Studies Using Antibodies against Filaggrin and Filaggrin 2 in Canine Normal and Atopic Skin Biopsies.

Filaggrin is important for the skin barrier and atopic dermatitis. Another filaggrin-like protein, filaggrin 2, has been described. We evaluated antibodies against both filaggrins in normal and atopic skin biopsies from dogs before and after allergen challenges (D0, D1, D3 and D10). Filaggrins expression was evaluated by immunohistochemistry and Western blot. We used PCR to investigate changes in filaggrin gene expression. Effects of group (p = 0.0134) and time (p = 0.0422) were shown for the intensity of filaggrin staining. Only an effect of group was found for filaggrin 2 (p = 0.0129). Atopic samples had higher intensity of staining than normal dogs [filaggrin on D3 (p = 0.0155) and filaggrin 2 on D3 (p = 0.0038) and D10 (p < 0.0001)]. Atopic samples showed increased epidermal thickness after allergen exposure (D3 vs. D0, p = 0.005), while normal dogs did not. In atopic samples, significant increased gene expression was found for filaggrin overtime but not for filaggrin 2. Western blot showed an increase in filaggrin 2 on D3. A small size band (15 kD) containing a filaggrin sequence was found in Western blots of atopic samples only. We conclude that atopic skin reacts to allergen exposure by proliferating and increasing filaggrin production but that it also has more extensive filaggrin degradation compared to normal skin.

Immunolocalization and Expression of JAK1 and JAK3 in the Skin of Dust Mite-Sensitive Beagle Dogs before and after Allergen Exposure.

Janus kinase (JAK) pathways have emerged as targets of treatment, yet localization and expression of JAK1 and JAK3 in canine atopic skin have not been studied. This study aimed to compare the localization and expression of JAK1 and JAK3 in the skin of atopic dogs before and after allergen exposure. Skin biopsies taken from atopic beagles sensitized to house dust mites (HDM) before (D0) and after four weeks (D28) of allergen exposure were stained. Staining was subjectively scored by examiners unaware of the source of the slides. Image J was used for the semiquantitative assessment of staining intensity. JAK1 and JAK3 staining was epidermal and dermal. JAK1 staining was cytoplasmic, primarily found in basal keratinocytes and dermal cells, while JAK 3 was nuclear (all epidermal levels and on dermal inflammatory cells). Epidermal thickness was significantly higher on D28 than on D0 (p < 0.0001). For JAK1, epidermal staining divided by epithelial thickness was significantly lower on D28 (p = 0.0002) compared to D0. For JAK3 staining, intensity in the dermis was significantly higher on D28 (p = 0.0405) compared to D0. We conclude that decreased expression of JAK1 in the epidermis and increased expression of JAK3 in the dermis of atopic dogs occur after allergen exposure.

Topical Bacteriophage Therapy for Staphylococcal Superficial Pyoderma in Horses: A Double-Blind, Placebo-Controlled Pilot Study.

Increased antimicrobial resistance highlights the need for alternatives to antibiotics. Bacteriophages, which are benign viruses that kill bacteria, are promising. We studied the efficacy of topical bacteriophages for treating equine staphylococcal superficial pyodermas. Eight Staphylococcus aureus isolates were tested against a bacteriophage bank, and a cocktail consisting of two bacteriophages was prepared. Twenty horses with clinical and cytological evidence of superficial pyoderma and confirmed S. aureus infection based on swabbed culture were enrolled in the study. Each horse received both the bacteriophage cocktail and the placebo at two different infection sites, once daily for four weeks. Clinical lesions and cytology were evaluated weekly by an investigator who was unaware of the treatment sites. All infection sites were swabbed and cultured at the end of the study. A linear mixed model showed no significant differences between the placebo and treatment sites in terms of clinical signs, cytological scores of inflammation, and bacterial counts at the end of the study. It is possible that the bacteriophage cocktail killed S. aureus, but cytology scores did not change as new populations of cocci took over. The study limitations included a small sample size and inconsistent control of the underlying causes of pyodermas.

Skin biopsy guidelines: tips and advice from veterinary pathologists to practitioners.

