Proof of concept of the preventive efficacy of high-dose recombinant mono-allergen immunotherapy in atopic dogs sensitized to the Dermatophagoides farinae allergen Der f 2.

BACKGROUND: Allergen immunotherapy is currently the only intervention proposed to specifically prevent clinical flares after allergen challenges. The low molecular weight Der f 2 (Df2) is a major allergen in Japanese dogs sensitized to Dermatophagoides farinae house dust mites. OBJECTIVES: Pilot, blinded, placebo-controlled experiment testing the efficacy of subcutaneous immunotherapy (SCIT) with high doses of recombinant Df2 conjugated to the maltotriose pullulan (rDf2-P). METHODS: Eight Maltese beagle atopic dogs were sensitized to rDf2 then randomized to SCIT with rDf2-P (six dogs) or placebo (two). The immunotherapy consisted of six weekly injections of increasing doses (0.1-10.0 µg) of rDf2-P followed by four monthly injections of 10 µg of this allergen. Epicutaneous rDf2 challenges, rDf2-specific IgE serology and intradermal reactivity, as well as serum cytokine level measurements, were performed throughout the study. RESULTS: Subcutaneous injections of placebo did not alter the cutaneous reactivity after rDf2 challenge, while that of the dogs treated with rDf2-P SCIT disappeared in five of six dogs (83%) and was reduced in one of six (17%). During SCIT maintenance, skin lesion scores were significantly lower in dogs receiving SCIT compared to those treated with placebo. This clinical improvement was accompanied by a concurrent, yet not significant, decrease in rDf2-specific IgE serology and immediate intradermal reactivity. Cytokine serum levels were inconclusive. There were no adverse events seen with rDf2-P SCIT. CONCLUSIONS AND CLINICAL IMPORTANCE: The new mono-allergen SCIT appears safe and effective for reducing skin lesions after allergen challenges; it deserves further testing in dogs with spontaneous atopic dermatitis.

The novel high molecular weight Dermatophagoides farinae protein Zen-1 is a major allergen in North American and European mite allergic dogs with atopic dermatitis.

BACKGROUND: Atopic dogs with hypersensitivity to Dermatophagoides farinae (Df) have IgE recognizing high molecular weight (MW) allergens more often than the low MW Der f 1 and 2. A new high MW Df allergen, Zen-1, has been identified recently. OBJECTIVES: To determine the IgE reactivity of American and European Df-hypersensitive dogs to Zen-1, Der f 1 and Der f 2. METHODS: We tested sera from 33 Df-reactive dogs from the USA, 29 from Europe and 15 experimentally sensitized to Df, by ELISA against crude Df, Der f 1, Der f 2 and Zen-1. ELISA inhibition was performed with sera reactive to Zen-1. Intradermal testing (IDT) was also done with the same allergens in 25 other American atopic dogs. RESULTS: Altogether, IgE seropositivity to Zen-1 was more prevalent (86%) than that to Der f 1 (17%) or Der f 2 (19%). The IgE reactivity to Zen-1 was correlated to that against crude Df; this allergen alone inhibited a high percentage (median: 50%; range: 22-84%) of the binding to the crude mite extract. The seropositivity to low MW allergens was highest in experimentally sensitized dogs. Serum IgE recognition of Der f 1 was low in dogs with AD; that to Der f 2 was significantly lower in American dogs (6%) than in European ones (28%). A high prevalence of positive immediate IDT reactions to Zen-1 confirmed the likely relevance of serological results. CONCLUSIONS AND CLINICAL IMPORTANCE: This study establishes Zen-1 as a major allergen in atopic dogs sensitized to Df.

Evaluation of intraepidermal nerve fibres in the skin of normal and atopic dogs.

