Long-term effects of ciclosporin and oclacitinib on mediators of tolerance, regulatory T-cells, IL-10 and TGF-ß, in dogs with atopic dermatitis.

BACKGROUND: Atopic dogs often are managed with allergen-specific immunotherapy (AIT) and concurrent dosages of ciclosporin (CSA) or oclacitinib to alleviate their clinical signs. Both drugs might affect proper tolerance induction by inhibiting regulatory T-cell (Treg) induction. HYPOTHESIS/OBJECTIVES: We evaluated Treg cell numbers and serum interleukin (IL)-10 and transforming growth factor-beta (TGF-ß)1 levels in dogs diagnosed with atopic dermatitis (AD) and successfully treated with either CSA or oclacitinib for nine or more months. ANIMALS: We included 15 dogs receiving oclacitinib, 14 dogs treated with CSA, 15 healthy dogs, 13 dogs with untreated moderate-to-severe AD and 15 atopic dogs controlled with AIT. MATERIALS AND METHODS: Peripheral blood CD4+CD25+FOXP3+ T-cell percentages were determined using flow cytometry. Serum concentrations of IL-10 and TGF-ß1 were measured by enzyme-linked immunosorbent assay. RESULTS: The percentage of Treg cells in the CSA group was significantly lower in comparison with the healthy group (p = 0.0003), the nontreated AD group (p = 0.0056) or the AIT group (p = 0.0186). There was no significant difference in Treg cell percentages between the CSA and oclacitinib groups, nor between the oclacitinib and the healthy, nontreated AD or AIT-treated dogs. No significant differences were detected in IL-10 and TGF-ß1 serum concentrations between the five groups. CONCLUSIONS AND CLINICAL RELEVANCE: Lower Treg cell percentages in the CSA-treated dogs suggest an impact of this drug on this cell population; however, it does not necessarily mean that it diminishes tolerance. Functionality and cytokine production may be more important than the number of Treg cells. Further studies evaluating the treatment outcome of dogs receiving AIT and concurrent drugs are needed to show clinical relevance.

Higher prevalence of seizure activity in a small population of atopic dogs: a retrospective breed- and age-matched study.

BACKGROUND: Atopic dermatitis (AD) is considered to be a systemic disease in people shown to have an association with epilepsy. However, so far, no data about the association of epilepsy and atopy have been reported in dogs. OBJECTIVES: Given the homology between human and canine AD, and the increased incidence of epilepsy in atopic people, we investigated the association between AD and seizure-associated activity in a small canine population. ANIMALS: We included 34 atopic dogs and 34 breed- and age range-matched nonatopic dogs. METHODS AND MATERIALS: We investigated the association between canine AD and signs of seizures in a retrospective, breed- and age range-matched, case-controlled study. Dog owners were interviewed using a standardized questionnaire. The presence or absence of signs of seizure activity and possible comorbidities were questioned. RESULTS: Seven of the 34 atopic dogs also suffered from seizure activity. By contrast, only one dog affected with seizure signs could be identified among the 34 nonatopic dogs. Atopic dermatitis was associated with a higher frequency of seizure activity (McNemar test, P = 0.035; one-sided) and atopic dogs had a higher odds ratio to develop seizures [(95% CI) 7 (0.9-56.9)] compared to the age- and breed-matched nonatopic control group. No other comorbidities were detected. CONCLUSION AND CLINICAL IMPORTANCE: In our small retrospective study, we observed an increased prevalence of seizure activity in the atopic dog population. Further larger and prospective studies are needed to confirm these results.

Canine junctional epidermolysis bullosa due to a novel mutation in LAMA3 with severe upper respiratory involvement.

BACKGROUND: Junctional epidermolysis bullosa (JEB) is a group of congenital blistering skin diseases characterized by clefting through the lamina lucida of the basement membrane zone. OBJECTIVES: To characterize the clinical and morphological features of a congenital mechanobullous disease in a litter of puppies with severe upper respiratory involvement, and to identify an associated genetic variant. ANIMALS: Five of eight puppies in an Australian cattle dog cross-bred litter showed signs of skin fragility. Three were stillborn and one died at one month of age. The two surviving puppies were presented with blistering skin disease and severe respiratory distress. Additionally, one unaffected sibling was examined and blood was obtained for genetic testing. METHODS AND MATERIALS: Post-mortem examination, histopathological evaluation and electron microscopy were performed. Whole genome sequencing (WGS) of one affected puppy was compared to a database of 522 dogs of 55 different breeds for variant analysis. Sanger sequencing of one additional affected and one unaffected sibling confirmed the variant. RESULTS: Clinically, severe mucocutaneous ulcers occurred in frictional areas with claw sloughing. Histopathological results revealed subepidermal clefts and electron microscopy confirmed the split in the lamina lucida. Post-mortem examination documented extensive pharyngeal and laryngeal lesions with granulation tissue and fibrinous exudate obscuring the airway. Moderate tracheal hypoplasia contributed. The WGS revealed a novel missense variant in the laminin α3-chain XP_537297.2p(Asp2867Val), with an autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: A novel variant in LAMA3 caused a generalized and severe phenotype of JEB with an unique clinical presentation of upper airway obstruction.