Skin diseases are one of the most common problems seen in veterinary practices around the world. Many patients are presented with severe and/or chronic lesions, often refractory to treatment, and collection of skin biopsies is often beneficial to obtain or confirm a diagnosis and to help guide a management plan for patients. To obtain valuable information from skin biopsies, practitioners should follow recommended guidelines based on drug withdrawal and washout period, identification, and proper collection of skin lesions, which should be at different stages of progression, as well as include a thorough clinical history and differential list. These different steps taken prior to the submission of samples will often increase the chances of a more accurate diagnosis. Practitioners should also understand it may not always be possible for pathologists to provide a definitive diagnosis, but the information provided with skin biopsies can often be used to guide an appropriate treatment plan. This review will present general guidelines and suggestions to help obtain the most diagnostic skin samples for histopathological evaluation.

Transcription of interleukin 31 and its receptor by leukocytes after Culicoides sp stimulation is dose dependent but is not exaggerated in allergic horses or correlated with pruritus.

OBJECTIVE: Preliminary evidence supports a role for IL-31 in equine insect bite hypersensitivity (IBH) and pruritus. Our studies investigated IL-31 and IL-31 receptor-α (IL-31RA) transcription in leukocytes from normal and IBH horses in response to Culicoides nubeculosus. ANIMALS: 19 normal and 15 IBH horses were recruited in the summer of 2019 (low-dose study) and 8 normal and 10 IBH horses in the winter of 2022 to 2023 (high-dose study). Normal horses had no history or signs of allergic skin disease, while IBH horses had a history and clinical signs compatible with IBH. Pruritus was scored using a visual analog score or a 1 to 6 grading system. PROCEDURES: Whole blood leukocytes were incubated with saline (0.9% NaCl) solution or C nubeculosus (0.26 µg/mL [low dose]; 5 µg/mL [high dose]). Transcription of IL-31 and IL-31RA was measured by quantitative RT-PCR. RESULTS: Transcription of IL-31 and IL-31RA significantly increased in leukocytes from normal and IBH horses following high-dose C nubeculosus, and no differences were found between populations. Following low-dose C nubeculosus IL-31RA, transcription was increased in both normal and IBH horses, but IL-31 transcription was reduced in normal horses. No positive correlation was found between pruritus scores and IL-31 transcription after low- or high-dose C nubeculosus stimulation. CLINICAL RELEVANCE: Exaggerated IL-31 transcription was not identified in IBH horses, suggesting that dysregulation in IL-31 signaling occurs downstream or in localized tissues or involves regulation by yet unidentified receptor splice variants or IL-31-induced increased sensitivity to other pruritogens. Further studies to understand IL-31 signaling in equine allergic skin disease are needed.

Oclacitinib 10 years later: lessons learned and directions for the future.

Oclacitinib was approved in the United States 10 years ago for the management of atopic dermatitis (AD) and allergic skin disease in dogs. Many studies and case reports have been published in the past 10 years on the efficacy and safety of this medication, both at labeled doses to treat allergic dogs and off label to treat other diseases and given to other species. Concerns and confusion have occurred for both clinicians and owners regarding the long-term safety of this drug. The purpose of this review is to present the current knowledge on the efficacy, speed of action, effects on the immune system, and clinical safety of oclacitinib, based on evidence and published literature. We also aim to summarize the lessons learned in the past 10 years and to propose directions for the future.

Evolution of the Prevalence of Antibiotic Resistance to Staphylococcus spp. Isolated from Horses in Florida over a 10-Year Period.

Previous studies documented antibiotic resistance in horses but did not focus on skin specifically. We investigated antibiotic resistance and correlations between resistance patterns in skin infections. Records from 2009 to 2019 were searched for Staphylococcal infection and susceptibility results. Seventy-seven cases were included. Organisms identified were S. aureus (48/77), S. pseudintermedius (7/77), non-hemolytic Staphylococcus (8/77), beta-hemolytic Staphylococcus (6/77), and other species (8/77). Samples included pyoderma (36/77), wounds (10/77), abscesses (15/77), incision sites (5/77), nose (8/77), and foot (3/77). A trend analysis using non-parametric Spearman's test showed significant upward trend of resistance (p < 0.05) for 3/15 antibiotics (ampicillin, cefazolin, penicillin). Susceptibility was significantly different by Staphylococcal species for 8/15 antibiotics. Gentamicin showed significant susceptibility differences based on source (all abscesses were susceptible to gentamicin). Steel-Dwass test showed statistically significant (p = 0.003) difference between incision sites and abscesses. A non-parametric Kendall's T-test found significantly negative correlation between cefazolin and amikacin sensitivity (p = 0.0108) and multiple positive correlations of resistance (p < 0.05). This study confirms increasing resistance in dermatologic samples. It is unlikely that the sample source affects resistance, but Staphylococcus species may affect it. Study limitations include lack of information about previous antibiotic use and small sample size.

