Food-specific sublingual immunotherapy is well tolerated and safe in healthy dogs : a blind, randomized, placebo-controlled study

Background: Food allergies are increasing in prevalence but no treatment strategies are currently available to cure dogs with food allergy. Over the past decade, experimental food allergen-specific sublingual immunotherapy (FA-SLIT) has emerged as a potential treatment for food allergies in human medicine. However, FA-SLIT has not been investigated in dogs. Therefore, the objective of this study was to prospectively evaluate the safety, tolerability and dispenser sterility of FA-SLIT in healthy dogs before testing it in food allergic dogs. Eight experimental healthy beagle dogs, never orally exposed to peanut, were randomized in two groups to receive SLIT with peanut or placebo for 4 months. Subjects were monitored daily for local and systemic adverse effects. Blood samples for complete blood count and serum biochemistry, and urine for urinalysis were collected and the dogs' body weight was recorded at day 0, 35 and 119 of the SLIT treatment. Sera for the determination of peanut-specific IgG and IgE were collected at day 0, 35, 49, 70, 91, 105 and 119. Intradermal tests were performed before (day 0) and after (day 119) the experiment. The content of each dispenser used to administer treatment or placebo was tested for sterility after usage. In order to assess the presence or absence of sensitization, dogs were challenged 6 months after the end of the study with 2000 mu g of peanut extract daily for 7 to 14 days. Results: All dogs completed the study. The treatment did not provoke either local or systemic side-effects. Peanut-specific IgG significantly increased in treatment group. Even though a significant increase in peanut-specific IgE was also seen, intradermal tests were negative in all dogs before and after the experiment, and the challenge test did not trigger any adverse reactions in the treated dogs, which shows the protocol did not cause sensitization to peanut, but nevertheless primed the immune system as indicated by the humoral immune response. All dispenser solutions were sterile. Conclusions: Our results demonstrate that the used peanut-SLIT protocol is well tolerated and safe in healthy dogs. Further studies should evaluate tolerability, safety and efficacy in dogs with food allergy

Allergie bij de hond : een bevraging van dierenartsen en eigenaars over de toestand in Vlaanderen

In human medicine, the prevalence of allergies has increased during the last decades. A similar ten-dency is suspected in small animal veterinary medicine. Unfortunately, only little information is avai-lable about the prevalence of allergies in dogs in Flanders. In this study, veterinarians and owners were asked about the five most common types of allergy, i.e. atopy, flea allergy, food allergy, contact allergy and allergy to medication and injections. The median prevalence, estimated by the veterinarians, was respectively 15%, 10%, 5%, 2% and 1%. Some remarkable differences with the literature were noticed. A blood test was mentioned by the owners as the most frequently used test to diagnose food allergies. Living in an urban environment could not be linked with an increased risk to develop allergies, and 11% of the veterinarians reported washing-powder as a contact allergen. In the opinion of more than half of the veterinarians, the number of dogs with an allergy has increased during his/her career

Adjuvanting allergen extracts for sublingual immunotherapy : calcitriol downregulates CXCL8 production in primary sublingual epithelial cells

Application of allergens onto the sublingual epithelium is used to desensitize allergic individuals, a treatment known as sublingual immunotherapy. However, the response of sublingual epithelial cells to house dust mite allergen and potential tolerance-promoting adjuvants such as Toll-like receptor (TLR) ligands and calcitriol has not been investigated. In order to study this, primary sublingual epithelial cells were isolated from dogs and culturedin vitro. After 24-h incubation with aDermatophagoides farinaeextract, aDermatophagoides pteronyssinusextract, TLR2 ligands (FSL-1, heat-killed Listeria monocytogenes, Pam3CSK4), a TLR3 ligand (poly I:C), a TLR4 ligand [lipopolysaccharide (LPS)], and calcitriol (1,25-dihydroxyvitamin D-3), viability of the cells was analyzed using an MTT test, and their secretion of interleukin 6 (IL-6), IL-10, CXCL8, and transforming growth factor beta 1 (TGF-beta 1) was measured by enzyme-linked immunosorbent assay. Additionally, to evaluate its potential effect as an adjuvant, sublingual epithelial cells were incubated with calcitriol in combination with aD. farinaeextract followed by measurement of CXCL8 secretion. Furthermore, the effect ofD. farinaeand calcitriol on the transcriptome was assessed by RNA sequencing. The viability of the sublingual epithelial cells was significantly decreased by poly I:C, but not by the other stimuli. CXCL8 secretion was significantly increased byD. farinaeextract and all TLR ligands apart from LPS. Calcitriol significantly decreased CXCL8 secretion, and coadministration withD. farinaeextract reduced CXCL8 concentrations to levels seen in unstimulated sublingual epithelial cells. Although detectable, TGF-beta 1 secretion could not be modulated by any of the stimuli. Interleukin 6 and IL-10 could not be detected at the protein or at the mRNA level. It can be concluded that aD. farinaeextract and TLR ligands augment the secretion of the proinflammatory chemokine CXCL8, which might interfere with sublingual desensitization. On the other hand, CXCL8 secretion was reduced by coapplication of calcitriol and aD. farinaeextract. Calcitriol therefore seems to be a suitable candidate to be used as adjuvant during sublingual immunotherapy

