Evaluation of canine antimicrobial peptides in infected and noninfected chronic atopic skin

Background – Antimicrobial peptides (AMPs) are small immunomodulatory peptides produced by epithelial and immune cells. β‐Defensins (BDs) and cathelicidins (Caths) are the most studied AMPs. Recently, increased cutaneous expression of AMPs was reported in atopic humans and in beagles with experimentally induced atopy. Hypothesis/Objectives – Our goal was to analyse mRNA expression and protein levels of canine (c)BD1‐like, cBD2‐like/122, cBD3‐like, cBD103 and cCath in healthy and naturally affected atopic dogs, with and without active skin infection, along with their distribution in the epidermis using indirect immunofluorescence. Animals – Skin biopsies were taken from 14 healthy and 11 atopic privately owned dogs. Methods – The mRNA levels of cBD1‐like, cBD2‐like/122, cBD3‐like, cBD103 and cCath were quantified using quantitative real‐time PCR. The protein levels of cBD3‐like and cCath were analysed by relative competitive inhibition enzyme‐linked immunosorbent assay, while the distributions of cBD2‐like/122, cBD3‐like and cCath were detected by indirect immunofluorescence. Results – Dogs with atopic dermatitis had significantly greater mRNA expression of cBD103 (P = 0.04) than control dogs. Furthermore, atopic skin with active infection had a higher cBD103 mRNA expression (P = 0.01) and a lower cBD1‐like mRNA expression (P = 0.04) than atopic skin without infection. No significant differences in protein levels (cBD3‐like and cCath) or epidermal distribution of AMPs (cBD2‐like/122, cBD3‐like and cCath) were seen between healthy and atopic dogs. Conclusions and clinical importance – Expression of cBD103 mRNA was greater, while expression of cBD1‐like mRNA was lower in dogs with atopic dermatitis that had active infections. Work is needed to clarify the biological mechanisms and possible therapeutic options to maintain a healthy canine skin.     

Expression of thymic stromal lymphopoietin in canine atopic dermatitis

Background – In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). Hypothesis/Objectives – Our aim was to characterize canine TSLP and to assess its expression in CAD. Methods – Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT‐PCR. Real‐time quantitative RT‐PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. Results – Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full‐length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll‐like receptor ligands lipopolysaccharide and poly I:C. Conclusions and clinical importance – Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.     

Owner assessment of therapeutic interventions for canine atopic dermatitis: a long-term retrospective analysis

Background – Canine atopic dermatitis is a frequent diagnosis in veterinary medicine; however, the long‐term prognosis for canine atopic dermatitis has not been evaluated in a systematic fashion. Hypothesis/Objectives – To compare the relative efficacy of commonly used therapies for canine atopic dermatitis in two groups of dogs over 5 and 10 year time periods. Animals – Dogs were identified from the medical record database of a privately owned veterinary dermatology practice in the USA. Methods – Clients completed a four‐part, 28‐question, Internet‐based survey. Surveys were included in the analysis if one entire section was completed. Each question was completed independently of the answers to other questions. Results – Several respondents failed to complete all questions. Some respondents answered similar questions with contradictory answers. Each question was analysed individually. A total of 136 owner surveys were completed, 39 from the 10 year and 97 from the 5 year study dogs. Eighty‐five of 135 respondents indicated that their pet was receiving some form of medical therapy for atopic dermatitis at the time of the survey. Thirty of 90 respondents (33.3%) indicated that their dog improved during a dietary trial. Five dogs met the study’s definition for clinical cure. All five of these dogs had been treated with allergen‐specific immunotherapy. Conclusions and clinical importance – This study revealed that clients believe antihistamines can be a useful part of multimodal therapy for canine atopic dermatitis. The results also demonstrated that a significant number of canines benefited from dietary modification. In addition, allergen‐specific immunotherapy was the only treatment to induce true clinical remission of atopic dermatitis.     