Differences in Behavior between Normal and Atopic Keratinocytes in Culture: Pilot Studies.

Skin barrier dysfunction is important in atopic dermatitis and can be secondary to inflammation. Observation of keratinocytes in culture may show intrinsic differences. TransEpithelial Electrical Resistance (TEER) measures epithelial permeability. We cultured normal and atopic keratinocytes and found that TEER of atopic keratinocytes was significantly lower (p < 0.0001) than that of normals. Atopic keratinocytes grew upwards, first creating isolated dome-like structures and later horizontally into a monolayer. At time of confluence (D0), atopic keratinocytes were more differentiated, with higher filaggrin gene expression than normals. No differences existed between groups for TJ proteins (claudin, occludin, and Zonula Occludens-1) on D0 and D6. On D6, claudin and occludin were higher than D0, in normal (p = 0.0296 and p = 0.0011) and atopic keratinocytes (p = 0.0348 and 0.0491). Immunofluorescent staining showed nuclear location of filaggrin on D0 and cytoplasmic on D6. ANOVA showed increased cell size from D0 to D6 in both groups (effect of time, p = 0.0076) but no differences between groups. Significant subject effect (p = 0.0022) was found, indicating that cell size was subject-dependent but not disease-dependent. No difference for continuity for TJ protein existed between groups. These observations suggest that decreased TEER in atopics is not linked to TJ differences but is possibly linked to different growth behavior.

Canine Epidermal Keratinocytes (CPEK) Grown in Monolayer Are Not Representative of Normal Canine Keratinocytes for Permeability Studies: Pilot Studies.

Canine progenitor epidermal keratinocytes (CPEK) are used as canine keratinocyte cell line. Their suitability for skin barrier studies is unknown. Measurement of transepithelial electric resistance (TEER) evaluates epithelial permeability. We compared TEER and tight junction (TJ) expression in CPEKs and normal keratinocytes (NK) harvested from biopsies of normal dogs. CPEKs and NK were grown until confluence (D0) and for 13 additional days. Slides were fixed on D0 and stained with ZO-1 and claudin-1 antibodies. Five images/antibody were taken, randomized and evaluated blindly by three investigators for intensity, staining location, granularity, and continuousness. Cell size and variability were evaluated. TEER increased overtime to 2000 Ohms/cm in NK, while remained around 100-150 Ohms/cm in CPEK. ANOVA showed significant effect of time (p < 0.0001), group (p < 0.0001) and group x time interaction (p < 0.0001) for TEER. Size of CPEKs was significantly (p < 0.0001) smaller and less variable (p = 0.0078) than NK. Intensity of claudin-1 staining was greater in CPEKs (p < 0.0001) while granularity was less in CPEKs (p = 0.0012). For ZO-1, cytoplasmic staining was greater in CPEK (p < 0.0001) while membrane continuousness of staining was greater in NK (p = 0.0002). We conclude that CPEKs grown in monolayer are not representative of NK for permeability studies.

Topical κ-opioid receptor agonist asimadoline improves dermatitis in a canine model of atopic dermatitis.

This prospective, 4-week, placebo-controlled, cross-over study aimed to investigate the efficacy of 1% topical κ-opioid agonist, asimadoline, in a model of canine atopic dermatitis (AD). Fourteen beagles were challenged with house dust mites every 3-4 days for a total of 9 challenges. Severity of dermatitis was assessed, and pruritus was monitored using GoPro HERO cameras. Pruritus scoring was evaluated at 10 time periods; baseline, 4 h post allergen challenge and the last day of the study on Day 28. Scoring was done blindly by personnel using BORIS software. A global subjective score was also given using a visual analogue scale (VAS). A 4-week washout period occurred and dogs were crossed-over, the study was repeated, and the results were analysed using combined data. Gel was applied once daily on inguinal area (0.6 ml/dog). ANOVA showed significant effect of time (p < 0.0001) and group (p = 0.0001) on dermatitis scores. Overall, no statistically significant effect on pruritus was found due to a crossing of scores on Day 17. Overtime the placebo scores increased while the active ingredient showed decrease after first 3 weeks. It is concluded that this approach is promising in dogs with AD and longer studies with more frequent application may be beneficial.

