criteria: 1) erosive, ulcerative, and crusted lesions seen on the face, in the oral cavity, and on the extremities, 2) dermoepidermal clefting present in the basement membrane lamina lucida without inflammation or with mild neutrophilic infiltration, 3) basement membrane-fixed IgG and/or IgA antibodies, and 4) circulating IgA and IgG autoantibodies that target the 120-kd soluble protein LAD-1. The present study establishes unequivocally the existence of a naturally occurring canine model of LAD of humans.

Protein targets of monocrotaline pyrrole in pulmonary artery endothelial cells.

A single administration of monocrotaline to rats results in pathologic alterations in the lung and heart similar to human pulmonary hypertension. In order to produce these lesions, monocrotaline is oxidized to monocrotaline pyrrole in the liver followed by hematogenous transport to the lung where it injures pulmonary endothelium. In this study, we determined specific endothelial targets for (14)C-monocrotaline pyrrole using two-dimensional gel electrophoresis and autoradiographic detection of protein metabolite adducts. Selective labeling of specific proteins was observed. Labeled proteins were digested with trypsin, and the resulting peptides were analyzed using matrix-assisted laser desorption ionization mass spectrometry. The results were searched against sequence data bases to identify the adducted proteins. Five abundant adducted proteins were identified as galectin-1, protein-disulfide isomerase, probable protein-disulfide isomerase (ER60), beta- or gamma-cytoplasmic actin, and cytoskeletal tropomyosin (TM30-NM). With the exception of actin, the proteins identified in this study have never been identified as potential targets for pyrroles, and the majority of these proteins have either received no or minimal attention as targets for other electrophilic compounds. The known functions of these proteins are discussed in terms of their potential for explaining the pulmonary toxicity of monocrotaline.

DNA damage cell checkpoint activities are altered in monocrotaline pyrrole-induced cell cycle arrest in human pulmonary artery endothelial cells.

Monocrotaline pyrrole (MCTP) causes cyto- and karyomegaly and persistent cell cycle arrest in the G2 stage of the cell cycle in cultured bovine pulmonary artery endothelial cells. To better characterize the cell cycle regulatory mechanisms of this process as well as determine whether this process would occur in cells of human origin, we treated human pulmonary artery endothelial cell (HPAEC) cultures with MCTP and determined, by flow cytometry, the expression of cyclin B1 and p53 in conjunction with DNA content. We also validated by Western blots that the persistence of cdc2 in its inactivated phosphorylated state, previously described in bovine cell cultures, occurred in HPAEC. Alterations in p53, cyclin A, cyclin B1, and cdc25c expression were also examined in Western blots of treated HPAEC extracts. The response of HPAEC to MCTP was compared with that of adriamycin and nocodazole, agents known to cause cell cycle alterations. Results of these experiments demonstrate that HPAEC treated with MCTP develop a population of cells in G2 that has increased cyclin B1 expression. These cells express increased amounts of cdc2 but not cdc25c. The ratio of inactive triphosphorylated cdc2 to the active monophosphorylated form increased moderately from control cultures in contrast to predominance of the active form in nocodazole-treated cultures. In addition, a second population of cells expressing cyclin B1 had continued incorporation of BrdU and DNA content consistent with 8 N chromosomes. A similar 8 N cell population was evident in nocodazole-treated cells but these cells had both cyclin B1 positive and negative components. Compared with adriamycin, a known inducer of p53, MCTP-treated HPAEC expressed p53 only at high concentrations and p53 expression was not coordinated with G2 arrest or polyploidy. We conclude that HPAEC treated with low concentrations of MCTP develop G2 arrest in association with persistent cyclin B1 expression, failure to completely activate cdc2, and continued DNA synthesis through a pathway that is unrelated to altered expression of p53.

Equine bullous pemphigoid IgG autoantibodies target linear epitopes in the NC16A ectodomain of collagen XVII (BP180, BPAG2).

