Effect of recombinant canine IFNγ on canine atopic dermatitis evaluated by clinical signs,histopathology and expression of Th1/Th2 cytokine mRNA

Atopic dermatitis (AD) is thought to be caused by immunologic abnormalities expressed as a Th1/Th2 cytokine imbalance in both humans and dogs. Several studies have focused on the therapeutic effects of IFNγ in human AD with successful results; however, the mechanism of action of IFNγ is not fully understood. We investigated the effect of recombinant canine interferon gamma (rCaIFNγ) on 10 dogs with AD and evaluated the ratio of IL‐4 mRNA to IFNγ mRNA in peripheral blood mononuclear cells, serum total IgE levels, and histological changes in skin. After six injections of rCaIFNγ over a span of 2 weeks, seven of the 10 dogs showed improvement, and six of these seven dogs exhibited decreased IL‐4:IFNγ mRNA ratios. Two of the three cases that did not improve had increased IL‐4:IFNγ mRNA ratios. Total serum IgE levels were significantly decreased in nine of 10 cases. The number of IgE‐positive cells detected by immunostaining and the number of mast cells in skin biopsy samples were decreased. A reduction of epidermal cell layers was demonstrated by histopathology after treatment. These results demonstrated that rCaIFNγ may be a novel safe and effective therapeutic option for the treatment of canine AD, and the mechanism of action of rCaIFNγ may be related to the modulation of Th2 cytokines to Th1 cytokines with the reduction of serum IgE production. Funding: Self‐funded.     

FC‐51 Comparison of intradermal test and antigen‐specific IgE test in 22 cases of feline allergic dermatitis

The usefulness of the intradermal test (IDT) and the serological allergy test (SAT) for detecting antigen‐specific IgE in allergic cats has not yet been established. In this study, we compared the results of IDT with those of SAT and evaluated the clinical usefulness of the two tests for detecting possible allergens in allergic cats. IDT and SAT using eight antigens were performed on 22 cats with intense pruritus after excluding ectoparasites and performing diet elimination tests. Approximately 50% of the cats reacted to at least one allergen by either IDT or SAT, and 36.4% of the cats reacted on both IDT and SAT. In contrast, seven healthy cats did not show any reactions on IDT or SAT. The most commonly detected allergen in both tests was house dust mites (IDT, 36.4%; SAT, 40.9%). Five cats reacted to one allergen and the others reacted to more than one allergen with IDT. Three cats reacted to one allergen with SAT. The following percentage agreement between the results of the two tests was calculated: house dust mites (86.4%), cat fleas (63.6%), grass mix (86.4%), common mugwort (81.8%), cat epithelia (90.9%), ragweed (86.4%), Japanese cedar (90.9%), and plantain (81.8%). The overall mean percentage agreement was 83.5%. In summary, the present study showed good agreement between IDT and SAT for cats, and SAT may be more sensitive than IDT, but less specific for detecting sensitized allergens. Funding: Self‐funded.     

Cyclosporine A inhibits the mRNA expressions of IL-2, IL-4 and IFN-gamma, but not TNF-alpha, in canine mononuclear cells

The effects of the calcineurin inhibitors cyclosporine A (CsA) and FK506 on the mRNA expressions of various cytokines were evaluated in dogs to determine whether the effects of CsA and FK506 in dogs were similar to those in humans. The mRNA expression levels of the cytokines IL-2, IL-4, IFN-gamma and TNF-alpha were measured in PHA-stimulated canine PBMC using real-time RT-PCR after incubation with CsA or FK506 for 5 hr. Both reagents inhibited IL-2, IL-4 and IFN-gamma mRNA expressions in a dose-dependent manner. However, CsA hardly inhibited the mRNA expression of TNF-alpha. These findings are important for assessing the indications of CsA treatment in dogs.     