Serum anti‐Staphylococcus pseudintermedius IgE and IgG antibodies in dogs with atopic dermatitis and nonatopic dogs

Background – Dogs and humans with atopic dermatitis (AD) are predisposed to colonization and recurrent infection with Staphylococcus spp. Studies in humans suggest that staphylococcus‐specific immunoglobulin E (IgE) plays a key role in disease pathogenesis. Few such studies have been undertaken in dogs. Hypothesis/Objectives – The aim of this study was to compare levels of staphylococcus‐specific IgE and immunoglobulin G (IgG) in dogs with AD, nonatopic dogs with staphylococcal pyoderma, and nonatopic and noninfected control dogs. Animals – Sera were collected from 108 dogs with AD, 39 nonatopic dogs with staphylococcal pyoderma secondary to different underlying conditions, 67 age‐matched nonatopic control dogs, and nine control dogs reared in minimal disease conditions. Methods – Serum Staphylococcus pseudintermedius‐specific IgE and IgG antibodies were measured by enzyme‐linked immunosorbent assay. Results – Dogs with AD had significantly higher levels of anti‐staphylococcal IgE than nonatopic dogs with staphylococcal pyoderma and the two groups of control dogs. Levels of anti‐staphylococcal IgG were significantly higher in atopic dogs and nonatopic dogs with pyoderma compared with nonatopic control dogs and control dogs reared in minimal disease conditions, but there was no significant difference in levels of anti‐staphylococcal IgG between dogs with AD and nonatopic dogs with pyoderma. Conclusions and clinical importance – A significantly increased IgE response to S. pseudintermedius antigens in atopic dogs suggests an immunopathogenic role for anti‐staphylococcal IgE. The finding of elevated IgE and IgG in atopic dogs is also important as a prelude to studies on antigenic specificity and possible correlations with disease phenotype.     

Ciclosporin therapy is associated with minimal changes in calcium metabolism in dogs with atopic dermatitis

Background – Ciclosporin is widely used in the management of canine atopic dermatitis. In humans, ciclosporin therapy has been linked to disturbances in calcium metabolism and resultant skeletal disorders. Objectives – The objective of this study was to assess calcium homeostasis in dogs before and after a 6 week course of once daily oral ciclosporin at the licensed dose (5 mg/kg). Animals – Sixteen client‐owned dogs with spontaneous atopic dermatitis. Methods – Serum concentrations of calcium, phosphate, creatinine, 25‐hydroxyvitamin D, 1,25‐dihyroxyvitamin D and plasma concentrations of ionized calcium and parathyroid hormone (PTH) were measured, together with the urinary fractional excretion of calcium and phosphate. The extent of skin lesions was scored using the Canine Atopic Dermatitis Extent and Severity Index (CADESI)‐03 and the degree of pruritus by the Edinburgh Pruritus Scale prior to and at the end of the study. Results – The CADESI‐03 and the Edinburgh Pruritus Scale scores decreased satisfactorily in all dogs by the end of the study. Plasma PTH concentrations were significantly increased (P = 0.02) following ciclosporin treatment, whereas all other biochemical parameters were not significantly different from their starting values. The increase in PTH was mild in most cases and the proportion of dogs that had a PTH concentration above the reference range was not significantly different following treatment. Conclusions and clinical importance – This study indicates that ciclosporin has minimal impact on calcium metabolism in dogs with atopic dermatitis when used at the licensed and clinically effective dosage for 6 weeks.     

FC‐54 Evaluation of IgE‐mediated late‐phase reactions in the skin of normal placebo‐ and prednisolone‐treated dogs: cellular,cytokine and chemokine responses

