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.

Clinical and microscopic characteristics of canine toxic epidermal necrolysis.

Canine toxic epidermal necrosis (TEN), a rare and life-threatening cutaneous drug reaction, traditionally has been described as full-thickness devitalization of the epidermis with minimal dermal inflammation; however, few reports detail the histologic findings. We characterize the clinical features and histologic variations of 3 canine TEN patients. Clinically, irregular erythematous and purpuric macules evolved into widespread and severely painful erosions. The number of eroded mucosae varied; however, periocular and perilabial mucocutaneous junctions frequently were affected. Thirteen of 17 biopsies were evaluated. Apoptosis at multiple epidermal levels was the most common pattern of epidermal necrosis (12/13 biopsies, 92%). In contrast, full-thickness coagulation necrosis was present less often (7/13 biopsies, 52%). Lymphocytic interface dermatitis was the predominant inflammatory pattern, and intraepidermal lymphocytes, along with fewer histiocytes, were present to some degree in all samples along with lymphocytic satellitosis of apoptotic keratinocytes. The sequence of changes points to lymphocyte-mediated keratinocyte apoptosis as an early step in lesion development with subsequent variation in progression to coagulation necrosis among patients. Histopathologic changes overlapped with those reported for erythema multiforme, in contrast to traditional histologic descriptions of canine TEN. A specific algorithm for assessment of drug causality in epidermal necrolysis (ALDEN) was applied for each patient; carprofen was associated with a probable score for causality in 1 dog. Clinicians should be encouraged to take multiple biopsies in TEN suspect cases as nearly 25% of all biopsies lacked epithelium and were not diagnostic.

Bilaterally symmetrical alopecia with reticulated hyperpigmentation: a manifestation of cutaneous lupus erythematosus in a dog with systemic lupus erythematosus.

An adult castrated male Doberman Pinscher was presented with a 6-month history of well-demarcated alopecic patches with reticulated hyperpigmentation and fine peripheral scaling on the axillae, thorax, abdomen, inguinal region, and thighs. The dog later developed hyperthermia, lethargy, apparent joint pain, peripheral lymphadenomegaly, vomiting, and diarrhea. Relevant laboratory tests results included anemia, thrombocytopenia, proteinuria, and an elevated antinuclear antibodies serum titer. Histologically, skin biopsy specimens had a lymphocyte-rich interface dermatitis and interface mural folliculitis ending in follicular destruction. Altogether, these signs were consistent with a unique alopecic variant of chronic cutaneous lupus erythematosus, eventually associated with the development of systemic lupus erythematosus. This rare form of chronic cutaneous lupus needs to be added to the expanding list of lymphocyte-mediated autoimmune alopecias in dogs.

Evaluation of Plasma C-Reactive Protein as a Biomarker in Dogs with Atopic -Dermatitis Receiving Allergen-Specific Immunotherapy: A Pilot Study.

INTRODUCTION: In this pilot study, we wished to determine if C-reactive protein (CRP) levels could be a useful severity or treatment biomarker for canine atopic dermatitis (AD). Nine atopic dogs received allergen immunotherapy for 1 year. Blood was collected before and at four re-evaluation visits. At each time point, the skin lesions were graded with the Canine Atopic Dermatitis Extent and Severity Index (CADESI) 4, and the plasma CRP levels were measured by Enzyme-linked Immunosorbent Assay (ELISA). We found a significant yet minimal correlation between the CRP levels and the CADESI4 scores. The CRP levels were not significantly different between dogs with AD of increasing severity. Finally, there was no correlation between the percentage change in CADESI4 and CRP values during immunotherapy. In conclusion, the lack of significant difference in CRP levels between dogs of increasing AD severity and lack of correlation between percentage changes in skin lesion and CRP values suggest that this protein would not be a clinically-useful biomarker in atopic dogs.