BACKGROUND: Interest in intraepidermal nerve fibres (IENFs) is rising in human medicine, because variations in fibre density occur in some diseases and these neurites might contribute to disease pathogenesis. An increase in IENF density is seen in human atopic dermatitis (AD); there are no such data in atopic dogs. OBJECTIVES: To compare the prevalence of IENFs in normal and atopic canine skin. METHODS: Eight millimetre skin punch biopsies were taken from six sites of 25 healthy dogs without dermatitis and compared to lesional and nonlesional skin samples of dogs with AD (23 and 14 dogs, respectively). Thirty micrometre-thick paraffin-embedded sections were stained by indirect immunofluorescence for neuronal beta-3 tubulin. Only sections with detectable dermal nerves were then screened for the presence of IENFs. RESULTS: IENFs were identified in all 25 normal nasal planum sections, but in only one biopsy collected from each of the normal canine haired skin (NCHS) sites. As there was no significant difference in IENF prevalence between NCHS areas, they were grouped together. The rate of detection of IENFs was significantly higher (one-tailed Fisher's test, P = 0.004) in lesional AD specimens (18 of 23; 78%) than in nonlesional AD (four of 14; 29%) and NCHS specimens (four of 111; 4%, P < 0.0001). The prevalence of IENF detection in nonlesional AD samples was significantly higher than in normal canine skin (P = 0.006). CONCLUSIONS AND CLINICAL IMPORTANCE: IENFs are detected more commonly in canine AD than in normal haired skin; these results are comparable to those seen for human AD.

Expression patterns of superficial epidermal adhesion molecules in an experimental dog model of acute atopic dermatitis skin lesions.

BACKGROUND: The stratum corneum is critical for providing a functional skin barrier, especially in humans and dogs with atopic dermatitis. An effective barrier also depends upon intact corneodesmosomes and superficial epidermal tight junctions. HYPOTHESIS/OBJECTIVES: To study the expression of selected corneodesmosome, desmosome, tight and adherens junction proteins in an experimental model of acute atopic dermatitis skin lesions in dogs. METHODS: Control and house dust mite (HDM) allergen-containing patches (two types of patches) were applied to the skin of six Maltese-beagle atopic dogs hypersensitive to HDM. Patches were left on for 48 h, and biopsies were collected 24 h after removal. Frozen skin sections were stained by indirect immunofluorescence for corneodesmosin, desmoglein-1, desmocollin-1, claudin-1 and E-cadherin. Immunostains were assessed for their extent, intensity and patterns; they were compared between HDM and control patches on the same dogs. RESULTS: The immunostaining for E-cadherin, desmocollin-1 and desmoglein-1 was homogeneous, intercellular and continuous in all control and HDM patches. The immunoreactivity of corneodesmosin and claudin-1 was heterogeneous and reduced in intensity in 12 of 12 and eight of 12 HDM patches, respectively, in contrast to a normal expression seen in all control samples (Fisher's test, P < 0.001). CONCLUSIONS AND CLINICAL IMPORTANCE: These observations suggest that HDM allergens, via proteolytic digestion and/or because of induced allergic inflammation, affect the expression and possible function of corneodesmosomal and tight junction proteins. Ensuing intercellular junction alterations might promote an abnormally increased penetration of allergens through the epidermis.

Canine epidermolysis bullosa acquisita: a retrospective study of 20 cases.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a rare autoimmune subepidermal blistering disease of dogs and humans. OBJECTIVES: Our objectives were to describe clinical phenotypes, histopathology and treatment outcomes of canine EBA. ANIMALS: Twenty dogs diagnosed with EBA based on a subepidermal blister formation and collagen VII autoreactivity. RESULTS: Most dogs were young (median: 1.2-year-old) with a male-to-female ratio of 2.3:1. Nine of 20 dogs (45%) developed lesions before one year of age and 11 of 20 dogs (55%) were great danes. Tense vesicles and bullae (18 of 20; 90%) and deep erosions and ulcers (20 of 20; 100%) were the most common lesions and these affected predominantly the oral cavity (19 of 20; 95%), pinnae (16 of 20; 80%), axillae (15 of 20; 75%) and footpads (14 of 20; 70%). Histopathology identified neutrophilic perivascular dermatitis (17 of 17; 100%) without or with (12 of 17; 71%) eosinophils, which occasionally equalled (four cases) or outnumbered neutrophils (two cases). Subepidermal vesicles were either devoid of inflammation or contained neutrophils with or without eosinophils, fibrin and/or haemorrhage. A complete remission of skin lesions was obtained in 14 dogs with a median time of 58 days. Glucocorticoids were used in these dogs either as a monotherapy (3 of 14; 21%) or in combination with other immunomodulating drugs (11 of 14; 79%). The median dose of prednisone was 3 mg/kg/day. The remaining six dogs were euthanized. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine EBA is a rare subepidermal blistering disease with an inflammatory phenotype and a predilection for young great danes and male dogs. The outcome of treatment appears more favourable than assumed previously.

