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.

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.

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.

A pilot study on the effect of oclacitinib on epicutaneous sensitization and transepidermal water loss in a colony of atopic beagle dogs.

BACKGROUND: Dogs with atopic dermatitis are prone to sensitization to environmental allergens due to increased skin permeability; the effect of treatments on epicutaneous sensitizations is unknown. HYPOTHESIS/OBJECTIVES: To evaluate if oclacitinib (i) prevents new sensitizations and (ii) affects skin barrier function. ANIMALS: Atopic beagle dogs. METHODS: Aim 1. Ten dogs were randomly assigned to placebo or oclacitinib while exposed epicutaneously to a novel allergen. Sensitization was assessed using serum allergen-specific IgE and clinically by development of skin reactions at the site of allergen application. Time to develop dermatitis and allergen-specific IgE were compared between groups. Aim 2. Eight dogs were randomly assigned to placebo or oclacitinib for four weeks and challenged with an allergen known to trigger flares. After a four week wash-out, dogs were crossed-over and the protocol repeated. Transepidermal water loss (TEWL) was measured on days 0 and 28 of each arm. RESULTS: Aim 1. Oclacitinib significantly increased (P = 0.006) time to develop skin reactions compared to placebo. Four (of five) dogs receiving oclacitinib failed to develop skin reactions, whereas all placebo dogs developed dermatitis. There were no significant differences in allergen-specific IgE between groups. Aim 2. TEWL results were difficult to interpret. Significantly higher values were detected from the axilla in placebo compared to oclacitinib-treated dogs (P = 0.047). TEWL values were significantly higher from the inguinal area in oclacitinib (P = 0.039) treated dogs but not placebo at the end of the study. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinically, oclacitinib delayed development of dermatitis at the site of allergen application. TEWL results were difficult to interpret and additional studies are required for clarification.

Single blinded, randomized, placebo-controlled study on the effects of ciclosporin on cutaneous barrier function and immunological response in atopic beagles.

Ciclosporin (CsA) is a common treatment for canine atopic dermatitis (cAD). cAD is a very common skin disease with a multifactorial pathogenesis due to complex interactions between the host and the environment. The purpose of this study was to describe the physical and immunological effects of CsA in cAD using a canine model of AD. Fourteen beagles were enrolled; seven received CsA orally every 24 h for 28 days, and seven received placebo. All dogs were exposed to relevant allergens, house dust mite solution, one day prior to treatment and once weekly thereafter for 28 consecutive days. Canine atopic dermatitis extent and severity index-03 (CADESI-03) and skin biopsies were performed on day 0, 14, and 28. Quantitative RT-PCR was used to determine levels of cutaneous cytokines and barrier function markers. Indirect immunofluorescence was used to determine protein expression and distribution of nuclear messengers, barrier function and inflammatory [thymic stromal lymphopoietin (TSLP)] markers. The data were tested for normality and then the upaired two samples Student's t-test and the repeated measurements ANOVA, followed by the Dunnett's Multiple Comparison Test as post-hoc analysis, were performed. A P value of <0.05 was considered statistically significant. A significant decrease in CADESI-03 occurred for the treatment group compared to placebo (p = 0.023) on day 28. On day 14, a significant increase in TSLP protein expression [p = 0.019 (placebo); p = 0.02 (CsA)] and a significant decrease in Transforming Growth Factor (TGF)-ß mRNA [p = 0.01 (placebo); p = 0.015 (CsA)] were noted in both groups compared to baseline. On day 28, a significant increase in canine beta defensin (cBD)103 [p = 0.012 (placebo)] and cBD3-like mRNAs [p = 0.044 (placebo)], and filaggrin [p = 0.035 (CsA)] and TSLP protein expressions [p = 0.0092 (CsA)] were seen compared to baseline. In contrast, a significant decrease in mRNA of Tumor Necrosis factor (TNF)-α [p = 0.013 (CsA)], Interleukin (IL)-10 [p = 0.038 (CsA)], TGF-ß [p = 0.017 (CsA)], and caspase 14 [p = 0.014 (CsA)] was seen on day 28 compared to baseline. Comparison of the groups revealed no significant effect on skin immunologic milieu or barrier markers despite evident improvement of physical signs in the treatment group. Although this study confirmed the usefulness of CsA for the treatment of cAD, a clear involvement of CsA on some of the currently known immunological alterations present in cAD was not determined. However, it is important to note that there was no measurable exacerbation of skin barrier dysfunction secondary to CsA administration in this model.