INTRODUCTION: Dans cette étude pilote, nous avons souhaité déterminer si les niveaux de protéine C-réactive (CRP) pourraient être un biomarqueur de gravité ou de traitement utile pour la dermatite atopique canine (DA). Neuf chiens atopiques ont reçu une immunothérapie allergénique spécifique pendant un an. Du sang a été prélevé avant et lors de quatre visites de réévaluation. À chacune de ces visites, les lésions cutanées ont été classées au moyen de l'indice d'étendue et de gravité de la dermatite atopique canine (CADESI) 4 et les taux plasmatiques de CRP ont été mesurés par dosage immuno-enzymatique (ELISA). Nous avons trouvé une corrélation significative mais minime entre les niveaux de CRP et les scores CADESI4. Les niveaux de CRP n'étaient pas significativement différents entre les chiens atteints de DA de gravité croissante. Enfin, il n'y avait pas de corrélation entre le pourcentage de changement des valeurs de ­CADESI4 et de CRP pendant l'immunothérapie. En conclusion, l'absence de différence significative des taux de CRP entre les chiens de gravité croissante de DA et le manque de corrélation entre les changements de ­pourcentage de lésion cutanée et les valeurs de CRP suggèrent que cette protéine ne serait pas un biomarqueur cliniquement utile chez les chiens atopiques.

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.

Epidermolysis bullosa acquisita in a Great Dane.

Autoimmune subepidermal blistering diseases in dogs were all classified as bullous pemphigoid until 1998. Since then, refinements in reagents and immunological techniques have allowed diseases which are histologically similar but which have a different molecular pathogenesis to be described. This report describes the first case of one such disease, epidermolysis bullosa acquisita, to be documented in the UK. The dog presented with a severe blistering and ulcerative disease affecting the oral cavity, pinnae and distal limbs. The diagnosis was confirmed by histopathology and direct and indirect immunofluorescent demonstration of immunoglobulin G reactivity to basement membrane antigens. Treatment with glucocorticoids, azathioprine, colchicine and an intravenous infusion of immunoglobulins resulted in complete resolution. The drugs were discontinued 12 months after the start of treatment and the dog remained in remission.

Acute necrotising pulmonary vasculitis and pulmonary hypertension in a juvenile dog.

A five-month-old female Jack Russell terrier was presented for investigation of acute lethargy, anorexia, coughing, respiratory distress and weakness. Examination findings included cyanosis, a grade 3 of 6 systolic heart murmur and prolonged capillary refill time. Radiography and echocardiography revealed severe pulmonary hypertension, cor pulmonale and right-sided heart failure. Indirect measurement of the systolic pulmonary artery pressure estimated pressures over 100 mmHg. Despite treatment the patient died. Post-mortem examination did not identify a congenital cardiovascular anomaly. Histopathology confirmed acute necrotising pulmonary arteritis and immunohistochemistry failed to identify any immune complex or complement deposition.

A Toxocara canis infection influences the immune response to house dust mite allergens in dogs.

BACKGROUND: The "hygiene hypothesis" suggests that a western way of life, including the extended use of anti-infective drugs, a high standard of hygiene and the resulting reduced exposure to microorganisms, could be one of the possible explanations for the increasing prevalence of allergic diseases in humans and animals. OBJECTIVES: we wished to evaluate if a nematode infection influenced IgE sensitization and allergic reactions to house dust mites in an experimental atopic dog model. METHODS: Twelve 10-week-old beagles were included: six of them were inoculated orally withToxocara canis (Tc) while six served as non-infected. Tc-specific IgE and IgG against Tc L3 E/S antigen (TcE/S antigen) were measured before and after Tc infection. All twelve dogs were sensitized epicutaneously to Dermatophagoides farinae (Df) house dust mites and then challenged twice epicutaneously with the mite. Total IgE and Df-specific IgE were measured before/after sensitization and after challenge. Local skin lesion scores were assessed before/after sensitization and after challenge while the duration of pruritus manifestations was measured by video after the second challenge. RESULTS: Toxocara canis -infected dogs exhibited higher levels of IgG and IgE levels against Tc, Df-specific IgE, total IgE but lower skin lesion scores and pruritus durations after challenge, compared to dogs not infested with this nematode. CONCLUSION & CLINICAL RELEVANCE: These observations suggest that a Tc infection increases the sensitization to Df in dogs. The possible protective effect against Df-induced clinical signs after allergen challenge should be confirmed in larger studies.

Molecular cloning of canine bullous pemphigoid antigen 2 cDNA and immunomapping of NC16A domain by canine bullous pemphigoid autoantibodies.