Serum autoantibody profiles of IgA, IgE and IgM in canine pemphigus foliaceus.

BACKGROUND: Pemphigus foliaceus (PF) is the most common IgG-mediated autoimmune skin disease in dogs. Studies of human PF have revealed the presence of other antigen-specific autoantibody isotypes, thereby uncovering new avenues of investigation of the disease pathomechanism. HYPOTHESIS/OBJECTIVES: The aim was to obtain information about the autoantibody isotype response in canine PF. METHODS: Sera from 34 dogs with PF were tested for the presence of antikeratinocyte, anti-desmocollin-1 and anti-desmoglein-1 IgA, IgE and IgM using indirect immunofluorescence. RESULTS: Using our indirect immunofluorescence technique, IgA, IgE and IgM autoreactivities were detected in six, one and zero of 34 sera from PF-affected dogs, respectively. Two of the six IgA-positive sera contained antikeratinocyte and anti-desmocollin-1 IgA, while the four remaining sera tested positive either for antikeratinocyte IgA (two of six) or for anti-desmocollin-1 IgA (two of six). A single serum contained anti-desmocollin-1 IgE. None of the six sera from healthy dogs contained detectable IgA, IgE or IgM autoantibodies. CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings suggest that sera from dogs with PF rarely contain IgA or IgE autoantibodies at levels detectable by indirect immunofluorescence, while IgM autoreactivity appears not to be a feature of this disease. Considering these findings, it appears that canine PF is aetiologically and immunologically similar to that of the classic human PF, in which the IgG autoantibody response is also the predominant type.

Stratum corneum removal facilitates experimental sensitization to mite allergens in atopic dogs.

In humans with atopic dermatitis and in mouse models of IgE-mediated allergic diseases, evidence is mounting that the stratum corneum (SC) provides an important barrier against environmental allergens. At this time, it is not known whether the SC has a similar role in dogs, especially in those with atopic dermatitis. The objectives of this pilot study were to determine whether SC removal led to earlier and stronger sensitization of atopic dogs to Dermatophagoides farinae (Df) house dust mites. Five Maltese-beagle atopic (MBA) dogs were sensitized epicutaneously after the SC was removed with ten tape strips (TS group), while sensitization was done without tape strips in five other MBA dogs (nontape stripping; NTS group). During this 16 week study, sensitization was assessed with allergen-specific IgE serology, intradermal testing with Df allergens and determination of stimulation indices of blood mononuclear cells cultured with Df and stained for CD4 and the activation markers CD25 or CD30. Compared with dogs from the NTS group, those of the TS group exhibited earlier rises in Df-specific IgE serum levels, usually had higher allergen-specific IgE titres, showed higher intradermal test reactivity and had earlier increases and higher percentages of CD25- or CD30-positive activated allergen-specific peripheral CD4-positive T lymphocytes. These observations implicate a role of the SC as a barrier limiting sensitization to exogenous allergens in this experimental atopic dog model.

Clinical and immunological heterogeneity of canine subepidermal blistering dermatoses with anti-laminin-332 (laminin-5) auto-antibodies.

Laminin-332 (laminin-5) is a basement membrane heterotrimeric protein composed of alpha-3, beta-3 and gamma-2 laminin chains. Laminin-332 polypeptides are targeted by auto-antibodies in human patients with mucous membrane (cicatricial) pemphigoid or, more rarely, subepidermal vesicular diseases that resemble epidermolysis bullosa acquisita (EBA) or bullous pemphigoid (BP). The objectives of this report were to characterize the clinical, histopathological and immunological characteristics of nine dogs with auto-antibodies targeting laminin-332. Immunological investigations consisted of direct immunofluorescence (IF), indirect IF with intact and salt-split canine gingival, and salt-split normal or laminin-332-deficient human skin, immunoblotting with purified human laminin-332 and immunoblotting with recombinant NC1 domain of human collagen VII. All dogs exhibited varying degrees of skin blistering and ulceration associated with microscopic subepidermal vesiculation with or without inflammatory cells. Indirect IF established that circulating IgG auto-antibodies bound the dermal side of salt-split canine lip and human skin. In five dogs, IgG variably recognized the basement membrane of laminin-332-deficient human skin (three dogs negative, two dogs positive). In all nine dogs, IgG auto-antibodies detected purified human laminin-332 by immunoblotting. In two dogs, additional targeting of collagen VII-NC1 was present. These observations establish laminin-332 as a novel basement membrane antigen in dogs with autoimmune blistering diseases with variable clinical phenotypes. The names 'acquired junctional epidermolysis bullosa', 'anti-laminin-332 mucous membrane pemphigoid (MMP)' and 'mixed auto-immune subepidermal blistering dermatosis' are proposed for dogs with clinical signs reminiscent of EBA, MMP or BP respectively.