IgE‐mediated late‐phase reactions can be induced in the skin of normal and atopic dogs by intradermal injections of anti‐IgE antibody. The histology of these reactions is very similar to that of naturally occurring atopic dermatitis. To characterize the cellular, cytokine and chemokine responses in the skin of placebo‐ and prednisolone‐treated dogs, normal beagles received either placebo or 0.5 mg/kg prednisolone twice daily for three days prior to intradermal injection of polyclonal rabbit anti‐canine IgE. Eight‐millimetre punch biopsy skin samples were taken before injection and at the injection sites after 6, 24 and 48 h. Histological and immunohistochemical examination revealed a rapid cellular influx. Eosinophil and neutrophil numbers increased from <1 to 61.4 ± 14.1, and from 7 to 62.2 ± 10.8 cells/mm², respectively, within 6 h after injection, and remained moderately elevated 48 h later. The numbers of CD1c+, CD3+ and CD4+ mononuclear cells were also increased by 6 h. Taqman analysis demonstrated 2.5‐ to 72‐fold increases in mRNA expression for IL‐13, IL‐5, MCP (CCL2), RANTES (CCL5) and TARC (CCL17). Levels of mRNA for IL‐2, IL‐4, IL‐6, and IFNγ remained negligible. Prednisolone administration suppressed the influx of neutrophils and eosinophils, and the expression of IL‐13, CCL2, CCL5 and CCL17 (33, 97, 58, 86, 73 and 90%, respectively), as well as the influx of CD1c+ and CD3+ cells. These data document the cytokine and chemokine response to anti‐IgE injection and demonstrate the anti‐inflammatory effect of prednisolone. Funding: Schering‐Plough Animal Health.     

Interleukin‐31: its role in canine pruritus and naturally occurring canine atopic dermatitis

Background – Interleukin‐31 (IL‐31) is a member of the gp130/interleukin‐6 cytokine family that is produced by cell types such as T helper 2 lymphocytes and cutaneous lymphocyte antigen positive skin homing T cells. When overexpressed in transgenic mice, IL‐31 induces severe pruritus, alopecia and skin lesions. In humans, IL‐31 serum levels correlate with the severity of atopic dermatitis in adults and children. Hypothesis/Objective – To determine the role of IL‐31 in canine pruritus and naturally occurring canine atopic dermatitis (AD). Animals – Purpose‐bred beagle dogs were used for laboratory studies. Serum samples were obtained from laboratory animals, nondiseased client‐owned dogs and client‐owned dogs diagnosed with naturally occurring AD. Methods – Purpose‐bred beagle dogs were administered canine interleukin‐31 (cIL‐31) via several routes (intravenous, subcutaneous or intradermal), and pruritic behaviour was observed/quantified via video monitoring. Quantitative immunoassay techniques were employed to measure serum levels of cIL‐31 in dogs. Results – Injection of cIL‐31 into laboratory beagle dogs caused transient episodes of pruritic behaviour regardless of the route of administration. When evaluated over a 2 h period, dogs receiving cIL‐31 exhibited a significant increase in pruritic behaviour compared with dogs that received placebo. In addition, cIL‐31 levels were detectable in 57% of dogs with naturally occurring AD (≥13 pg/mL) but were below limits of quantification (<13 pg/mL) in normal, nondiseased laboratory or client‐owned animals. Conclusions – Canine IL‐31 induced pruritic behaviours in dogs. Canine IL‐31 was detected in the majority of dogs with naturally occurring AD, suggesting that this cytokine may play an important role in pruritic allergic skin conditions, such as atopic dermatitis, in this species.     

Electron microscopic observations of stratum corneum intercellular lipids in normal and atopic dogs.

The barrier function of mammalian skin is maintained by intercellular stratum corneum lipids. In human patients with atopic dermatitis, an abnormal lipid barrier results in dry skin and increased transepidermal water loss. At this time, it is not known if a defective lipid barrier is present in atopic dogs. Normal and atopic canine skin were postfixed in ruthenium tetroxide and studied using transmission electron microscopy to determine structural differences within stratum corneum lipids. Intercellular lipid lamellae were graded on a semiquantitative scale. The deposition of stratum corneum lipid lamellae in atopic canine skin appeared markedly heterogeneous compared with that seen in normal canine skin. When present, the lamellae often exhibited an abnormal structure. The continuity and thickness of the intercellular lipid lamellae were significantly less in nonlesional atopic than in normal canine skin. These preliminary observations suggest that the epidermal lipid barrier is defective in atopic canine skin. Additional studies are needed to further characterize the biochemical defect and to possibly correct it with nutritional and/or pharmacologic intervention.     

