Judy Paps

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.

Lack of preventing effect of systemically and topically administered histamine H1 or H4 receptor antagonists in a dog model of acute atopic dermatitis

As there is evidence for an anti‐inflammatory efficacy of histamine H4 receptor (H4R) selective antagonists, we aimed at testing the efficacy of the H4R antagonists JNJ7777120 and JNJ28307474 in comparison with histamine H1 receptor (H1R) antagonists hydroxyzine and cetirizine for skin lesion prevention in a canine model of acute atopic dermatitis. Six atopic Maltese‐beagle dogs experimentally sensitized to Dermatophagoides farinae (Df) house dust mites were selected for this study. Twenty‐four hours after challenge by epicutaneous application of Df, erythematous skin lesions were scored. In this blinded, placebo and active controlled study, topical JNJ7777120 or JNJ28307474 was applied as a 1% solution before allergen challenge. The latter was also given orally at 15 mg/kg before and after allergen challenge. A 0.015% triamcinolone acetonide solution was used as a positive control. The H1R antagonists hydroxyzine and cetirizine were administered orally before challenge in a second experiment. Twenty‐four hours after challenge, placebo‐treated animals had a median lesional score of 2. Treatment with topical and oral JNJ28307474 resulted in a median score of 2.5. After topical administration of JNJ7777120, the median lesional score was 2. Hydroxyzine and cetirizine did also not reduce the median score of the placebo treatment. Triamcinolone acetonide prevented all dogs from having any lesions. Determination of histamine in lesions revealed that only during the initiation increased concentrations of histamine were detected. In conclusion, the preventive administration of H1R or H4R antagonists has no impact on the development of acute skin lesions in this experimental canine atopic dermatitis model.

Effect of a novel topical diester glucocorticoid spray on immediate- and late-phase cutaneous allergic reactions in Maltese-beagle atopic dogs: a placebo-controlled study.

The inhibitory effect of 0.0584% hydrocortisone aceponate spray on immediate- and late-phase skin reactions and the duration of inhibition after medication withdrawal were studied in 10 Maltese-beagle atopic dogs. All subjects were sprayed on axillary and inguinal regions and on one randomly chosen side of the thorax once daily for 14 (phase 1) or 7 days (phase 2). Intradermal injections (IDT) of histamine and anticanine IgE antiserum were performed bilaterally on the thorax before, 7 and 14 days after treatment. During phase 2, IDT was performed once weekly for 5 weeks. Each IDT was evaluated by an investigator blinded to the site of active treatment. Skin biopsies of 24-h anti-IgE-associated late-phase reactions were collected from both thoracic sides before and 14 days after treatment to determine the number of inflammatory cells and dermal thickness. Phase 1: Histamine and anti-IgE-induced global wheal scores at treated sites were significantly lower after 7 and 14 days with negative reactions present in >90% of dogs. Late-phase reactions at both sides were also significantly decreased compared with that at baseline, and this was associated with reduced inflammatory cell influx. Moreover, a significant decrease in dermal thickness was recorded at treated sides after 14 days. Phase 2: Histamine reactions became positive at untreated sides in all dogs 2 weeks after treatment. In conclusion, the 0.0584% hydrocortisone aceponate spray significantly decreased immediate- and late-phase IDT reactions, and prolonged application caused skin atrophy at treated sites. A 2-week withdrawal period prior to IDT is proposed.

Early Activation of Th2/Th22 Inflammatory and Pruritogenic Pathways in Acute Canine Atopic Dermatitis Skin Lesions

Determining inflammation and itch pathway activation in patients with atopic dermatitis (AD) is fraught with the inability to precisely assess the age of skin lesions, thus affecting the analysis of time-dependent mediators. To characterize inflammatory events occurring during early experimental acute AD lesions, biopsy samples were collected 6, 24, and 48 hours after epicutaneous application of Dermatophagoides farinae house dust mites to sensitized atopic dogs. The skin transcriptome was assessed using a dog-specific microarray and quantitative PCR. Acute canine AD skin lesions had a significant up-regulation of genes encoding T helper (Th) 2 (e.g., IL4, IL5, IL13, IL31, and IL33), Th9 (IL9), and Th22 (IL22) cytokines as well as Th2-promoting chemokines such as CCL5 and CCL17. Proinflammatory (e.g., IL6, LTB, and IL18) cytokines were also up-regulated. Other known pruritogenic pathways were also activated: there was significant up-regulation of genes encoding proteases cathepsin S (CTSS), mast cell chymase (CMA1), tryptase (TPS1) and mastin, neuromedin-B (NMB), nerve growth factor (NGF), and leukotriene-synthesis enzymes (ALOX5, ALOX5AP, and LTA4H). Experimental acute canine house dust mite-induced AD lesions exhibit an activation of innate and adaptive immune responses and pruritogenic pathways similar to those seen in humans with acute AD, thereby validating this model to test innovative therapeutics modalities for this disease.