Atopic Dermatitis in Domestic Animals: What Our Current Understanding Is and How This Applies to Clinical Practice.

Atopic dermatitis is a clinical syndrome that affects both people and animals. Dogs closely mimic the complexity of the human skin disease, and much progress has been made in recent years in terms of our understanding of the role of skin impairment and the identification of new treatments. Cats and horses also develop atopic syndromes which include both cutaneous and respiratory signs, yet studies in these species are lagging. It is now recognized that atopic dermatitis is not a single disease but a multifaceted clinical syndrome with different pathways in various subgroups of patients. Appreciating this complexity is clinically relevant as we develop more targeted treatments which may work well in some patients but not in others. Different phenotypes of atopic dermatitis have been described in dogs, and it is possible that phenotypes related to breed and age may exist in other animals similar to how they are described in people. The awareness of different mechanisms of disease leads to the desire to correlate different phenotypes with specific biomarkers and responses to treatment. In this review, the current understanding and updated information on atopic syndrome in animals are described, highlighting opportunities for further studies in the future.

Advances in our understanding of canine atopic dermatitis.

Canine atopic dermatitis (cAD) is a genetically inherited clinical syndrome that encompasses a diversity of mechanisms and can have a variety of triggers. Development of clinical disease is the result of genetic factors and environmental conditions, which shape the resulting immunological response. Clinical disease becomes evident once a threshold of inflammatory response is achieved. Skin barrier impairment plays a role in promoting cutaneous dysbiosis and increased allergen penetration. Keratinocytes shape the response of dendritic cells and subsequent lymphocytic response. Thymic stromal lymphopoietin is one of the links between the damaged skin barrier and the modulation of a T-helper (Th)2 response. It is still unclear whether mutations in skin barrier genes exist in atopic dogs, as they do in humans, or whether the observed alterations are purely secondary to inflammation. A dysregulated immune response with increased Th2, Th17 and CD4+ CD25+ regulatory T cells has been reported. A variety of cytokines [interleukin(IL)-31, IL-34, Macrophage migration inhibitory factor] are proposed as potential biomarkers and treatment targets because they are increased in the serum of atopic dogs when compared to controls, although a correlation between serum levels of these factors and severity of disease is not always present. The main issue with many published studies is that atopic dogs are always only compared to normal controls. Thus, it is unclear whether the changes that we find are truly a signature of cAD or merely a manifestation of nonspecific broad inflammatory responses. Studies considering comparison with other inflammatory diseases different from cAD are urgently needed to correctly identify what is specific to this complicated syndrome.

La dermatite atopique canine (cAD) est un syndrome clinique à prédisposition génétique qui passe par une variété de mécanisme et peut avoir une variété de facteurs déclencheurs. Le développement de la maladie clinique est le résultat de facteurs génétiques et de conditions environnementales qui façonnent la réponse immunologique résultante. La maladie clinique devient évidente une fois que le seuil de la réponse inflammatoire est atteint. Le défaut de barrière cutanée joue un rôle dans la promotion de la dysbiose cutanée et augmente la pénétration des allergènes. Les kératinocytes façonnent la réponse des cellules dendritiques et la réponse lymphocytaire subséquente. La lymphopoiétine stromale thymique est un des liens entre la barrière cutanée endommagée et la modulation de la réponse Th-2. Il n'est toujours pas certain que les mutations des gènes de la barrière cutanée existent chez le chien atopique, comme chez l'homme, ou si les altérations observées sont purement secondaires à l'inflammation. Une réponse immunitaire déréglée avec augmentation des cellules T régulatrices Th2, Th17 et CD4+ CD25+ a été rapportée. Une variété de cytokines [interleukine(IL)-31, IL-34, Macrophage migration inhibitory factor] est proposée comme biomarqueurs potentiels et cibles de traitement parce qu'ils sont augmentés dans le serum des chiens atopiques quand comparés aux contrôles, bien qu'une corrélation entre les taux sériques de ces facteurs et la sévérité de la maladie ne soit pas toujours présente. La principale théorie, appuyée par de nombreuses publications, est que les chiens atopiques sont toujours comparés aux contrôles sains. Ainsi, il n'est pas clair si les changements que nous trouvons sont une véritable signature de cAD ou plutôt une manifestation de réponses inflammatoires non spécifiques. Les études considérant une comparaison avec d'autres maladies inflammatoires différentes de cAD sont urgemment nécessaires pour correctement identifier ce qui est spécifique de ce syndrome complexe.