Clinical use of a ceramide-based moisturizer for treating dogs with atopic dermatitis

In humans, skin barrier dysfunction is thought to be responsible for enhanced penetration of allergens. Similar to conditions seen in humans, canine atopic dermatitis (CAD) is characterized by derangement of corneocytes and disorganization of intercellular lipids in the stratum corenum (SC) with decreased ceramide levels. This study was designed to evaluate the effects of a moisturizer containing ceramide on dogs with CAD. Dogs (n = 20, 3~8 years old) with mild to moderate clinical signs were recruited and applied a moisturizer containing ceramide for 4 weeks. Transepidermal water loss (TEWL), skin hydration, pruritus index for canine atopic dermatitis (PICAD) scores, and canine atopic dermatitis extent and severity index (CADESI) scores of all dogs were evaluated. Skin samples from five dogs were also examined with transmission electron microscopy (TEM) using ruthenium tetroxide. TEWL, PICAD, and CADESI values decreased (p < 0.05) and skin hydration increased dramatically over time (p < 0.05). Electron micrographs showed that the skin barrier of all five dogs was partially restored (p < 0.05). In conclusion, these results demonstrated that moisturizer containing ceramide was effective for treating skin barrier dysfunction and CAD symptoms.     

Small Demodex populations colonize most parts of the skin of healthy dogs

Background – It is unproven that all dogs harbour Demodex mites in their skin. In fact, several microscopic studies have failed to demonstrate mites in healthy dogs. Hypothesis/Objectives – Demodex canis is a normal inhabitant of the skin of most, if not all, dogs. This hypothesis was tested using a sensitive real‐time PCR to detect Demodex DNA in the skin of dogs. Animals – One hundred dogs living in a humane society shelter, 20 privately owned and healthy dogs and eight dogs receiving immunosuppressive or antineoplastic therapy. Methods – Hair samples (250–300 hairs with their hair bulbs) were taken from five or 20 skin locations. A real‐time PCR that amplifies a 166 bp sequence of the D. canis chitin synthase gene was used. Results – The percentage of positive dogs increased with the number of sampling points. When a large canine population was sampled at five cutaneous locations, 18% of dogs were positive for Demodex DNA. When 20 skin locations were sampled, all dogs tested positive for mite DNA. Our study indicates that Demodex colonization of the skin is present in all dogs, independent of age, sex, breed or coat. Nevertheless, the population of mites in a healthy dog appears to be small. Demodex DNA was amplified from all 20 cutaneous points investigated, without statistically significant differences. Conclusions and clinical importance – Using a real‐time PCR technique, Demodex mites, albeit in very low numbers, were found to be normal inhabitants of haired areas of the skin of healthy dogs.     

Are transepidermal water loss and clinical signs correlated in canine atopic dermatitis? A compilation of studies

Background – Cutaneous impairment plays a crucial role in atopic dermatitis (AD). Transepidermal water loss (TEWL) measurement is an indirect assessment of skin barrier function and correlates with disease severity in humans. Skin impairment also exists in canine AD; however, concerns exist regarding variability and reliability of TEWL measurements in dogs. Hypothesis/Objectives – The purposes of this retrospective study were twofold: first, to investigate the correlation between severity of dermatitis [measured by Canine Atopic Dermatitis Extent and Severity Index (CADESI)] and TEWL; and second, to evaluate whether increased TEWL at a young age correlates with disease severity later in life. Methods – Data from atopic beagles and dogs with natural AD were analysed. Transepidermal water loss was measured in atopic beagles (n = 24) with an open chamber and in dogs with naturally occurring AD with a closed chamber device (two studies, with n = 14 and n = 18). Pearson product–moment correlation was used for analyses. Transepidermal water loss of the inguinal region, axilla, antebrachial flexure and pinna was analysed. Correlations were investigated for each study, separately first and then jointly. They included CADESI and TEWL of individual regions, total CADESI and total TEWL of all measured regions, and total CADESI and TEWL of key regions. Results – In atopic beagles, TEWL measured at 1 year of age pre‐ and post‐allergen challenge was correlated with CADESI at 1, 3 and 6 years of age. Overall, low correlation coefficients were found; therefore, a biologically relevant connection could not be demonstrated. The main significant positive correlation was found between TEWL in the pinna and total CADESI. Conclusions and clinical importance – It is concluded that TEWL does not correlate with disease severity.     