Detection of allergen-specific antibody-secreting cells in dogs by ELISPOT

Current laboratory tests are unable to distinguish healthy from allergic dogs. Unlike serum antibody responses, circulating antibody-secreting cells (ASC) are temporarily induced after each contact with the antigen. These ASC can be identified using ELISPOT and the observation of allergen-specific ASC might correlate with the causative allergens in dogs with an allergic dermatitis. In this study, blood was sampled from six privatelyowned allergic dogs and six non-allergic laboratory beagles to determine the frequency of circulating allergenspecific ASC for common allergens. Blood IgE+, IgA + and IgG + cells were magnetically isolated to determine the number of allergen-specific ASC with ELISPOT for Dermatophagoides farinae, Dermatophagoides pteronyssinus, Alternaria alternata, birch, timothy grass, wheat, cow’s milk, bovine, chicken and lamb meat. For IgA and IgG, allergen-specific spots were observed, however for IgE, no spots were detected for any of the allergens. ELISPOT could not differentiate allergic from non-allergic dogs. When the responses to the different allergens were compared, more IgA ASC for D. pteronyssinus were observed compared to some of the other allergens which was statistically significant for the non-allergic dogs and approached significance in the allergic dogs. These findings indicate that ELISPOT can be used to identify circulating allergen-specific IgA- and IgG-secreting cells. The technique did however not detect allergen-specific IgE ASC and was unable to distinguish allergic from nonallergic dogs. Only a small number of studies have studied allergen-specific IgA in dogs. The finding that dogs have higher numbers of D. pteronyssinus-specific IgA ASC points out that apart from IgE and IgG, it might be interesting to include IgA measurements for certain allergens to analyse the complete spectrum of both the protective and pro-allergic antibody responses

Immortalised canine buccal epithelial cells' CXCL8 secretion is affected by allergen extracts, Toll-like receptor ligands, IL-17A and calcitriol

Epithelial cells are known to produce mediators which can influence the behaviour of neighbouring immune cells. Although the oral mucosa has gained increased interest as a route to induce allergy desensitisation and mucosal pathogen immunisation in dogs, there is only limited knowledge on the factors which impact mediator secretion by canine oral epithelial cells. The study's objective was to enlarge the knowledge on the stimuli that can influence the secretion of some pro- and anti-inflammatory cytokines and the chemokine CXCL8 by canine buccal epithelial cells. To investigate this, buccal epithelial cells were isolated from a biopsy of a dog and immortalised by lentiviral transduction of the SV40 large T antigen. The cells were stained with a CD49f and cytokeratin 3 antibody to confirm their epithelial origin. Cells were incubated with allergen extracts, Toll-like receptor ligands (TLRL), recombinant cytokines and vitamin A and D metabolites. Subsequently, the secretion of the cytokines interleukin (IL)-4, IL-6, IL-10, IL-17A, IFN-gamma, TGF-beta 1 and the chemokine CXCL8 was assayed by ELISA. Immortalised canine buccal epithelial cells stained positive for CD49f but not for cytokeratin 3. The cells produced detectable amounts of CXCL8 and TGF-beta 1. A Dermatophagoides farinae extract, an Alternaria alternata extract, Pam3CSK4, heat-killed Listeria monocytogenes, FSL-1, flagellin and canine recombinant IL-17A significantly increased CXCL8 secretion, while the vitamin D metabolite calcitriol significantly suppressed the production of this chemokine. This study showed that certain allergens, TLRL, IL-17A and calcitriol modulate CXCL8 secretion in a cell line of canine buccal epithelial cells