Bullous pemphigoid (BP) is an autoimmune subepithelial blistering dermatosis of humans, dogs, cats and pigs. It is characterized by skin-fixed and circulating IgG autoantibodies that target one or both BP antigens. An immunological homologue of BP in humans was diagnosed in two horses with cutaneous and mucosal ulcerations as well as microscopic subepithelial vesiculation. Immunological investigations revealed similar findings for both the horses. Direct immunofluorescence demonstrated the presence of IgG deposited linearly at the dermoepidermal junction in mucosal and skin biopsy specimens. Indirect immunofluorescence testing confirmed the existence of circulating basement membrane-specific IgG autoantibodies. Using intact and salt-split epithelial substrates, serum IgG were shown to target antigens situated not only at the basal, but also at the lateral and apical aspects of stratum basale keratinocytes. Immunoblotting and ELISA corroborated that the IgG from affected horses, but not those from normal controls, exhibited high immunoreactivity against the NC16A extracellular domain of type XVII collagen (BPAG2, BP180). Equine BP could be proposed, therefore, as another spontaneous model of this most common basement membrane autoimmune dermatosis of humans.

A spontaneously arising porcine model of bullous pemphigoid.

Bullous pemphigoid (BP) is an IgG-mediated autoimmune blistering disease targeting the hemidesmosomal proteins bullous pemphigoid antigens 1 and 2. Currently, there is no active animal model in which to dissect the immunopathogenic mechanism. We noticed that cutaneous blistering arose spontaneously in 12 adult Yucatan minipigs. Skin lesions consisted of turgid, isolated or clustered vesicles that occasionally evolved from erythematous and pruritic patches. Histopathological examination revealed subepidermal vesicles rich in intact and degranulated eosinophils. Antigen mapping and transmission electron microscopy confirmed that dermoepidermal separation took place in the lamina lucida of the epidermal basement membrane zone. Direct immunofluorescence revealed the presence of IgG deposited linearly at the dermoepidermal junction in seven of nine skin specimens examined. Indirect immunofluorescence testing confirmed the presence, in the serum from eight of eight affected pigs, of circulating basement membrane-specific IgG autoantibodies (titers 1 : 50 to 1 : 250). Using uncleaved and salt-split lip substrates, the autoantibodies were shown to target antigens situated not only at the basal, but also at the lateral and apical aspects of stratum basale keratinocytes. Immunoelectron microscopy confirmed that circulating IgG autoantibodies recognized hemidesmosomal antigen(s). ELISA, immunoblotting and immunoadsorption demonstrated that five of eight serum samples exhibited high immunoreactivity against BPAG2-NC16A peptides. This novel porcine acquired blistering dermatosis could be proposed as a valuable model to conduct immunomechanistic studies on the natural progression of BP, correlation of autoreactive T cells or autoantibodies with disease activity, and the role of eosinophils in the blistering process, as these diseases cannot be modeled easily in human patients or in murine passive transfer models.

Effects of a 1 per cent hydrocortisone conditioner on the prevention of immediate and late-phase reactions in canine skin.

Ten laboratory beagles were used to determine whether a 1 per cent hydrocortisone conditioner applied topically for three consecutive days would inhibit IgE-mediated immediate and late-phase reactions induced in their skin. The trial was blinded, controlled with the product's vehicle and designed with a crossover. It consisted of three phases: one period without treatment (control phase) and two periods of treatment with either the active ingredient or the vehicle. Immediate and late-phase reactions were induced by the intradermal injection of rabbit anti-canine IgE polyclonal antibodies. Twenty minutes after the intradermal challenge, the diameter of the wheal, but not the erythematous flares, were significantly reduced after the application of the active product. In contrast, IgE-mediated cutaneous late-phase reactions, evaluated by measurements of dermal thickness and eosinophil counts six hours after challenge and the numbers of dermal CD3-positive T lymphocytes after 24 hours, were not reduced by its application.