A comparison of the clinical manifestations of feeding whole and hydrolysed chicken to dogs with hypersensitivity to the native protein

Twenty-six dogs with known adverse food reactions were fed whole chicken for 14 days. From this group, 12 dogs with cutaneous manifestations following exposure to chicken meat were selected and randomly divided into two groups (n = 6). Each group was then fed hydrolysed chicken or hydrolysed soy for 14 days in a blinded crossover design with a 17-day washout period between each diet. Assessments of a CADESI (Canine Atopic Dermatitis Extent and Severity Index) score and pruritus were performed throughout the entire study, and combined in a global score (GS). Serum was collected weekly for the measurement of chicken- and soy-specific IgG and IgE. Dogs displayed the most severe clinical response when eating whole chicken compared to baseline (P < 0.001). The GS was significantly reduced in 11 of the 12 dogs when fed hydrolysed chicken were compared to those fed whole chicken (3.58 ± 2.81 versus 20.38 ± 14.65, P < 0.01). Serum immunoglobulin G and E responses were variable and did not show relationship with specific dietary exposure.

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.

A pilot open trial evaluating the efficacy of low-dose aminopterin in the canine homologue of human atopic dermatitis.

SIR, Methotrexate and aminopterin (AMT) are antifolate agents that also exhibit anti-inflammatory effects stemming from several mechanisms that include the extracellular release of adenosine, a potent anti-inflammatory autocoid. Because of such properties, methotrexate has been used in the last decade for treatment of inflammatory conditions such as psoriasis. Results from two open trials suggested that methotrexate benefited human patients with atopic dermatitis (AD). Compared with methotrexate, AMT appears to be more potent and to have a higher bioavailability and lower toxic effects. In this proof-of-concept open two-phase trial, we report the efficacy of AMT in the spontaneous canine model of human AD. In the first phase that lasted 4 weeks, nine dogs with perennial AD diagnosed with conventional methods were given oral AMT 0Æ010 mg kg once weekly. During this phase, subjects did not receive any other interventions outside of weekly antimicrobial shampoos and monthly flea preventatives. Before, and 2 and 4 weeks after beginning AMT, investigators graded skin lesions using the validated Canine Atopic Dermatitis Extent and Severity Index (CADESI). Additionally, owners rated the degree of pruritic manifestations using a 10-point visual analogue scale (PVAS). A global score (GS) combined previous grades as follows: GS = (CADESI · PVAS) ⁄100. At the end of this phase, investigators and owners evaluated subjectively the overall efficacy of AMT using a scale from 0 (no efficacy; < 10% reduction in signs) to 4 (very good efficacy; 75–90% reduction in signs). During the second phase, which could last up to 11 months, dogs received AMT 0Æ010 mg kg once weekly, but this dosage could be increased to 0Æ015 and 0Æ020 mg kg once weekly in case of need. In this extension phase, anti-infectious and ⁄or anti-inflammatory medications were allowed if necessary, and were recorded. Patients were seen monthly and evaluated as above. Weekly during the first month, and every 2–4 weeks thereafter, blood was drawn for full blood counts and serum chemistry panels. Eight of nine dogs completed the 4-week induction phase; one dog (no. 7) was withdrawn because of bacterial folliculitis. CADESI and GS values, but not PVAS numbers, were significantly lower after 4 weeks compared with pretreatment (repeated-measures ANOVA, P < 0Æ05; Fig. 1). After 4 weeks, the median reductions in CADESI, PVAS and GS were 36%, 2Æ8 ⁄10 points and 62%, respectively. At that time, a ‡ 50% reduction from baseline CADESI and GS values was achieved in three of eight and six of eight dogs, respectively (Table 1), and the overall efficacy rating was 2Æ2, a value slightly above the ‘fair efficacy; 25–50% reduction’ mark. Seven dogs continued to receive AMT for up to 40 weeks. One dog (no. 5) was withdrawn after 8 weeks because of lack of efficacy despite doubling the dosage of AMT. Altogether, six of seven dogs (86%) achieved a ‡ 80% reduction in baseline GS values at one time point during the extension phase (Tables 1 and 2), and the disease neared or was in complete Fig 1. Evolution of global scores over 4 weeks in nine dogs treated with aminopterin. In the upper panel, each line corresponds to a different patient. In the lower panel, boxes correspond to the 95% confidence interval, the line is the median and the whiskers highlight the range of values. CADESI, Canine Atopic Dermatitis Extent and Severity Index; PVAS, pruritus visual analogue scale.

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.

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.

Cowhage can induce itch in the atopic dog

Itch is a cardinal symptom of atopic dermatitis in humans and dogs. Until now, experimental induction of itch in dogs has proven difficult. The objectives of this study were to determine whether protease‐rich spicules, protein extracts and the protease mucunain of the tropical legume cowhage provoked itch and inflammation when rubbed onto canine skin. Native spicules variably induced itch manifestations in about half of the dogs, while challenges with protease‐deactivated spicules remained negative. The epicutaneous application of cowhage extract and mucunain after microneedle roller usage also induced pruritus and inflammation. Importantly, there was an interindividual inconsistency in pruritus and inflammation induction and also marked differences in pruritus intensity after challenge. In conclusion, cowhage spicules, protein‐rich extracts and mucunain can all induce pruritus and inflammation in dogs as in other species, but the inconsistency of provocation is currently a limitation of this challenge type for future studies of pruritus in dogs.