The autoantibody-mediated subepidermal blistering skin disease bullous pemphigoid affects both humans and dogs. We previously demonstrated that canine bullous pemphigoid patient's autoantibodies targeted skin basement membrane component and a 180-kDa keratinocyte protein. We extend our works to partially isolate the cDNA encoding canine bullous pemphigoid antigen 2 (BPAg2, BP180). Total RNA extracted from a papillomavirus-immortalized canine keratinocyte cell line and a cultured canine squamous carcinoma cell line SCC 2/88 were used to isolate fragments of cDNA encoding BPAg2 by reverse transcription-PCR and 5'-rapid amplification of cDNA end. The isolated sequence included the 5'-untranslated region, the entire intracellular, transmembranous, and extracellular NC16A autoantigenic domains, plus a small segment of the collagenous domain. Sequence analyses of the isolated cDNA showed 87 and 85% identities between canine and human at the nucleotide sequence and at the deduced amino acid sequence levels, respectively. The canine BPAg2 sequence was confirmed by a rabbit antibody raised against a 18-amino acid peptide deduced from the canine NC16A nucleotide sequence. Autoantibodies from canine bullous pemphigoid patients' sera recognized epitopes within the human NC16A domain. The cloning of the cDNA encoding this disease-associated protein may allow us to develop a canine model in dissecting the immunopathologic mechanism underlying bullous pemphigoid.

Langerhans cell hyperplasia and IgE expression in canine atopic dermatitis.

Langerhans cells appear to be critical for IgE-mediated allergen capture and presentation in human atopic dermatitis. The present study sought to determine whether epidermal (i.e Langerhans cells) and dermal dendritic cells in the skin of dogs with atopic dermatitis are hyperplastic and expressed surface IgE. Frozen sections of lesional or non-lesional atopic and normal control canine skin were immunostained with CD1a-, CD1c-, and IgE-specific monoclonal antibodies. The enumeration of cells was performed by morphometry in both the epidermis and the dermis. Cell counts were compared with each individual's total serum IgE levels. Higher numbers of epidermal and dermal dendritic cells were present in atopic dogs than in normal control animals. Epidermal Langerhans cell counts were significantly higher in lesional than in non-lesional atopic specimens. IgE+ dendritic cells were observed in lesional atopic epidermis and dermis, and non-lesional atopic dermis, but not in normal control skin specimens. The percentages of IgE+ dendritic cells were correlated with each patient's total serum IgE levels. These results demonstrate dendritic cell hyperplasia and IgE expression in canine atopic dermatitis. Increased epidermal Langerhans cell counts in lesional specimens suggest an epidermal allergen contact in canine atopic dermatitis.

Investigation of epidermotropism in canine mycosis fungoides: expression of intercellular adhesion molecule-1 (ICAM-1) and beta-2 integrins.

In human mycosis fungoides (MF), interactions between LFA-1 (CD11a/CD18) and ICAM-1 (CD54) are involved in lymphocyte adhesion to keratinocytes. The purpose of this study was to evaluate the expression of ICAM-1, beta-2 integrins and class II major histocompatibility complex molecules (MHC II) on keratinocytes and infiltrating lymphocytes in canine MF. Sections of frozen skin biopsy specimens from normal dogs (n = 3) and dogs with MF (n = 17) were evaluated by immunohistochemistry for expression of ICAM-1, beta-2 integrins, and class II MHC molecules. Our results demonstrated that in canine MF, ICAM-1 was expressed variably on epidermal and follicular keratinocytes. The extent of keratinocyte ICAM-1 expression did not correlate with the degree of lymphocyte epithelial infiltration, nor with lymphocyte LFA-1 expression. This was especially evident in cases of Pagetoid reticulosis-like disease in which prominent lymphocyte epidermotropism was not accompanied by keratinocyte ICAM-1 expression. Keratinocyte class II MHC molecule expression did not correlate with keratinocyte ICAM-1 expression. In conclusion, in canine MF, the lack of statistically significant correlations between epithelial lymphocyte infiltration and keratinocyte ICAM-1 expression, and between keratinocyte ICAM-1 and lymphocyte LFA-1 staining, suggests that the LFA-1/ICAM-1 pathway is not the major adhesion mechanism between lymphocytes and keratinocytes. It is suspected that different ligands of the LFA-1 integrin (e.g. ICAM-2) or other adhesion molecules (e.g. CD2/LFA-3, VLA-1) might be involved in the epitheliotropism phenomenon in canine MF. These hypothesis cannot be evaluated in the dog at this time owing to the lack of specific monoclonal antibodies.

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.

The ACVD task force on canine atopic dermatitis (VII): mediators of cutaneous inflammation.

Controversy still exists on the role of various inflammatory mediators in the pathogenesis of canine atopic dermatitis. The objective of this article is to review the most recent information available on the inflammatory mediators that have been implicated in the pathogenesis of this disease. Studies on the role of histamine, serotonin, leukotrienes and various cytokines are presented in a comparative manner reviewing the experimental evidence for a role in the pathogenesis and the arguments against it.