Is the skin barrier abnormal in dogs with atopic dermatitis?

In mammalian skin, the stratum corneum exerts a barrier function that protects from transepidermal water loss and the penetration of exogenous molecules, such as allergens, from the environment. Recently, skin barrier defects have been shown to be of prime importance in the pathogenesis of human atopic dermatitis. In this review, we summarize the latest research data pertinent to the stratum corneum and barrier function of dogs with atopic dermatitis. At the time of this writing, there is increasing evidence that a skin barrier defect likely exists in dogs with atopic dermatitis. This barrier dysfunction is characterized by abnormal intercellular stratum corneum lipid lamellae, abnormal stratum corneum morphology, reduced and abnormal ceramide content and, in some but not all dogs, abnormal filaggrin expression. In association with these changes, there is higher transepidermal water loss in atopic than in normal canine skin. Furthermore, atopic inflammation appears to worsen transepidermal water loss and filaggrin expression. It remains unknown, however, if the changes observed are primary (i.e. of genetic origin) or secondary to atopic inflammation that also exists even in clinically normal skin. Finally, whether or not a therapeutic intervention aimed at restoring a dysfunctional skin barrier is of any clinical benefit to atopic dogs has not yet been proven unequivocally.     

Heterogeneity of Staphylococcus pseudintermedius isolates from atopic and healthy dogs

Staphylococcus pseudintermedius is part of the normal canine flora but frequently causes pyoderma in canine atopic dermatitis (AD). This study aimed to determine whether particular S. pseudintermedius strains were associated with AD and/or pyoderma. Ninety‐six S. pseudintermedius isolates from the ear, nares, perineum and lesions of 21 atopic and 16 healthy dogs were lysed with proteinase K and digested with 40 U SmaI. Restriction products were separated using pulsed‐field gel electrophoresis (PFGE) with an Oxford S. aureus control and lambda‐ladder DNA concatomer markers. A dendrogram was constructed by the unweighted pair group method. All isolates showed a ≥56% similarity coefficient. Nine distinct PFGE clusters were identified, as follows: five from both atopic and healthy dogs; three from atopic dogs only; and one from healthy dogs only. Nine clusters were isolated from the nares, eight from the perineum, five from the ears and six from pyoderma lesions. There were no significant differences in the frequency of isolation from atopic or healthy skin, body sites or infected lesions for any of the clusters. Two of six healthy dogs and 18 of 20 atopic dogs with multiple isolates had closely related isolates (less than three band differences) at more than one sampling site. Isolates from pyoderma lesions were closely related to at least one mucosal isolate in 11 of 16 dogs. Staphylococcus pseudintermedius isolates appear to be heterogeneous, and colonization or infection of atopic skin was not associated with any particular strain or cluster of strains.     

Transmission electron microscopy studies in an experimental model of canine atopic dermatitis

Impairment of skin barrier function has been hypothesized in canine atopic dermatitis (AD). In this prospective, controlled study, the ultrastructure of the upper epidermal layers was investigated using an experimental model of canine AD. Seven atopic Beagles sensitized to Dermatophagoides farinae and four healthy Beagles were used as controls. Both normal and atopic dogs were challenged with D. farinae for 3 days. Clinical signs were scored and skin biopsies were taken from the inguinal area before and 3 days after allergen exposure. Samples were processed to enhance lipid visibility and evaluated by Transmission Electron Microscopy. Emphasis was placed on evaluation of the lipid lamellae (LL), and lamellar bodies (LB) of the stratum corneum.
After allergen challenge, atopic Beagles developed severe pruritic dermatitis while no skin lesions were noted in the controls. Ultrastructurally, before allergen challenge, atopic Beagles displayed focally severe abnormalities in LL organization and wider intercellular spaces containing abnormal lipid material. In atopic Beagles, LBs were frequently found inside corneocytes while this finding was not observed in the controls. After allergen challenge, further increase of intercellular spaces was observed in the stratum corneum of atopic Beagles while no appreciable changes were observed in the normal dogs. Intercellular spaces in atopic Beagles were filled with abundant amounts of abnormal lipid material and highly disorganized LL. It is concluded that baseline differences in the ultrastructure of the skin exist between normal and experimentally sensitized atopic Beagles and that these changes are aggravated by allergen challenge and the resulting flare-up of dermatitis.     