A case of hyperplastic dermatosis of the West Highland White Terrier controlled by recombinant canine interferon-gamma therapy

A 3.5-year-old, male West Highland White Terrier was diagnosed as having hyperplastic dermatosis by clinical and histopathological findings. Controlling of Malassezia overgrowth by antifungal drugs provided a temporal improvement of the skin lesions, but the disease was deteriorated within the next 2 months despite the negative demonstration of the yeasts. Induction of recombinant canine interferon-gamma (rCaIFN-gamma) therapy resulted in almost complete cure of the skin lesions within 2 months after the initiation of the therapy. No adverse effects were detected during the therapy. Our results suggested that the rCaIFN-gamma therapy is potential to be a novel therapeutic option for controlling the breed-specific hyperplastic skin disease.     

A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.     

Generation of canine dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.     

Successful treatment of two dogs with allergic dermatitis by anti-allergic peptides (MS-antigen)

The effects of non-specific immunotherapy with anti-allergic peptides extracted from the urine of human allergic patients (MS-antigen), in two dogs with allergic dermatitis (AD) have been described. Clinically, severe pruritus accompanied by secondary bacterial pyoderma did not respond to conventional therapy with systemic antibiotics. The first clinical change appeared as a significant reduction in pruritus within 3 months, around the time of the 15th injection in both cases. The clinical condition was stabilized after 5 months, allowing the gradual withdrawal of concurrent therapies and an increase of injection intervals. The correlation between the results of intradermal skin tests before and after treatment and the improvement of clinical signs was not obvious.     

Pemphigus foliaceus in association with systemic lupus erythematosus, and subsequent lymphoma in a cocker spaniel

A seven-year-old neutered male cocker spaniel was presented with an 11-month history of generalised bacterial dermatitis. There were skin lesions over the entire body, which were round, slightly raised and encrusted. Skin biopsies were collected and the histological findings were consistent with pemphigus foliaceus. Immunohistochemical staining by the indirect immunoperoxidase method was positive, with desmosomal deposition of immunoglobulin (Ig) G. Haematological analysis revealed a regenerative anaemia and profound thrombocytopenia, while a Coombs' test was positive for polyvalent canine Coombs' reagent and anti-dog IgG. An antinuclear antibody test was positive, with a titre of 10,240. An ophthalmic examination demonstrated low tear production (keratoconjunctivitis sicca). Seven months after initial referral, the dog was re-presented with severe generalised peripheral lymphadenopathy. Radiographic evaluation of the thorax and abdomen revealed enlarged cranial mediastinal and sublumbar lymph nodes. Tru-Cut biopsy from an enlarged lymph node confirmed the diagnosis of lymphoma, which was phenotyped as a B-cell tumour. The diagnosis in this case was systemic lupus erythematosus, with the unusual feature of pemphigus foliaceus, and subsequent development of B-cell lymphoma. The case adds further to knowledge of the protean clinical presentations of canine autoimmune diseases and provides additional evidence for the potential association between autoimmunity and immune-system neoplasia in this species.     

Comparison of response to immunotherapy by intradermal skin test and antigen-specific IgE in canine atopy

The intradermal skin test (IDST) and serologic allergy test (SAT) has been developed for confirming a diagnosis of canine atopy and determining allergens for immunotherapy. To determine the prevalence of causative allergens for canine atopic dermatitis in Japan, IDST and SAT were performed with the CMG Immunodot strips on 95 atopic dogs using 9 allergens. In addition, we compared agreement rate, sensitivity and specificity between them (using IDST as the standard). The allergen most commonly positive in both tests was house dust mites (IDST: 69.5%, SAT: 48.4%). Moreover, Japanese cedar, mugwort and grass mix were detected as attendant causative allergens. Agreement rates between the two tests ranged from 67.4% to 96.8%; the overall mean agreement rate were 81%. SAT was shown to have sensitivity to IDST ranging from 16.7 to 68.2%. The specificities were very high for all allergens, on the order of 94.9-100% (median=98.7%). Finally, the efficacy of immunotherapy was evaluated on 27 atopic dogs based on IDST (15 dogs) and SAT (12 dogs) results. Overall, 60% (9/15) of the IDST group and 66.8% (8/12) of the SAT group experienced a 50% to 100% reduction in their symptomatology. No significant differences were found in response to immunotherapy during the follow-up period between allergen selection methods. These results indicate the value of serologic tests as an aid to identifying an allergen solution for immunotherapy.