Shortened immunotherapy dose-escalation saves time, but is it safe? A case-control study comparing the rates of adverse reactions between conventional and fast-escalation subcutaneous immunotherapy protocols during the induction phase.

BACKGROUND: Allergen immunotherapy (AIT) is the only intervention believed to change the course of atopic diseases. As dogs appear to have fewer severe adverse events (AEs) compared to people receiving AIT, a prolonged dose-escalation induction phase might not be needed. OBJECTIVES: To report the incidence and characteristics of AEs induced by a fast-escalation subcutaneous immunotherapy (f-SCIT) protocol compared to a conventional (c-SCIT) regimen. ANIMALS: One hundred dogs treated with either f- SCIT (Centre 1, 50 dogs) or c-SCIT (Centre 2, 50 dogs). METHODS AND MATERIALS: A case-control study retrospectively evaluating AEs during the induction of AIT. We determined the incidence and type of AEs in each SCIT group; we also assessed factors such as self-limitation and the need for AE-associated protocol changes. RESULTS: Twelve of 100 dogs (12%) developed a SCIT-attributable AE during the induction phase, with one dog having a local and 11 having systemic reactions (nine Grade I, two Grade II, none of grades III or IV). Dogs treated with the f-SCIT had a significantly higher rate of AEs (11 of 50; 22%) compared to those receiving the c-SCIT (one of 50; 2%). Most of the AEs (10 of 11; 91%) in the f-SCIT group were mild and self-limiting. CONCLUSIONS AND CLINICAL IMPORTANCE: The induction phase of f-SCIT is simpler, and the maintenance phase is reached faster than that of the c-SCIT. Despite its higher rate of AEs than with the c-SCIT regimen, the majority of f-SCIT-associated AEs were mild and self-limiting. Whether or not this f-SCIT protocol leads to a faster time-to-efficacy needs to be determined.

Cutaneous polyautoimmunity in two unrelated dogs: pemphigus foliaceus and generalized discoid lupus erythematosus.

BACKGROUND: Polyautoimmunity, the concurrent expression of two or more distinct autoimmune diseases (ADs) in a single individual, is a known phenomenon in humans and has been rarely reported in dogs. To the best of the authors' knowledge, comorbid pemphigus foliaceus (PF) and generalized discoid lupus erythematosus (GDLE) has not been reported in dogs. HYPOTHESIS/OBJECTIVES: To describe the clinical, histological and immunological features and treatment outcome of two unrelated dogs with comorbid PF and GDLE. ANIMALS: One 10-year-old, spayed German shepherd dog and one 8-year-old, castrated American Staffordshire terrier presented for evaluation of a symmetrical, facial- and/or pedal-dominant pustular dermatitis with concurrent, truncal scaly plaques. METHODS: For each dog, clinicopathological characterization included physical examination, lesion cytological evaluation, bacterial culture and sensitivity testing, skin histopathological investigation and direct and indirect immunofluorescence testing. Additional diagnostic imaging and haematological testing was performed to exclude extracutaneous disease. RESULTS: Both dogs exhibited lesions clinically and histologically compatible with PF and GDLE. Moreover, one dog exhibited generalized leucotrichia and chronic superficial keratitis. Remission was achieved with immunosuppressive dosages of prednisolone [high-dose pulse (Case 1) or standard immunosuppressive dosage (Case 2)] and ciclosporin (5-6 mg/kg/day). Tissue-bound antikeratinocyte immunoglobulin (Ig)G and IgM were detected in both dogs. A weak basement membrane zone deposit of C3 was seen in one dog. Circulating antikeratinocyte and anti-desmocollin-1 IgG were detected in one dog. CONCLUSIONS AND CLINICAL IMPORTANCE: Cutaneous polyautoimmunity can occur in the dog. Depending on the specific disease combinations, overlapping clinical features may present diagnostic and/or therapeutic challenges. Moreover, these cases should be monitored for development of additional cutaneous or extra-cutaneous AD(s).

Rapid response of hyperkeratotic erythema multiforme to oclacitinib in two dogs.