Evaluation on the effects of 0.1% Peumus boldus leaf and Spiraea ulmaria plant extract combination on bacterial colonization in canine atopic dermatitis: A preliminary randomized, placebo controlled, double-blinded study.

Defective skin barrier characterize canine atopic dermatitis (AD). Pyoderma is the most common complication. Herbal compounds have been suggested as alternatives to control bacterial colonization for their effect on natural antimicrobial peptides (AMPs). This study evaluated the effects of 0.1% Peumus boldus leaf and Spiraea ulmaria plant extract combination on clinical signs, bacterial colonization and AMPs secretion in atopic dogs compared to placebo. Twenty privately-owned atopic dogs were randomly divided in 2 groups (treatment: n = 10; placebo: n = 10) and their abdomen was sprayed every 24 h for 4 weeks. Total and inguinal clinical scores (CADESI-03), manual bacterial count, and skin washes for AMPs (cBD3-like and cCath) were performed on days 0, 14 and 28. AMPs were detected using in-house, previously-validated, canine-specific ELISAs. Data were statistically analyzed and a p < 0.05 was considered significant. Clinical scores and AMPs secretion did not differ significantly between the two groups at any time point. A significant reduction of the clinical scores was seen in the placebo group at 14 and 28 days (p < 0.04). On days 14 and 28, a reduction in the bacterial count was seen in the treated group compared with placebo (p < 0.009 and p = 0.04, respectively). Compared to baseline, a reduction in Staphylococcus spp. was seen in the treated group after 14 days of treatment (p < 0.03). These results show the efficacy of this plant extract combination against bacterial colonization, suggesting its potential usefulness in preventing bacterial infection in atopic dogs. The influence of this compound on AMPs secretion or other mechanisms should be further evaluated.

Investigation of the correlation of serum IL-31 with severity of dermatitis in an experimental model of canine atopic dermatitis using beagle dogs.

BACKGROUND: IL-31 is a cytokine that is believed to play an important role in atopic dermatitis (AD). IL-31 levels positively correlate with disease severity in children with AD. Currently, there is no study that has investigated such a correlation in atopic dogs. HYPOTHESIS/OBJECTIVE: The purpose of this study was to evaluate the correlation between IL-31 serum levels and severity of dermatitis. It was hypothesized that a positive correlation exists between severity of AD and circulating levels of IL-31. ANIMALS: Sixteen atopic beagles experimentally sensitized to house dust mites. METHODS: Atopic beagles were exposed to dust mites epicutaneously twice weekly for four weeks. Severity of dermatitis was scored by the Canine Atopic Dermatitis and Extent Severity Index, 3rd iteration (CADESI-03) on days 0 and 28. Blood samples were taken on days 0 and 28 to measure serum IL-31 using a commercially available ELISA. RESULTS: Correlation between CADESI-03 scores and serum IL-31 levels was not detected on day 0 (Pearson, r = -0.2609, P = 0.3291). After flare-up of dermatitis was induced with allergen exposure, a significant positive correlation was detected between serum IL-31 and CADESI-03 on Day 28 (r = 0.6738, P = 0.004). CONCLUSIONS AND CLINICAL IMPORTANCE: Positive correlation was detected in active disease between severity of dermatitis and circulating levels of IL-31. Additional studies are needed to investigate this correlation in other breeds of dogs and to test whether circulating levels of IL-31 may predict clinical response to biological agents aimed at IL-31.

Randomized, double-blinded, placebo-controlled pilot study on the effects of topical blackcurrant emulsion enriched in essential fatty acids, ceramides and 18-beta glycyrrhetinic acid on clinical signs and skin barrier function in dogs with atopic dermatitis.