Development and validation of the Canine Atopic Dermatitis Lesion Index,a scale for the rapid scoring of lesion severity in canine atopic dermatitis

Background – The third iteration of the Canine Atopic Dermatitis Extent and Severity Index (CADESI‐03) is the only tool rigorously validated for canine atopic dermatitis (CAD) lesion scoring. The CADESI‐03 requires 248 evaluations, limiting its widespread use. Hypothesis/Objectives – The goal of the study was to develop and validate a practical method of grading CAD lesions that requires scoring only the frequently affected body regions. Animals – Fifty‐seven privately owned atopic dogs were used in the study. Methods – The Canine Atopic Dermatitis Lesion Index (CADLI) was evaluated in an open, multicentre reliability study. Validity was assessed with expert opinion (content validity) and comparison of CADLI with existing disease severity measures (construct and criterion validity). Reliability was evaluated by analysing repeated observations of each dog. Convenience was assessed in terms of the time required to complete the scale. Results – The CADLI scores correlated with overall assessment scores (r = 0.60, P < 0.001, linear mixed model) and pruritus severity scores (r = 0.53, P < 0.001, linear mixed model), establishing construct validity. The CADLI was strongly correlated with CADESI‐03 (r = 0.84, P < 0.001, linear mixed model), establishing criterion validity. The CADLI values obtained by two observers correlated very strongly (r = 0.91, P < 0.001), as did the repeat values for the same observer (r = 0.98, P < 0.001). The mean time to complete the CADLI was less than that required for CADESI‐03 (1.9 and 12.6 min, respectively), a highly significant difference (P < 0.001). Conclusion and clinical importance – The CADLI was found to be an effective measure of CAD lesion severity, strongly correlating with CADESI‐03. The convenience of CADLI makes it suitable for use in both clinical research and practice.     

Skin mast cell histamine release following stem cell factor and high-affinity immunoglobulin E receptor cross-linking in dogs with atopic dermatitis

Stem cell factor (SCF) influences mast cell activation and inflammatory mediator release, and is elevated in tissues undergoing allergic inflammation. Wheal formation in response to the injection of SCF or anti-immunoglobulin (Ig)E antibody injection was compared between normal (n = 10) and nonlesional atopic (n = 10) canine skin. In situ SCF secretion was compared between lesional and nonlesional skin using immunohistochemistry. Histamine release by skin cell suspensions after stimulation with SCF, concanavalin A (ConA) or rabbit anticanine IgE antibodies was compared between normal and atopic dogs. All dogs exhibited strong responses to intradermal SCF injection at 10 and 50 ng mL(-1). Atopic dogs had significantly (P = 0.002) larger wheal responses to anti-IgE than normal dogs; but there was no difference in numbers of skin mast cells bearing IgE as detected by immunohistochemistry. Only atopic dogs exhibited interstitial deposition of SCF in both lesional and nonlesional skin specimens. Median histamine release stimulated by SCF in the absence of IgE from lesional skin cells was higher in atopic than normal dogs (P = 0.04). These experiments suggest that dermal SCF secretion could potentiate histamine release following IgE receptor cross-linking and thus, could be one of the explanations for the inherent mast cell hyperexcitability observed in canine atopic dermatitis.     