Investigations on the nature and pathogenicity of circulating antikeratinocyte antibodies in dogs with pemphigus foliaceus.

In humans with pemphigus foliaceus (PF), pathogenic autoantibodies are principally of IgG4 subclass and they cause superficial vesiculation when injected into neonatal mice. The objectives of this study were to determine the isotypes of circulating antikeratinocyte antibodies in dogs with PF, to assess whether serum antikeratinocyte antibody titres decreased during successful treatment, and to study whether such antibodies were pathogenic in passive transfers. Using indirect immunofluorescence with neonatal mouse skin substrates, circulating antikeratinocyte IgG antibodies were detected in 36 of 44 dogs with PF (82%). Serum autoantibodies belonged predominantly to IgG4 (three of 44; 80%) and IgG1 (30 of 44; 68%) subclasses. Antikeratinocyte IgG antibodies were detected in 16 of 20 normal dogs (80%), and these antibodies were IgG1 (16 of 20, 80%) but rarely IgG4 (two of 20; 10%) isotypes. In four dogs, IgG4 antikeratinocyte antibody titres decreased concomitantly to lesions nearing or reaching complete remission. In contrast, IgG or IgG1 titres remained stable or increased when lesions abated. Antikeratinocyte antibodies targeted mainly intercellular autoantigen(s) in the stratum granulosum, while in fewer dogs, such antibodies bound to cytoplasmic basal antigen(s). Intradermal injections of PF or pemphigus vulgaris (PV) IgG into neonatal mice caused subgranular or suprabasal acantholytic vesiculation without granulocyte infiltration, respectively. Similar transfers of normal dog IgG did not cause vesiculation. These observations suggest that antikeratinocyte IgG4 antibodies could be relevant to disease pathogenesis. Importantly, canine PF or PV IgG appear to be pathogenic when transferred passively into mice, causing vesiculation at epidermal levels similar to those of the natural disease.

Lack of detection of circulating skin-specific IgE autoantibodies in dogs with moderate or severe atopic dermatitis.

Human patients with atopic dermatitis (AD) commonly exhibit IgE reactivity to cutaneous self-antigens. The presence of serum IgE autoantibodies appears to correlate with disease severity, and it is suspected to reflect or contribute to tissue damage. The objective of this study was to determine whether IgE autoantibodies specific for cutaneous antigens could be detected in the serum of dogs with AD. Serum was collected from 19 dogs with untreated moderate to severe AD and four specific-pathogen free (SPF) dogs. Indirect immunofluorescence was performed using normal canine skin collected at four different locations (concave ear, nose, medial thigh and lateral thorax), while Western immunoblotting was done using normal canine ear pinna epidermal and dermal extracts and reducing conditions. In both methods, IgE was detected using a monoclonal antibody specific for heat stable epitopes of canine IgE. At 1:10 dilution, specific IgE autoantibodies against cutaneous autoantigens were not detected, with either method, in AD and SPF canine sera. Either IgE autoreactivity is not associated with moderate to severe AD in dogs, or the methods employed herein were not sensitive enough to permit IgE autoantibody detection.

Desmoglein-1 is a minor autoantigen in dogs with pemphigus foliaceus.