BACKGROUND: Hyperkeratotic erythema multiforme (HKEM) is a clinically distinct dermatosis and poorly characterized syndrome, comprised of hyperkeratotic plaques with variable symmetry and apoptosis similar to "classic" erosive canine EM. Hyperkeratotic EM has a protracted clinical course and, although treatments with glucocorticoids, azathioprine and/or ciclosporin have been tried, rates of remission are low. OBJECTIVES: To describe successful treatment of HKEM in two dogs using oclacitinib. ANIMALS: A 7-year-old, spayed Havanese dog (Case 1) and a 1-year-old, intact cryptorchid Dachshund dog (Case 2). METHODS: Case characterization and clinical diagnoses were based on lesion character, surgical biopsy, cytological evaluation, culture, direct immunofluorescence (DIF) and expected responses to treatments. RESULTS: Both cases exhibited multifocal, often symmetrical hyperkeratotic plaques with adherent scale. Histological findings revealed prominent epidermal hyperplasia, parakeratotic hyperkeratosis, lymphocytic dermatitis and transepidermal apoptosis with lymphocytic satellitosis. DIF revealed fine, patchy IgG, IgM and IgA basement membrane deposits (Case 2). Both dogs exhibited rapid improvement with oral oclacitinib (0.6-0.9 mg/kg twice daily) with a complete remission of clinical signs observed in 12 and seven weeks in cases 1 and 2, respectively. CONCLUSION AND CLINICAL IMPORTANCE: Oclacitinib could be considered as a fast-acting and effective treatment option for HKEM in dogs.

Efficacy and Safety of Subcutaneous Allergen-Specific Immuno-Therapy in Horses with Allergic Cutaneous and Respiratory Diseases-A Systematic Review.

Allergen-specific immunotherapy (AIT) is the only current intervention that has the ability to modify the immune response toward a tolerogenic state. This study aimed to assess the efficacy and safety of AIT in horses with allergic diseases in a systematic manner. Three databases were searched to identify articles reporting clinical outcomes and adverse events associated with AIT. The articles were evaluated for beneficial responses to AIT, defined as a ≥50% reduction in clinical signs, and clinical remission. Horses with respiratory diseases, urticaria, and pruritic dermatitis receiving insect monotherapy or multi-allergen AIT were included. All adverse events were graded, and analytical and confounding biases were assessed. The results showed that multi-allergen AIT had a beneficial response in 75% of horses with respiratory diseases, 88% with urticaria, and 56% with pruritic dermatitis. However, horses treated solely with insect AIT for pruritic dermatitis had a lower response rate (36%). Self-limiting local reactions were the most common adverse events, with systemic reactions grade II accounting for 11% of reported events. Analytical and confounding biases were identified as major limitations in the available studies. Further research is needed to address these biases and provide stronger evidence on the efficacy and safety of AIT in horses with allergic diseases.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Canine macrophages can like human macrophages be in vitro activated toward the M2a subtype relevant in allergy.

The M2a subtype of macrophages plays an important role in human immunoglobulin E (IgE-mediated allergies) and other Th2 type immune reactions. In contrast, very little is known about these cells in the dog. Here we describe an in vitro method to activate canine histiocytic DH82 cells and primary canine monocyte-derived macrophages (MDMs) toward the M2a macrophages using human cytokines. For a side-by-side comparison, we compared the canine cells to human MDMs, and the human monocytic cell line U937 activated towards M1 and M2a cells on the cellular and molecular level. In analogy to activated human M2a cells, canine M2a, differentiated from both DH82 and MDMs, showed an increase in CD206 surface receptor expression compared to M1. Interestingly, canine M2a, but not M1 derived from MDM, upregulated the high-affinity IgE receptor (FcεRI). Transcription levels of M2a-associated genes (IL10, CCL22, TGFß, CD163) showed a diverse pattern between the human and dog species, whereas M1 genes (IDO1, CXCL11, IL6, TNF-α) were similarly upregulated in canine and human M1 cells (cell lines and MDMs). We suggest that our novel in vitro method will be suitable in comparative allergology studies focussing on macrophages.

Bidirectional Regulation of COX-2 Expression Between Cancer Cells and Macrophages.

BACKGROUND/AIM: Our aim was to investigate the crosstalk between tumor and immune cells (M2 macrophages) and its effects on cyclo-oxygenase-2 (COX2) regulation in canine mammary tumors (CMT). MATERIALS AND METHODS: Sh1b CMT cells and human BT474 mammary or HT29 colon cancer cells were co-cultured with canine peripheral blood mononuclear cells (PBMCs) or with macrophage-like differentiated THP1 monocytes (dTHP1). Intracellular COX2 expression by PBMCs, dTHP1 and cancer cells was evaluated by flow cytometry. RESULTS: Co-culturing of Sh1b and canine PBMCs induced COX2 overexpression in CMT cells. In turn, COX2 expression by PBMCs, mostly CD68+ macrophages, was attenuated by co-culture with Sh1b (p=0.0001). In accordance, co-culture with dTHP1 prompted intracellular production of COX2 in both Sh1b CMT cells and HT29 human colon cancer cells and reduced production of COX2 in BT474 human mammary cancer cells. The intracellular COX2 expression from dTHP1 decreased when treated with conditioned medium from cultured Sh1b and HT29 cancer cells. CONCLUSION: Bidirectional COX2 regulation between cancer and monocytes/macrophages might shape a tolerogenic tumor microenvironment in CMT.

