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.

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.

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.

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.