The majority of human patients with pemphigus foliaceus (PF) have circulating IgG autoantibodies that target conformational epitopes on the desmosomal cadherin desmoglein-1 (dsg1). Limited studies using immunoblot techniques suggested that the principal autoantigen in dogs with PF might also be dsg1. It was the objective of this study to test this hypothesis. A comprehensive survey of canine PF sera was conducted using a novel screening strategy that detects conformational epitopes. This method consists of the ectopic expression of canine dsg1 at the surface of human 293T epithelial kidney cells and their live screening, i.e. prior to fixation. Out of seven control human PF sera that bound to canine epidermis, three (57%) contained IgG autoantibodies that recognized ectopically expressed canine dsg1 with a membrane and punctate pattern. Out of 83 canine PF sera only five (6%) contained IgG that recognized canine dsg1. Consistent with findings for human PF sera obtained in this study, autoantibody binding was conformation- and glycosylation-dependent as demonstrated by calcium chelation with EDTA and tunicamycin or wheat germ agglutinin treatment, respectively. In conclusion, these studies establish canine dsg1 as a minor autoantigen for canine PF. Antigenic epitopes appear to be conformation- and glycosylation-dependent.

Allergic inflammation is augmented via histamine H4 receptor activation: The role of natural killer cells in vitro and in vivo.

BACKGROUND: Natural Killer cells (NK cells) are identified as pivotal mediators in allergic skin diseases and accumulate in lesions of atopic dermatitis (AD) patients. Histamine levels are increased in these lesions and histamine is involved in chemotaxis in dendritic cells and NK cells. OBJECTIVE: The aim of this study was to determine if the histamine H4 receptor (H4R) mediates NK cell chemotaxis and whether it influences interplay between NK cells and dendritic cells during the early phase of allergic inflammation. METHODS: Chemotactic function of the H4R as well as the influence of the H4R on the cytokine profile of an NK cell-dendritic cell co-culture was studied in vitro. The effect of H4R activation on NK cell migration, NK cell-dendritic cell interaction and cytokine levels in the skin was further characterized in the murine TDI model of allergic dermatitis. Additionally, the impact of the H4R on dermal NK cells was determined in the ovalbumin (OVA)- induced allergic dermatitis model, comparing wild type and H4R knockout mice. RESULTS: The selective H4R agonist ST-1006 induced NK cell chemotaxis in vitro, which was inhibited with the H4R antagonist JNJ7777120. In vivo, mice treated with TDI plus ST-1006 topically onto the ear, showed significantly enhanced ear swelling and an increased number of NK cells compared to just allergen challenged ears. CCL17 levels in the ear were also significantly increased 8h after allergen challenge. Histology revealed that the main source for increased CCL17 were dendritic cells. These effects could be blocked using the H4R antagonist JNJ7777120. In the chronic model of allergic dermatitis, OVA induced NK cell migration into lesional skin sites. The number of NK cells was lower in OVA-sensitized H4R knockout mice compared to wild type mice. CONCLUSIONS: These results identify the H4R as a new target controlling NK cell migration and NK cell-dendritic cell interaction in the skin during early allergic inflammation. These results further suggest that blocking the H4R in the skin might be beneficial in diseases like AD.

Laminin-5 is targeted by autoantibodies in feline mucous membrane (cicatricial) pemphigoid.

In human and canine patients with mucous membrane (cicatricial) pemphigoid (MMP), circulating autoantibodies have been shown to target multiple epidermal basement membrane antigenic epitopes. These autoantigens include collagen XVII in humans and dogs, as well as laminin-5, laminin-6 or integrin alpha-6/beta-4 in human beings. The purpose of this study was to determine if autoantibodies targeted laminin-5 in a cat exhibiting clinical and microscopic lesions resembling those of MMP in humans. In this patient, an indirect immunofluorescence (IF) assay revealed circulating IgG and IgA autoantibodies that bound to the basement membrane zone on the dermal side of salt-split gingiva (titer 1:1000 for IgG and 1:50 for IgA). Immunoblotting, performed with affinity-purified human laminin-5, demonstrated that the autoantibodies bound the alpha-3 chain of this heterotrimer. These observations identify laminin-5 as one of the antigens recognized by circulating autoantibodies in this feline homologue of MMP in humans and dogs.

Absent expression of collagen XVII (BPAG2, BP180) in canine familial localized junctional epidermolysis bullosa.