Gender aspects in allergies of pets - A secondary publication and update.

Allergies need not only affect humans; this multifactorial and complex disease can also affect animals. Comparative allergology investigates the many similarities between the pathogenesis, clinics, diagnosis, and therapy of the disorders in humans and pet animals. In contrast to human allergy research, the veterinary field lacks access to a central database, which means there are no cohort studies published. This limits not only the research on breed and regional differences in allergies, but also further studies on the impact of gender in allergies of domestic animals. Moreover, domestic cats, dogs and male horses are castrated in most cases, which neutralises any effects of sexual hormones. In this review article a few interesting findings regarding gender aspects in companion animals were extracted from current literature. In summary, there is a lack of data on gender effects on allergies in cats, dogs or horses.

Innate function of house dust mite allergens: robust enzymatic degradation of extracellular matrix at elevated pH.

BACKGROUND: Exposure to the house dust mite Dermatophagoides pteronyssinus (D.p.) increases the risk for developing allergic diseases in humans and their best friends, the dogs. Here, we explored whether this allergenic mite via its enzymes may impact the cutaneous extracellular matrix (ECM), which critically determines epithelial barrier integrity both structurally and functionally. METHODS: Two extracts obtained from either dust-purified or cultured D.p. bodies were used in the present study. To assess the potential impact of D.p. on protein components of the ECM, proteolytic activity of the D.p. extracts were determined by casein and gelatin gel zymography, and their N-acetyl-ß-hexosaminidase activity determined colorimetrically. In addition, IgE-dependent and innate degranulation potential of D.p. was examined in canine MPT-1 mast cells and neurite outgrowth assay using rat pheochromocytoma PC-12 cells. RESULTS: In gel zymography, both extracts digested the substrates casein and gelatin in a dose-dependent manner, especially at alkaline pH, and effective in a wide range of temperatures (30 °C-42 °C). In particular, a 25-kDa band corresponding to Der p 1, the major D.p. allergen for humans, was found enzymatically active in both casein and gelatin gels regardless of the presence of metal ions and of alkaline conditions. Besides protease activity, N-acetyl-ß-hexosaminidase activity was detected in both extracts, suggesting that D.p. affects the cutaneous ECM through deteriorating both proteins and glycosaminoglycans. While both D.p. extracts induced IgE-dependent mast cell degranulation, much less innate effects on mast- and neuronal cells were observed. CONCLUSIONS: Our data highlight that D.p. is a robust source of several distinct enzymes with protease- and N-acetyl-ß-hexosaminidase activities. In alkaline milieu they can degrade components of the ECM. Therefore, D.p. may contribute to epithelial barrier disruption especially when the skin surface pH is elevated.

Pollen Allergies in Humans and their Dogs, Cats and Horses: Differences and Similarities.

Both humans and their most important domestic animals harbor IgE and a similar IgE receptor repertoire and expression pattern. The same cell types are also involved in the triggering or regulation of allergies, such as mast cells, eosinophils or T-regulatory cells. Translational clinical studies in domestic animals could therefore help cure animal allergies and at the same time gather knowledge relevant to human patients. Dogs, cats and horses may spontaneously and to different extents develop immediate type symptoms to pollen allergens. The skin, nasal and bronchial reactions, as well as chronic skin lesions due to pollen are in principle comparable to human patients. Pollen of various species most often causes allergic rhinitis in human patients, whereas in dogs it elicits predominantly eczematous lesions (canine atopic dermatitis), in horses recurrent airway obstruction or hives as well as pruritic dermatitis, and in cats bronchial asthma and so-called cutaneous reactive patterns (eosinophilic granuloma complex, head and neck pruritus, symmetric self-induced alopecia). In human allergy-specific IgE detection, skin tests or other allergen provocation tests should be completed. In contrast, in animals IgE and dermal tests are regarded as equally important and may even replace each other. However, for practical and economic reasons intradermal tests are most commonly performed in a specialized practice. As in humans, in dogs, cats and horses allergen immunotherapy leads to significant improvement of the clinical symptoms. The collected evidence suggests that canines, felines and equines, with their spontaneous allergies, are attractive model patients for translational studies.