BACKGROUND: Lipid-based emulsions can be useful for the management of canine atopic dermatitis (cAD). 18-beta glycyrrhetinic acid (GRA), a component of liquorice root, has anti-inflammatory and anti-pruritic effects. HYPOTHESIS/OBJECTIVES: To evaluate the effects of a topical lipid emulsion containing ceramides, fatty acids and GRA on clinical signs of cAD and skin barrier in a randomized, double-blinded, placebo-controlled trial. METHODS: Client owned (n = 45) dogs with nonseasonal, mild/moderate AD, received either treatment or placebo for three months. Skin lesions, pruritus, transepidermal water loss (TEWL) and global assessment (GA) were evaluated. RESULTS: Fourteen dogs receiving treatment and 14 receiving the placebo completed the study. After one month ≥50% reduction in pruritus was seen in seven of 14 dogs (50%) in the Treatment group, and in two of 14 dogs (14.3%) in the Control group (P = 0.047). After two and three months, significant reduction in pruritus was not seen. For Canine Atopic Dermatitis Extent and Severity Index (CADESI), TEWL and GA, there were no significant findings over time or between groups. CONCLUSIONS AND CLINICAL RELEVANCE: The emulsion had some transient beneficial clinical effects. However, it was not effective in controlling pruritus as a monotherapy. Further studies should examine whether owner compliance was a factor in the steady decline of effect on pruritus scores. Further studies evaluating its role as an adjunctive therapy are indicated.

Evaluation of the in vitro effect of Boldo and Meadowsweet plant extracts on the expression of antimicrobial peptides and inflammatory markers in canine keratinocytes.

Dogs with allergies are prone to skin infections and treatments/preventatives to boost innate immune-defenses are beneficial. The aim of this study was to evaluate the effects of Boldo and Meadowsweet extracts on the expression of ß-defensins (cBD), cathelicidin (cCath), and pro-inflammatory cytokines in canine keratinocyte. This study had two phases. Phase I evaluated mRNA expression of cBD103 and cCath, and secretion of cCath, IL-8 and TNF-α by keratinocytes harvested from healthy (n=5) and atopic (n=5) age-matched beagles exposed to Boldo (2% to 0.2%) and Meadowsweet (1% to 0.2%) extracts. Phase II focused on atopic keratinocytes (n=14) exposed to 0.2% Boldo, 0.2% Meadowsweet, and a mixture of 0.1% of both extracts. Phase I: cBD103 mRNA (all concentrations) and TNF-α secretion (2% Boldo) were increased in atopic compared with healthy keratinocytes. In atopic keratinocytes, cBD103 was increased after exposure to 1.5% and 0.2% Boldo. In healthy keratinocytes, 1% and 0.2% Meadowsweet, and 2% Boldo increased and decreased IL-8 secretion, respectively. In atopic keratinocytes, IL-8 increased after exposure to 1% and 0.4% Meadowsweet extract. Phase II: cBD103 mRNA increased after exposure to 0.2% Meadowsweet and to 0.1% mixture. cCath was increased after 0.2% Boldo, but decreased after 0.2% Meadowsweet or the 0.1% mixture. TNF-α secretion was decreased after 0.2% Boldo. It is concluded that low concentrations of both extracts and their combination may have some effects on cCath and cBD103 without stimulating an inflammatory response. However, more studies are needed to clarify the effects of these extracts on the local immunity.

Decreased expression of caspase-14 in an experimental model of canine atopic dermatitis.

Alterations in skin barrier function and filaggrin expression have been reported in atopic dermatitis (AD). Caspase-14, a protease important for filaggrin processing, is decreased in human AD. Atopic Beagle dogs with skin barrier alterations have been validated as model for AD. This study aimed to investigate caspase-14 in normal and atopic Beagle dogs. Skin biopsies from non-lesional and control skin were analyzed for caspase-14 by immunofluorescence. Six images/sections were blindly scored for intensity. Data were tested with unpaired Student's t test. A P value of <0.05 was considered significant. Caspase-14 was decreased in atopic compared to normal skin both quantitatively (P <0.001) and qualitatively (P = 0.006; agreement = 0.93; consistency = 0.94). In conclusion, caspase-14 is decreased in this model similarly to reports in humans, highlighting the relevance of filaggrin metabolic defects in AD.

Use of a Canine Model of Atopic Dermatitis to Investigate the Efficacy of a CCR4 Antagonist in Allergen-Induced Skin Inflammation in a Randomized Study.

