Transient and reversible reduction of stratum corneum filaggrin degradation products after allergen challenge in experimentally mite-sensitised atopic dogs.

BACKGROUND: A defective skin barrier occurs in dogs with atopic dermatitis, and there is controversy over whether this defect pre-exists, or is secondary to allergic inflammation. OBJECTIVES: To study if an allergen challenge decreases the natural moisturising factor (NMF), which contains the main filaggrin degradation products. ANIMALS: Four house dust mite (HDM)-sensitised adult atopic dogs from a research colony. METHODS AND MATERIALS: Dogs were challenged epicutaneously with HDMs on the right lateral abdomen while the left thorax served as control. We swabbed the skin surface before, and at days (D)1, D2, D3, D7 and D28 after challenge, on both selected sites; swabs were soaked in detergent and frozen until assayed. The NMF components were measured by liquid chromatography-tandem mass spectrometry (LC/MS-MS). RESULTS: The allergen challenge induced moderate skin lesions at the application sites, and also mild erythema at the control areas. The allergen provocation led to significant decreases in the total NMF and its components trans-urocanic acid (t-UCA), pyrrolidone carboxylic acid (PCA) and serine on both sites. Lesion scores abated by D7 and the NMF concentrations had re-increased by D28. Skin lesion scores correlated negatively with the total NMF, t-UCA and PCA concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: In this experimental model, a single epicutaneous allergen challenge led to a transient and reversible decrease in skin surface NMF and its components, and concentrations were negatively correlated with skin lesion scores. These observations suggest that some of the skin barrier anomalies seen in atopic dogs likely develop secondarily to the underlying cutaneous allergic inflammation.

Proactive maintenance therapy of canine atopic dermatitis with the anti-IL-31 lokivetmab. Can a monoclonal antibody blocking a single cytokine prevent allergy flares?

BACKGROUND: Once the signs of canine atopic dermatitis (AD) are controlled, the proactive application of topical glucocorticoids can delay disease flares. OBJECTIVES: We wished to determine if the proactive administration of the anti-IL-31 lokivetmab would prevent or delay flares of canine AD. ANIMALS: We tested this strategy in four Maltese-beagle atopic dogs before enrolling 21 dogs with spontaneous AD. METHODS AND MATERIALS: In our acute AD model, house dust mite (HDM)-sensitized dogs were injected once with lokivetmab. After seven days, an HDM suspension was applied epicutaneously, and both skin lesions and pruritus manifestations were quantified for 24 h. In a second study, 21 dogs with spontaneous AD controlled with anti-allergic drugs were treated with lokivetmab per manufacturer's recommendations; all anti-allergic drugs were discontinued within four weeks after the first injection. All dogs were followed prospectively for at least one year and the time-to-flare (TTF) of AD after the last day of anti-allergic treatment was determined. RESULTS: In the experimental study, one injection of lokivetmab prevented nearly all expected allergen-induced pruritus manifestations but not skin lesion development. In dogs with spontaneous AD, the median TTF after lokivetmab proactive therapy was 63 days. One-fourth of dogs did not exhibit a flare for at least one year while receiving lokivetmab monotherapy. CONCLUSIONS: Although lokivetmab seems more effective to prevent pruritus than skin lesions in dogs with experimental AD' it also can delay disease flares in some dogs with the spontaneous disease. Studies are needed to identify those patients most likely to respond to such a proactive regimen.

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.

Validation of a novel epicutaneous delivery system for patch testing of house dust mite-hypersensitive dogs.

BACKGROUND: Patch tests with allergens are used for the evaluation of cellular hypersensitivity to food and environmental allergens in dogs and humans with atopic dermatitis. Viaskin is a novel allergen epicutaneous delivery system that enhances epidermal allergen capture by immune cells. OBJECTIVES: To compare the use of Viaskin and Finn chamber patch tests in dogs hypersensitive to mite allergens. METHODS: Empty control or Dermatophagoides farinae house dust mite-containing Viaskin or Finn chamber patches were applied to the thoracic skin of six mite-hypersensitive Maltese-beagle crossbred atopic dogs. Lesions were graded 49 and 72 h after patch test application, and skin biopsies were collected after 72 h. Overall microscopic inflammation, eosinophil and T-lymphocyte infiltrations were scored. RESULTS: Positive macroscopic patch test reactions developed at five of six Viaskin application sites and four of six Finn chamber application sites. Median microscopic epidermal and dermal inflammation, as well as eosinophil and CD3 T-lymphocyte dermal scores were always higher in biopsies collected at Viaskin than at Finn chamber sites. Microscopic inflammation scores were significantly higher after mite allergen-containing Viaskin compared with empty patches, but this was not the case for mite-containing Finn chambers compared with control chambers. Scores obtained using Viaskin were not significantly different from those obtained using Finn chambers. Macroscopic and microscopic scores were significantly correlated. CONCLUSIONS AND CLINICAL IMPORTANCE: In mite-allergic dogs, Viaskin epicutaneous delivery systems appear to induce stronger allergen-specific inflammation than currently used Finn chamber patch tests. Consequently, Viaskin patches might offer a better alternative for screening cellular hypersensitivity to food and environmental allergens.

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.

Periostin Activation of Integrin Receptors on Sensory Neurons Induces Allergic Itch.

Chronic allergic itch is a common symptom affecting millions of people and animals, but its pathogenesis is not fully explained. Herein, we show that periostin, abundantly expressed in the skin of patients with atopic dermatitis (AD), induces itch in mice, dogs, and monkeys. We identify the integrin αVß3 expressed on a subset of sensory neurons as the periostin receptor. Using pharmacological and genetic approaches, we inhibited the function of neuronal integrin αVß3, which significantly reduces periostin-induced itch in mice. Furthermore, we show that the cytokine TSLP, the application of AD-causing MC903 (calcipotriol), and house dust mites all induce periostin secretion. Finally, we establish that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Altogether, our results identify a TSLP-periostin reciprocal activation loop that links the skin to the spinal cord via peripheral sensory neurons, and we characterize the non-canonical functional role of an integrin in itch.

A blinded randomized controlled trial evaluating the usefulness of a novel diet (aminoprotect care) in dogs with spontaneous food allergy.

Aminoprotect Care (APC) is a novel diet composed of aminoacids, potato proteins and corn starch. The objectives of this study were to determine whether Maltese-Beagle atopic (MBA) dogs hypersensitive to corn exhibited clinical signs and changes in immunological markers after being fed APC. The study was designed as a blinded randomized controlled crossover experiment. Ten MBA dogs with signs of allergy within five days of ingesting corn were selected. Dogs were randomized to be fed either their maintenance diet with corn or APC for five days. After a washout of two weeks, diets were switched. Before and daily during each intervention, skin lesions were graded by an investigator while pruritus was assessed by another. Before and at the end of each intervention, the percentage of circulating CD4+CCR4+, corn-activated CD4+ T-lymphocytes and serum corn-specific IgE levels were measured and ratios of post:pre values calculated. During this trial, pruritus and skin lesions increased significantly in MBA dogs when ingesting corn while no such increase occurred when fed APC. Total, median and maximal pruritus values were significantly higher in MBA dogs ingesting corn compared to APC. There were no significant differences between interventions in the immunological parameters assessed. In summary, even though APC contains corn starch to which corn-sensitive MBA dogs often react, the ingestion of APC did not lead to significant increases in skin lesions or pruritus. Aminoprotect Care might prove valuable for management of food allergies. These experimental observations must be validated in large field studies.

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.

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.