La dermatitis atópica canina (cAD) es un síndrome clínico hereditario que abarca una diversidad de mecanismos y puede tener una variedad de desencadenantes. El desarrollo de la enfermedad clínica es el resultado de factores genéticos y condiciones ambientales, que dan origen a la respuesta inmunológica resultante. La enfermedad clínica se hace evidente una vez que se alcanza un umbral de respuesta inflamatoria. El deterioro de la barrera cutánea juega un papel en la promoción de la disbiosis cutánea y el aumento de la penetración de alérgenos. Los queratinocitos modulan la respuesta de las células dendríticas y la subsiguiente respuesta linfocítica. La linfopoyetina del estroma tímico es uno de los vínculos entre la barrera cutánea dañada y la modulación de una respuesta T-helper (Th)2. Todavía no está claro si existen mutaciones en los genes de la barrera cutánea en perros atópicos, como ocurre en los humanos, o si las alteraciones observadas son puramente secundarias a la inflamación. Se ha publicado la existencia de una respuesta inmune disregulada con aumento de linfocitos T reguladores Th2, Th17 y CD4+CD25+. Se han propuesto una variedad de citoquinas [interleucina (IL) -31, IL-34, factor inhibidor de la migración de macrófagos] como posibles biomarcadores y objetivos de tratamiento porque aumentan en el suero de perros atópicos en comparación con los controles, aunque no existe una correlación entre los niveles séricos de estos factores y la gravedad de la enfermedad. El principal problema de muchos estudios publicados es que los perros atópicos siempre se comparan solo con los controles normales. Por lo tanto, no está claro si los cambios que encontramos son realmente una firma de cAD o simplemente una manifestación de respuestas inflamatorias amplias inespecíficas. Se necesitan con urgencia estudios que consideren la comparación con otras enfermedades inflamatorias diferentes a la cAD para identificar correctamente qué es específico de este complicado síndrome.

A dermatite atópica canina (DAC) é uma síndrome clínica herdada geneticamente que compreende uma diversidade de mecanismos e pode ter uma variedade de gatilhos. O desenvolvimento da doença clínica é o resultado de fatores genéticos e condições ambientais que podem moldar a resposta imunológica resultante. A doença clínica se torna evidente uma vez que o limiar da resposta inflamatória é atingido. O comprometimento da barreira cutânea desempenha um papel na promoção da disbiose cutânea e no aumento na penetração de alérgenos. Os queratinócitos moldam a resposta das células dendríticas e resposta linfocítica subsequente. A linfopoietina estromal tímica é uma das ligações entre a barreira cutânea danificada e a modulação da resposta de T-helper (Th)2. Ainda não está claro se existem mutações nos genes da barreira cutânea em cães atópicos, como existem em humanos, ou se as alterações observadas são puramente secundárias à inflamação. Uma resposta imune desregulada com aumento de células Th2, Th17 e T reguladoras CD4+ CD25+ já foi relatada. Uma variedade de citocinas [interleucina (IL)-31, IL-34, fator inibidor da migração de macrófagos] são propostos como biomarcadores potenciais já que estão aumentados no soro de cães atópicos comparados aos controles, apesar de a correlação entre os níveis séricos destes fatores e a gravidade da doença nem sempre está presente. O principal problema de muitos estudos publicados é que cães atópicos são sempre comparados a controles normais. Assim, não está claro se as alterações que encontramos são realmente uma particularidade da DAC ou meramente uma manifestação de respostas inflamatórias amplas e não específicas. Estudos considerando a comparação com outras doenças inflamatórias diferentes da DAC são urgentemente necessários para identificar corretamente o que é específico desta síndrome complicada.

Investigation on the Effect of Dose, Frequency and Duration of Allergen Exposure on Development of Staphylococcal Infections in a Chronic Model of Canine Atopic Dermatitis.