Atopic dermatitis (AD) is an inflammatory skin disease characterized by infiltration of skin homing lymphocytes into the dermis. Most of these lymphocytes express the chemokine receptor CCR4, and the frequency of blood CCR4(+) lymphocytes correlates with AD disease severity. Canine AD is a pruritic inflammatory condition that shows many features of the human disease, including CCR4 overexpression. Therefore, we tested a potent selective CCR4 antagonist in an allergen challenge model of canine AD, both clinically and histologically, to investigate whether this chemokine pathway plays a role in the inflammatory response. Using a four-period randomized cross-over study design, 14 beagles were challenged with allergen and clinically monitored. Biopsy samples were taken before and after allergen challenge. A clear reduction of clinical scores was observed with oral prednisolone (P < 0.0001) but not for the CCR4 inhibitor. A subset of the dogs (5/13) showed partial inhibition (30-49%) of the clinical signs with CCR4 inhibitor treatment, and this finding was supported by the results of histopathologic analysis of skin biopsy samples. This partial response is consistent with redundancy in chemokine pathways and highlights the need for therapies blocking multiple pathways. This study shows the utility of this canine model of AD for testing new therapeutic agents.

A comparative study of epidermal tight junction proteins in a dog model of atopic dermatitis.

BACKGROUND: Tight junctions (TJ) are important for skin barrier function and could be relevant in modulating allergen penetration in atopic dermatitis (AD). Humans with AD have been described to have decreased expressions of some TJ proteins in the skin. HYPOTHESIS/OBJECTIVES: This study aimed to investigate TJ protein expression using an experimental AD model in dogs. METHODS: Skin biopsies from six atopic (nonlesional skin) and five normal beagle dogs were stained for TJ proteins [zonula occludens 1 (ZO-1), occludin, claudin-1] by immunohistochemistry. Staining intensity was evaluated both objectively using imaging software and subjectively. Six images/sections were randomized and blindly scored by six investigators for intensity, distribution, integrity and staining pattern. RESULTS: The intensity of ZO-1 was significantly decreased in the atopic group objectively (P = 0.010) and subjectively (P = 0.002) relative to the normal group. Occludin was decreased significantly subjectively (P = 0.027) but not objectively. Claudin was not significantly different between groups by either quantification. Additionally, only ZO-1 demonstrated a significantly patchier staining pattern in the atopic group. There was no consistent staining pattern in this study. CONCLUSIONS AND CLINICAL IMPORTANCE: ZO-1 and occludin, which have not been described to be associated with the development of AD in humans, could play a role in this atopic dog model. Further investigation on the expression and modulation of TJ proteins and their clinical relevance is needed.

First report in a dog model of atopic dermatitis: expression patterns of protease-activated receptor-2 and thymic stromal lymphopoietin.

BACKGROUND: Protease-activated receptor (PAR)-2 plays a crucial role in inflammation and the skin barrier. Protease-activated receptor-2 is activated by proteolytic enzymes of allergens and stimulates thymic stromal lymphopoietin (TSLP), promoting T-helper 2 cytokines. In humans with atopic dermatitis (AD), increased expression of PAR-2 and TSLP has been reported. HYPOTHESIS/OBJECTIVES: To compare the pattern of staining of PAR-2 and TSLP between normal and atopic beagle dogs. The hypothesis tested was that increased expression is present in atopic dog skin compared with healthy control skin. ANIMALS: Eight atopic and five normal dogs were challenged for 3 days with house dust mites. METHODS: Skin biopsies were taken to measure the intensity, distribution, integrity and cell staining pattern on days 0, 3 and 10, both objectively and subjectively. Clinical signs were scored and compared between groups. RESULTS: Atopic dogs showed a significant increase in clinical scores on days 3 (peak of challenge) and 10 (resolution) and a significant condensed staining pattern for TSLP in the stratum basale at all times in comparison to normal dogs. They showed a significant patchy pattern for PAR-2 on days 0 and 3 and for TSLP at all times compared with normal dogs. The intensity itself was not significantly increased in atopic dogs compared with normal animals for both PAR-2 and TSLP. CONCLUSIONS AND CLINICAL IMPORTANCE: These preliminary findings do not confirm a difference in the amount of expression but rather in its pattern. Studies using PAR-2 or TSLP inhibitors could shed light on their clinical relevance.