Hereditary junctional epidermolysis bullosa (JEB) represents a subset of mechanobullous diseases associated with defective hemidesmosome/anchoring filament proteins leading to cleavage in the lamina lucida of the epidermal basement membrane. In humans, most cases of JEB have been related to a deficiency of either laminin-5, collagen XVII (BPAG2, BP180) or integrin ß4. We describe the existence of a previously unreported form of familial localized non-lethal JEB in German Shorthaired Pointer littermates. Acral, auricular and oral erosions and ulcers were observed. Severe ulceration of the footpads was present. Skin biopsy specimens of non-lesional and lesional skin of affected dogs were screened for a defect in basement membrane proteins using indirect immunofluorescence and immunoperoxidase testing. Epidermal staining for laminin-5 and integrin α6ß4 was similar in affected and normal control dogs. Lack of expression of collagen XVII was uniquely identified in all sections of JEB probands compared with normal control dogs. The defective expression of collagen XVII is likely to be caused by mutation(s) of the COL17A1 gene, as previously reported in humans. This is, to date, the first report of a deficient basement membrane protein in canine JEB.

Canine epidermolysis bullosa acquisita: circulating autoantibodies target the aminoterminal non-collagenous (NC1) domain of collagen VII in anchoring fibrils.

The classification of autoimmune blistering skin diseases is based on the skin antigen(s) targeted by pathogenic autoantibodies. In humans and dogs, there is increasing evidence that autoimmune subepidermal bullous diseases represent different nosological entities. This study establishes the existence of the canine equivalent of epidermolysis bullosa acquisita (EBA) in humans. Canine EBA, like the inflammatory variant of its human counterpart, is characterized by spontaneous vesicles arising from an inflammatory eruption. Dermo-epidermal separation occurs in association with neutrophilic infiltration in the superficial dermis. Tissue-fixed and circulating IgA and IgG autoantibodies specific for the lower basement membrane zone can be detected by immunofluorescence methods. Using immunoelectron microscopy, autoantibodies are shown to target the distal end of anchoring fibrils in the sublamina densa. ELISA and immunoblotting utilizing eukaryotically expressed recombinant collagen VII subdomains confirm that the circulating autoantibodies are specific for the aminoterminal globular non-collagenous NC1 domain of type VII collagen.

Deficient plakophilin-1 expression due to a mutation in PKP1 causes ectodermal dysplasia-skin fragility syndrome in Chesapeake Bay retriever dogs.

In humans, congenital and hereditary skin diseases associated with epidermal cell-cell separation (acantholysis) are very rare, and spontaneous animal models of these diseases are exceptional. Our objectives are to report a novel congenital acantholytic dermatosis that developed in Chesapeake Bay retriever dogs. Nine affected puppies in four different litters were born to eight closely related clinically normal dogs. The disease transmission was consistent with an autosomal recessive mode of inheritance. Clinical signs occurred immediately after birth with superficial epidermal layers sloughing upon pressure. At three month of age, dogs exhibited recurrent superficial skin sloughing and erosions at areas of friction and mucocutaneous junctions; their coat was also finer than normal and there were patches of partial hair loss. At birth, histopathology revealed severe suprabasal acantholysis, which became less severe with ageing. Electron microscopy demonstrated a reduced number of partially formed desmosomes with detached and aggregated keratin intermediate filaments. Immunostaining for desmosomal adhesion molecules revealed a complete lack of staining for plakophilin-1 and anomalies in the distribution of desmoplakin and keratins 10 and 14. Sequencing revealed a homozygous splice donor site mutation within the first intron of PKP1 resulting in a premature stop codon, thereby explaining the inability to detect plakophilin-1 in the skin. Altogether, the clinical and pathological findings, along with the PKP1 mutation, were consistent with the diagnosis of ectodermal dysplasia-skin fragility syndrome with plakophilin-1 deficiency. This is the first occurrence of ectodermal dysplasia-skin fragility syndrome in an animal species. Controlled mating of carrier dogs would yield puppies that could, in theory, be tested for gene therapy of this rare but severe skin disease of children.

Patch testing of experimentally sensitized beagle dogs: development of a model for skin lesions of atopic dermatitis.