High allergen-specific serum immunoglobulin E levels in nonatopic West Highland white terriers

Human and canine atopic dermatitis (AD) share an association with IgE specific to environmental allergens, but few studies have evaluated serum allergen‐specific IgE in nonatopic dogs. This study compared serum allergen‐specific IgE levels in 30 atopic and 18 nonatopic West Highland white terriers. Atopic dermatitis was confirmed using standard criteria. Nonatopic dogs were over 5 years of age and had no clinical signs or history of AD. Serum allergen‐specific IgE levels were measured with Allercept^® IgE ELISAs using a 48‐allergen Australian panel. Positive reactions were defined as ≥150 ELISA absorbance units. Intradermal tests were performed in 16 atopic dogs, either at the time of or at various times prior to serum collection. In atopic dogs, the most common positive ELISA and intradermal test results were to Dermatophagoides farinae (11 of 30 dogs), but there were no statistically significant correlations between results from the two methods for any allergen. In nonatopic dogs, multiple high‐positive ELISA reactions were reported to 45 of 48 allergens, most commonly D. farinae and Tyrophagus putrescentiae (17 of 18 dogs each). Positive ELISA results in nonatopic dogs were statistically significantly higher than those in atopic dogs for 44 of 48 allergens, including two allergens (D. farinae and Dermatophagoides pteronyssinus) commonly regarded as significant in canine AD. In conclusion, positive allergen‐specific IgE ELISAs were not specific for canine AD, and high allergen‐specific IgE levels were seen in nonatopic dogs. The clinical significance of this and whether it characterizes a protective phenotype is unclear.     

FC‐36 The use of immunostimulatory bacterial DNA sequences in allergen‐specific immunotherapy of canine atopic dermatitis

The purpose of this study was to evaluate a combination of immunostimulatory bacterial DNA sequences and allergen‐specific immunotherapy for the treatment of canine atopic dermatitis. Seven dogs with nonseasonal atopic dermatitis diagnosed by history, clinical signs and exclusion of differential diagnoses were included. All dogs had been on allergen‐specific immunotherapy for at least 12 months with incomplete responses, were on additional antipruritic therapy and showed residual pruritus. Pruritus was marked by the owner on a visual analogue scale, lesions were determined by a clinician using the Canine Atopic Dermatitis Extent and Severity Index (CADESI), and concurrent medications were recorded before entering the study and after 14 weeks of treatment. Peripheral blood mononuclear cells were isolated and cultured; canine cytokine message for IFNγ, IL‐4, TNF and IL‐10 was quantitated using RT‐PCR. A mixture of allergen extract and liposome‐DNA complexes was injected intradermally at the beginning of the study and after 2, 4, 6, 10 and 14 weeks. CADESI, pruritus and medication scores, and cytokine messages at the beginning and end of the study were compared with a paired t‐test. There were significant improvements in pruritus scores (P = 0.0277). Reductions in medication scores and CADESI were not statistically significant. IL‐4 production decreased significantly (P = 0.0428); decreases in other cytokines were not significant. Although the number of dogs in this pilot study was small, the results warrant further investigation of a combination of immunostimulatory bacterial DNA sequences and allergen‐specific immunotherapy for the treatment of canine atopic dermatitis. Funding: Self‐funded.     

Engineered cross‐reacting nanobodies simplify comparative oncology between humans and dogs

Antibodies cross‐reacting with homologue antigens in different species would be essential reagents for the development of comparative oncology studies. In comparison with conventional immunoglobulin Gs, recombinant nanobodies (single‐domain variable regions of heavy‐chain only antibodies of Camelidae origin) can be easily isolated in vitro and engineered into a variety of reagents with optimized characteristics for different research and clinical applications. We recovered an anti‐human epidermal growth factor receptor 2 (anti‐HER2) nanobody from a naïve llama library by direct panning on whole cells and expressed it fused to Fc and green fluorescent protein. These immunoreagents were assessed by flow cytometry and immunofluorescence with both human and canine cells overexpressing HER2 and its canine homologue dog epidermal growth factor receptor 2. The reported data illustrate the potential of using this class of antibodies in comparative oncology and suggest some development perspectives enabled by in vitro panning of pre‐immune nanobody libraries.