Canine atopic dermatitis (CAD) is chronic and frequently complicated by Staphylococcal infections. Understanding the role of allergen dose, frequency and duration of exposure in triggering infections requires a model. Most models elicit acute inflammation and do not mimic real-life disease. Here we describe the effects of allergen exposures on development of infections in a model of chronic CAD. Diagnosis of pyoderma was based on clinical signs and consistent cytology. Study 1 evaluated the role of duration of exposure keeping the daily dose constant (25 mg/day). The one-week protocol involved three exposures, 3 days in a row. The one-month protocol involved twice-weekly challenges for 4 weeks. The three-month protocol involved twice-weekly challenges for 12 weeks. Study 2 evaluated different daily doses while keeping constant the total weekly dose (25 mg) and duration (3 weeks). Low-dose used 5 mg/day for 5 days, each week. High-dose used 12.5 mg/day twice-weekly. In Study 1, the longer the exposure, the more dogs developed pyoderma (6/9 in the three-month study, 2/9 in the one-month and 0 in the one-week). In Study 2, low-dose daily exposure caused more infections (5/8) than high-dose infrequent exposure (0/8). It is concluded that low-grade, daily exposure for a long time is most relevant for development of staphylococcal infections.

Comparison of various treatment options for canine atopic dermatitis: a blinded, randomized, controlled study in a colony of research atopic beagle dogs.

BACKGROUND: No study has directly compared the various treatment options for canine atopic dermatitis and their effects on skin barrier. HYPOTHESIS/OBJECTIVES: To compare prednisone, oclacitinib, ciclosporin and lokivetmab treatment of atopic dermatitis. ANIMALS: Nineteen atopic beagle dogs. METHODS AND MATERIALS: Controlled, blinded study. Dogs were challenged with allergen twice weekly and randomized to oclacitinib, ciclosporin, lokivetmab, prednisone or no treatment for four weeks. Dermatitis and pruritus were assessed at baseline and after each challenge. Transepidermal water loss (TEWL) and hydration were measured at baseline, Day (D)14 and D28 (pinnae, axilla, groin). Area under the curve (AUC) was calculated for Canine Atopic Dermatitis Extent and Severity Index, 3rd iteration (CADESI-03), pruritus, TEWL and hydration. For CADESI, the AUC of the first two weeks was compared to that of the last two weeks. RESULTS: For CADESI, restricted maximum-likelihood ANOVA showed effect of time (P = 0034) and group x time interaction (P = 0.0169). In the first two weeks, prednisone and oclacitinib were significantly lower than controls (P = 0.019 and P = 0.015, respectively). Lokivetmab prevented flares. Due to variability, no significance differences in pruritus were observed among groups. The TEWL increased with time in controls (P = 0.0237) and ciclosporin (P = 0.04, axilla, D28 versus D0) but not in the oclacitinib and lokivetmab groups. CADESI-03 correlated with TEWL (P = 0.0043) and pruritus (P = 0.0283). Hydration did not correlate with any parameters. Hydration decreased in controls and prednisone group (axilla, D14 versus D0, P = 0.004 and P = 0.027, respectively). AUC for hydration, over time, was higher for lokivetmab and oclacitinib than controls (P = 0.014 and P = 0.04, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Lokivetmab prevented flares when given before challenge. Oclacitinib and lokivetmab have some positive effects on skin barrier parameters.

Topical treatment with SPHINGOLIPIDS and GLYCOSAMINOGLYCANS for canine atopic dermatitis.

BACKGROUND: Skin barrier dysfunction plays a key role in atopic dermatitis (AD). This impairment is related to altered composition and metabolism of epidermal sphingolipids and a deficiency of ceramides. Glycosaminoglycans (GAGs), and especially hyaluronic acid, could be useful in the management of AD. This study aimed to evaluate the effects of a novel topical treatment consisting of sphingolipids and GAGs extracts in dogs with AD. This formulation is different from previously tested products because the sphingolipid extract contained high amounts of sphingomyelin, a precursor of ceramides, and this has been shown to enhance endogenous synthesis of ceramides and to increase lamellar-related structures in vitro. Thus, it was hypothesized that this formulation could improve clinical disease and skin barrier function in patients with AD. RESULTS: Twelve house dust mite (HDM) allergic atopic beagle dogs were randomized into two groups: control (n = 6; no treatment) or treatment (n = 6; topical sphingolipids and GAGs twice weekly for 8 weeks). Dogs were challenged with allergen twice weekly and the severity of dermatitis was scored using the canine atopic dermatitis and extent severity index (CADESI-03) once weekly. Skin barrier function (measurement of transepidermal water loss) and severity of pruritus (both pruritus visual analog scale [PVAS] and pruritus timed episodes) were assessed at 0, 4 and 8 weeks of treatment. Assessments were done by personnel unaware of group allocation. Complete blood count, serum biochemistry and stratum corneum (SC) lipidomics analyses were done at baseline and at week 8. Compared to baseline, significant increases in CADESI (P = 0.0003) and PVAS (P = 0.041) were observed only in the control group, and SC polyunsaturated fatty acids increased significantly only with treatment (P = 0.039). Compared to control, treatment group had a significantly lower CADESI after 1 week (P = 0.0078) and a significantly lower PVAS after 8 weeks (P = 0.0448). Treatment was well tolerated. CONCLUSIONS: In this study in dogs with AD, a new topical formulation containing sphingomyelin-rich sphingolipids plus GAGs extracts attenuated the clinical worsening induced by HDM, supporting its use in atopic patients, either as an adjunctive treatment or used as monotherapy in certain cases.