In humans with atopic dermatitis (AD), the epicutaneous application of allergens (atopy patch tests or APT) to which the patients are sensitized often results in the development of inflammation resembling that of spontaneous skin lesions. Dogs are affected with a natural homologue of human AD, but information on the induction of positive patch testing reactions is limited. The objectives of this pilot study were to determine the nature and cellular dynamics of inflammation occurring after APT in dogs hypersensitive to house dust mite and flea allergens. Laboratory Beagles were sensitized experimentally to Dermatophagoides farinae house dust mites (two dogs), Ctenocephalides felis flea saliva (one dog) or both (two dogs). Two other dogs served as nonsensitized controls. Both allergens and saline were applied epicutaneously. Macroscopic evaluations and skin biopsies were performed at 4, 24, 48 and 96 h after starting allergenic challenge. Biopsies were evaluated histologically and immunohistochemically with a panel of monoclonal antibodies specific for canine leucocyte antigens. Positive macroscopic reactions consisted of erythema, oedema and induration, and they occurred between 24 and 96 h after allergen application. Macroscopic and microscopic APT reactions developed only whenever serum IgE was present against tested allergens. Microscopically, positive APT was associated with epidermal hyperplasia, Langerhans' cell hyperplasia, and eosinophil and lymphocyte epidermotropism. Dermal inflammation was mixed and arranged in a superficial perivascular to interstitial pattern. Numerous IgE+-CD1+ dendritic cells and gamma-delta T-lymphocytes were observed. Macroscopically and microscopically, APT reactions in these experimentally sensitized animals resembled those seen in lesional biopsy specimens of dogs and humans with spontaneous AD. Therefore, APT in hypersensitive dogs provides a relevant experimental model to investigate the pathogenesis and treatment of both canine and human AD skin lesions.

Clinical, histopathological and immunological characteristics of exfoliative cutaneous lupus erythematosus in 25 German short-haired pointers.

Clinical, histopathological and immunological features of exfoliative cutaneous lupus erythematosus, an uncommon generalized exfoliative dermatitis occurring exclusively in German short-haired pointers, were characterized in 25 dogs. The disease affects young adult dogs and its familial incidence strongly suggests a hereditary origin. Lesions were characterized by scaling and alopecia affecting 100 (25/25) and 76% (19/25) of dogs, respectively. Follicular casts were present in 28% (7/25) of dogs. The muzzle, pinnae and dorsum were typically affected. Generalized skin lesions were described in 52% (13/25) of dogs. Systemic signs of pain and lameness affected several dogs. Anaemia and thrombocytopenia were detected in several dogs with a more severe clinical phenotype. The most common histopathological features were hyperkeratosis and a lymphocytic interface dermatitis. Direct immunostaining revealed IgG deposition in the epidermal and follicular basement membrane of 100 (19/19) and 41% (7/17) of dogs, respectively. Circulating antifollicular and antisebaceous gland IgG antibodies were demonstrated by indirect immunostaining in 57% (4/7) of dogs. This disease usually responds poorly to immunosuppressive therapy and it has a guarded prognosis. Where outcome was recorded, 85% (10/12) of dogs were euthanased due to either a failure to respond to, or complications associated with, immunomodulatory therapy. Two affected dogs are in remission and maintained on immunomodulatory dosages of prednisolone. This study demonstrates the existence of a cellular and humoral immune response directed against the epidermal basement membrane of dogs with exfoliative cutaneous lupus erythematosus. Additional studies are required to further characterize the immunological pathogenesis of this disease.

Isotype determination of circulating autoantibodies in canine autoimmune subepidermal blistering dermatoses.

The three most common canine autoimmune blistering skin diseases (AISBD), bullous pemphigoid (BP), mucous membrane pemphigoid (MMP) and epidermolysis bullosa acquisita (EBA) have recently been separated based on clinical, histological and immunological grounds. The objectives of this study were to determine the isotype profiles of circulating autoantibodies in these dermatoses. Serum was collected from 5 dogs with BP, 15 with MMP and 11 with EBA. All sera were tested using an indirect immunofluorescence method using salt-split canine gingiva as substrate. Anti-basement membrane IgG autoantibodies were detected in all patients. Among the IgG autoantibodies, IgG1 and IgG4 were encountered most frequently, while IgG2 and IgG3 were uncovered in some dogs. IgE autoantibodies were detected more often than IgA or IgM autoantibodies in any of the three entities. The predominance of IgG1, IgG4 and IgE autoantibody isotypes in dogs with AISBD is very similar to the situation found in humans with the homologous diseases.