The ACVD task force on canine atopic dermatitis (XXII): nonsteroidal anti-inflammatory pharmacotherapy.

The pharmacotherapy of canine atopic dermatitis has relied primarily on the use of glucocorticoids and anti-histamines. During the last decade, other anti-inflammatory drugs have been investigated in clinical trials. This paper will review the studies using misoprostol, cyclosporine, tacrolimus, phosphodiesterase inhibitors, capsaicin, leukotriene inhibitors and serotonin-reuptake inhibitors for treatment of dogs with atopic dermatitis. For each drug the mechanism of action, the rationale for use in atopic dermatitis, the clinical efficacy, reported adverse effects and strength of recommendation for treatment of canine atopic dermatitis are described. At the time of this writing, there is fair evidence to support the recommendation for using cyclosporine, misoprostol and pentoxifylline for treatment of canine atopic dermatitis. This recommendation can be strengthened by the performance of additional blinded randomized controlled trials with larger number of dogs. In contrast, there is insufficient evidence to recommend for or against treatment with tacrolimus, leukotriene inhibitors, serotonin-reuptake antagonists and capsaicin.

The ACVD task force on canine atopic dermatitis (V): biology and role of inflammatory cells in cutaneous allergic reactions.

Numerous inflammatory cells are thought to play a role in the pathogenesis of canine atopic dermatitis (AD) although, in the past, mast cells were considered the most important. However, evidence for this assumption is lacking. In this paper, we review the literature concerning the role of inflammatory cells in allergic reactions and conclude that a complex interplay exists between a wide variety of cell types. Thus, on the basis of the available evidence, the cells that appear to be the most important in the pathogenesis of canine AD are Langerhans' cells and dermal dendritic cells (both responsible for antigen processing and presentation), B-lymphocytes (responsible for reaginic antibody production), allergen-specific helper T-lymphocytes (responsible for cytokine production leading to activation of B-cells and other inflammatory cells) and mast cells (production of inflammatory mediators leading to inflammation).

The ACVD task force on canine atopic dermatitis (VIII): is the epidermal lipid barrier defective?

In humans with atopic dermatitis (AD), it is suspected that the epidermal lipid barrier is abnormal because of combined insufficient extrusion of lipid-containing organelles into the superficial epidermal intercellular spaces as well as skin lipid metabolic defects. To date, studies investigating skin hydration and lipids in atopic dogs are scarce and unfortunately have yielded conflicting data. Whether or not dogs with AD exhibit dry skin and an inadequate stratum corneum barrier, therefore, remains the subject of speculation.

The ACVD task force on canine atopic dermatitis (IX): the controversy surrounding the route of allergen challenge in canine atopic dermatitis.

For decades, the dogma that environmental allergens trigger cutaneous inflammation led to the denomination of canine atopic dermatitis as "allergic inhalant dermatitis". Definitive proof for a respiratory route of allergen challenge is lacking, however. Recent observations suggest, in fact, that skin inflammation could occur because of epidermal allergenic contact. The aim of this paper is to review the evidence published in favor and against the two suspected routes of allergen provocation.

The ACVD task force on canine atopic dermatitis (XVIII): histopathology of skin lesions.

For years, the histopathology of skin lesions of canine atopic dermatitis was deemed non-specific for this diagnosis. However, more recent studies have established that canine atopic skin lesions exhibit an inflammatory pattern characterized as a chronic, hyperplastic and spongiotic, mixed perivascular dermatitis. The nature of epidermal and dermal inflammatory cell infiltrates has now been characterized using modern immunological techniques. Epitheliotropic cells include Langerhans' cells, T-lymphocytes and rare eosinophils. Dermal cells are composed of mast cells, dermal antigen-presenting cells, T-lymphocytes and occasional intact and degranulated eosinophils. This paper provides an historical review of the landmark papers that have elucidated the pathology of canine atopic dermatitis.

The ACVD task force on canine atopic dermatitis (XIX): general principles of therapy.

The treatment of canine atopic dermatitis is multifaceted and consists of a combination of actions that include the use of allergen avoidance, anti-inflammatory agents, allergen-specific immunotherapy and antimicrobial drugs. The importance and order of these treatment steps vary from patient to patient. General recommendations for each of the therapeutic steps are highlighted in this paper. Specific details are covered in other papers of this issue.