Reduced anchoring fibril formation and collagen VII immunoreactivity in feline dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa was diagnosed in a cat with juvenile-onset epithelial sloughing of the oral mucosa, footpads, and haired skin. Dermoepidermal separation occurred in the absence of inflammation or cytolysis of basal epidermal cells. Collagen IV-specific immunostaining corroborated the fact that clefting took place below the epidermal basement membrane. Ultrastructural examination revealed that the proband's anchoring fibrils exhibited a filamentous morphology and were decreased in number compared with those in a normal cat. Finally, the attenuated immunoreactivity for collagen VII in our patient led us to suspect that its encoding gene, COL7A1, could be mutated in this case of feline dystrophic epidermolysis bullosa.

Bullous systemic lupus erythematosus (type I) in a dog.

In human patients with systemic lupus erythematosus, cutaneous subepidermal blistering can occur because of the production of antibodies specific for basement membrane antigens. This condition is referred to as bullous systemic lupus erythematosus (BSLE). A dog was diagnosed with BSLE because it fulfilled the following criteria: (i) a diagnosis of systemic lupus erythematosus by standard methods; (ii) an acquired, vesicular, erosive and ulcerative eruption; (iii) microscopical subepidermal vesicles with neutrophil-predominant inflammation at the dermo-epidermal junction; (iv) deposition of IgG at the epidermal basement membrane zone; and (v) circulating IgG autoantibodies against type VII collagen. Anti-collagen VII type I-BSLE therefore needs to be considered as a possible differential diagnosis for canine autoimmune subepidermal blistering diseases.

Novel feline autoimmune blistering disease resembling bullous pemphigoid in humans: IgG autoantibodies target the NC16A ectodomain of type XVII collagen (BP180/BPAG2).

In humans and dogs, bullous pemphigoid (BP) is an autoimmune blistering disease associated with the production of basement membrane autoantibodies that target the 180-kd type XVII collagen (BP180, BPAG2) and/or the 230-kd plakin epidermal isoform BPAG1e (BP230). In two adult cats, an acquired dermatosis and stomatitis was diagnosed as BP subsequent to the fulfillment of the following criteria: 1) presence of cutaneous vesicles, erosions, and ulcers; 2) histologic demonstration of subepidermal vesiculation with inflammatory cells, including eosinophils; 3) in vivo deposition of IgG autoantibodies at the epidermal basement membrane zone; and 4) serum IgG autoantibodies targeting a 180-kd epidermal protein identified as type XVII collagen. In both cats, the antigenic epitopes targeted by IgG autoantibodies were shown to be situated in the NC16A ectodomain of type XVII collagen, a situation similar to that of humans and dogs with BP. Feline BP therefore can be considered a clinical, histopathologic, and immunologic homologue of BP in humans and dogs.

Monocrotaline pyrrole interacts with actin and increases thrombin-mediated permeability in pulmonary artery endothelial cells.

One of the earliest morphologic changes evident in the monocrotaline (MCT) model of pulmonary hypertension in rats is microvascular leak. Whether this represents a direct effect of MCT metabolites or is secondary to inflammatory and thrombotic changes remains uncertain. To determine whether MCT directly affects endothelial cell permeability barrier function, we characterized the interaction of the reactive pyrrole intermediate of MCT (MCTP) with endothelial cell actin and characterized its effects on thrombin-mediated signal transduction and monolayer permeability. Bovine pulmonary endothelial cells (BPAEC) treated with MCTP had altered distribution of filamentous actin evident by fluorescence microscopy. Correlative Western blots and autoradiography of actin isolated from BPAEC treated with 14C-MCTP showed comigration of actin and MCTP-derived 14C. MCTP treatment did not alter cellular free Ca2+ concentrations nor did it interfere with thrombin-mediated intracellular Ca2+ signal. Pretreatment with MCTP significantly augmented the thrombin-mediated transudation of Evan's blue albumin in BPAEC monolayers apparently by increasing the size of intercellular gaps. We conclude that MCTP directly interacts with actin to alter its polymerization state but does not significantly affect endothelial cell response to contractile stimulus. Our results suggest that MCTP may affect endothelial cell barrier function through alterations in intracellular junctions.