Résumé Il a été récemment possible de différencier les trois plus fréquentes dermatoses bulleuses sous-épidermiques (pemphigoide bulleuse [BP], pemphigoïde des muqueuses [MMP] et épidermolyse bulleuse acquise [EBA]) sur la base de critères cliniques, histologiques et immunologiques. Les buts de cette étude étaient de déterminer les profils isotypiques des autoanticorps circulants dans ces maladies. Le sérum de 5 chiens à BP, 15 chiens à MMP et 11 chiens à EBA a été collecté et testé en utilisant une méthode d'immunofluorescence indirecte avec comme substrat une gencive canine clivée par le sel. Des autoanticorps IgG anti-membrane basale ont été détectés chez tous les patients. Parmi ces IgG, les IgG1 et IgG4 étaient rencontrées le plus souvent, alors que les IgG2 et IgG3 étaient absentes chez certains chiens. Des autoanticorps IgE étaient plus fréquemment rencontrés que des IgA ou des IgM pour les trois maladies. La prédominance d'IgG1, IgG4 et IgE chez les chiens présentant une dermatose auto-immune bulleuse sous-épidermique est très semblable à la situation rencontrée chez les patients humains souffrant de maladies équivalentes.

Resumen Las tres enfermedades ampollosas y autoinmunes de la piel más frecuentes en la especie canina, penfigoide bulloso (PB), penfigoide de las membranas mucosas (PMM) y epidermólisis bullosa adquirida (EBA), se han separado recientemente en términos clínicos, histológicos e immunológicos. Los objetivos del estudio fueron determinar el perfil de isotipos de autoanticuerpos circulantes en estas dermatosis. Se tomaron muestras serológicas de cinco perros con PB, 15 con PMM y 11 con EBA. Todos los sueros fueron analizados mediante un método indirecto de immunofluorescencia utilizando separación por NaCl (salt-split) y encía canina como substrato. Se detectaron autoanticuerpos IgG antimembrana basal en todos los pacientes. De los autoanticuerpos IgG, los isotipos IgG1 e IgG4, se hallaron con más frecuencia, mientras que los IgG2 e IgG3 fueron detectados sólo en algunos perros. Autoanticuerpos IgE fueron detectados más a menudo que los autoanticuerpos IgA o IgM en las tres entidades. El predominio de los isotipos de autoanticuerpos IgG1, IgG4 e IgE en perros con enfermedades ampollosas autoinmunes de la piel es muy similar a la situación encontrada en humana con enfermedades homólogas.

Immunopathology of vesicular cutaneous lupus erythematosus in the rough collie and Shetland sheepdog: a canine homologue of subacute cutaneous lupus erythematosus in humans.

Clinical and histological features of an erosive disease in the rough collie and Shetland sheepdog are most consistent with a vesicular variant of cutaneous lupus erythematosus (VCLE). This paper reports the immunopathological findings of canine VCLE using samples from 17 affected dogs. Lesional skin sections were stained with monoclonal antibodies specific for CD3 (11 dogs) or a panel of monoclonal antibodies specific for leukocyte antigens (two dogs). Apoptotic cells were detected using the TUNEL method in 12 cases. Direct (14 dogs) and indirect immunofluorescence tests (five dogs) were also performed. Circulating antibodies to extractable nuclear antigens (ENA) were surveyed in 11 dogs by immunoblotting and ELISA. The predominant cells at the dermal-epidermal interface were identified as CD3(+) T lymphocytes expressing CD4 or CD8 and CD1(+) dendritic antigen presenting cells. In 7/12 dogs (58%), apoptosis of basal keratinocyte nuclei was present. Up-regulation of MHCII and ICAM-1 was observed on basal keratinocytes from the two dogs examined. Direct immunofluorescence revealed deposition of immunoglobulins bound to the cytoplasm of keratinocytes (6/14 dogs; 43%), to the dermal-epidermal junction (7/14 dogs; 50%), or to superficial dermal venules (13/14 dogs; 93%). Circulating IgG auto-antibodies targeting one or more ENA were detected in nine (82%) and eight (73%) of 11 dogs by immunoblotting and ELISA, respectively. These auto-antibodies recognized Ro/SSA and/or La/SSB in four (36%) and six (55%) of 11 dogs respectively by these two methods. Altogether, results of these studies provide evidence supporting the hypothesis that canine VCLE is an immunological homologue of subacute cutaneous lupus erythematosus in humans.