Identification of differentially expressed microRNAs in the skin of experimentally sensitized naturally affected atopic beagles by next-generation sequencing.

Canine atopic dermatitis (AD) is a very common inflammatory skin disease, but limited data are available on the genetic characterization (somatic mutations, microarrays, and genome-wide association study (GWAS)) of skin lesions in affected dogs. microRNAs are good biomarkers in inflammatory and neoplastic diseases in people. The aim of this study was to evaluate microRNA expression in the skin of atopic beagles, before and after exposure to Dermatophagoides farinae. Four atopic and four unrelated age-matched healthy beagle dogs were enrolled. Total RNA was extracted from flash-frozen skin biopsies of healthy and atopic dogs. For the atopic dogs, skin biopsies were taken from non-lesional (day 0) and lesional skin (day 28 of weekly environmental challenge with Dermatophagoides farinae). Small RNA libraries were constructed and sequenced. The microRNA sequences were aligned to CanFam3.1 genome. Differential expressed microRNAs were selected on the basis of fold-change and statistical significance (fold-change ≥ 1.5 and p ≤ 0.05 as thresholds. A total of 277 microRNAs were sequenced. One hundred and twenty-one differentially regulated microRNAs were identified between non-lesional and healthy skin. Among these, two were increased amount and 119 were decreased amount. A total of 45 differentially regulated microRNAs between lesional and healthy skin were identified, 44 were decreased amount and one was increased amount. Finally, only two increased amount microRNAs were present in lesional skin when compared with that of non-lesional skin. This is the first study in which dysregulation of microRNAs has been associated with lesional and non-lesional canine AD. Larger studies are needed to understand the role of microRNA in canine AD.

Double blinded, vehicle controlled, crossover study on the efficacy of a topical endocannabinoid membrane transporter inhibitor in atopic Beagles.

The endocannabinoid system is important for skin homeostasis and alterations are linked to inflammatory diseases like atopic dermatitis (AD). Importantly, activation of cannabinoid receptor CB2 decreases pruritus and inflammation in mouse models. Reduction of inactivation of endogenous cannabinoids could, therefore, be a therapeutic option for AD. Dogs spontaneously develop AD, which closely mimics the human disease making them suitable to test new therapies. Our study aimed to test the effects of a topical endocannabinoid membrane transporter inhibitor (WOL067-531, 1% gel) on pruritus and dermatitis in a canine model of AD. Nineteen Beagles allergic to dust mites (DM) were randomized to receive either active ingredient or vehicle on inguinal area while challenged epicutaneously with DM twice weekly for 28 days. Treatment was administered twice daily and started after three challenges (day 8). Dermatitis and pruritus were scored weekly by personnel blinded to treatment allocation. Dermatitis was scored using a validated scoring system and pruritus was scored using camera recordings. After a 4-week washout, dogs were crossed over and the study was repeated. On days 15 and 22, dermatitis scores were significantly increased after DM challenge in the vehicle group (16.34, p = 0.0089 and 7.42, p = 0.04845, respectively) but not in the active ingredient group (p = 0.3177 and p = 0.3190, respectively). Significant decrease on pruritus both on inguinal area and overall (p = 0.048 and p = 0.032, respectively) occurred in the active ingredient group. No adverse effects were noted. In conclusion, the newly developed topical endocannabinoid membrane transporter inhibitor (WOL067-531) minimized allergic flares and pruritus in a canine model of AD.