Matrix metalloproteinase mRNA expression in canine anal furunculosis lesions

Although the aetiology of anal furunculosis (AF) in dogs is poorly understood, there is evidence for an underlying immune dysfunction. This is illustrated by the presence of a T helper type 1 cytokine mRNA profile in AF lesions and the clinical response to ciclosporin therapy. Expression of MMPs 2, 9 and 13 were evaluated in AF lesional biopsies by real-time quantitative RT-PCR. There was significantly increased expression of both MMP-9 and MMP-13 mRNA in AF biopsies compared to controls (p<0.001) but no significant difference in MMP-2 mRNA expression. Since MMP-9 and MMP-13 are primarily produced by macrophages, these data suggest that ulceration could be the result of aberrant activation of this cell type in the tissues. It is feasible that such pathological macrophage activity occurs in response to interferon-gamma secreted by T helper type 1 cells. This could explain why the lesions resolve following treatment with the immunosuppressive drug ciclosporin.     

Quantitative analysis of inflammatory and immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection

The present study sought to quantitatively examine mucosal inflammatory and immune responses in dogs with gastritis and the relationship of these responses to infection with Helicobacter. Gastric biopsies from 30 dogs were evaluated for B- and T-lymphocytes, neutrophils, eosinophils, macrophages, and mast cells. Mucosal atrophy, fibrosis, cellularity, and severity of gastritis were graded qualitatively. Messenger-RNA (mRNA) for actin, interleukin-1beta (IL-1beta), IL-4, IL-8, and IL-10, transforming growth factor beta (TGF-beta), and interferon gamma (IFN-gamma) was quantified by polymerase chain reaction (PCR). The presence of Helicobacter spp. was determined by urease activity, histology, PCR, and enzyme-linked immunosorbent assay. mRNA for IL-1beta, IL-8, IL-10, TGF-beta, and IFN-gamma was detected in most dogs. IL-4 mRNA was detected in only 1 dog. Correlations were observed for IL-1beta versus IL-8 and IL-10; IL-8 versus IL-10, IFN-gamma, and TGF-beta; and IL-10 versus IFN-y. Mucosal pathology was related to cytokine mRNA expression (neutrophils to IL-8 and IFN-gamma, macrophages and lymphocytes to IFN-gamma, and fibrosis to IL-1beta). Gastritis was categorized as lymphoplasmacytic in all dogs, and its histologic severity correlated with atrophy, infiltration with lymphocytes and macrophages, and expression of IL-10 and IFN-gamma. Of the dogs examined, 76.7% were infected with Helicobacter spp. Infection was associated with increased expression of TGF-beta and fibrosis. Circulating anti-Helicobacter immunoglobulin G titers were higher in uninfected than infected dogs. We conclude that lymphoplasmacytic gastritis in dogs is characterized by concurrent activation of proinflammatory and immunomodulatory cytokines, with increased mRNA expression related to mucosal pathology. No significant associations between Helicobacter infection and proinflammatory cytokine expression, severity of gastritis, or differences in the pathogenicity of different Helicobacter spp. were found.     

Quantification of mRNA encoding cytokines and chemokines in nasal biopsies from dogs with sino-nasal aspergillosis

Canine sino-nasal aspergillosis is usually caused by Aspergillus fumigatus and is similar to human chronic erosive non-invasive fungal sinusitis. The pathogenesis of the disease is poorly understood. We investigated the nature of the local immune response mounted in canine sino-nasal aspergillosis. Quantitative RT-PCR was carried out on RNA isolated from nasal biopsies from diseased and control dogs, using specific assays designed to amplify mRNA encoding a panel of cytokines and chemokines. Canine sino-nasal aspergillosis was associated with significantly increased expression of mRNA encoding MCP-1, -2, -3 and -4, IL-8, IL-10, IL-18 and TNF-alpha relative to controls (P<0.01) but there was no difference between groups with respect to IL-4, IL-5, IL-6, IL-12, TGF-beta, and eotaxin-2 and -3. The up-regulation of proinflammatory cytokines and chemokines related to the influx of phagocytic cells might account for the localisation of this infection to the upper respiratory tract. The up-regulation of the expression of the immunomodulatory cytokine IL-10 in nasal tissue from affected dogs might be important in limiting the extent of local tissue destruction, but might also account for the fact that infected dogs are generally unable to clear this infection spontaneously.     

Toxocara canis infection induces antigen-specific IL-10 and IFNgamma production in pregnant dogs and their puppies

Toxocara canis (T. canis) is originally a parasite of canine bitches and their pups. The pathogenicity of T. canis infection is enhanced during pregnancy and puppyhood. The aim of this study was to investigate if modification of IFNgamma and IL-10 secretion occurs during infection in pregnant dogs and puppies. Analysis of cytokines secreted could let us hypothesize a role for IL-10 and/or IFNgamma in T. canis infection. We tested T. canis-specific production of IFNgamma and IL-10 by lymphocytes of pregnant dogs and their puppies after in vitro re-exposure to purified excretory/secretory antigen (ESAg) from T. canis. Blood mononuclear cells (BMC) isolated from pregnant dogs and their puppies were cultured in the presence of ESAg. Cultures' supernatants were tested for cytokine levels by ELISA. Results obtained showed that IL-10 concentrations increased during pregnancy in infected animals and in the meantime IFNgamma production decreased. In puppyhood, we observed that, IL-10 concentration decreased with the age of puppies mainly in infected animals while IFNgamma increased. In conclusion, our data suggests that BMC of infected dogs have a particular modification of IL-10 and IFNgamma synthesis. These data could be the basis to design immunotherapeutic approaches.     

Immune dysregulation in flea allergy dermatitis--a model for the immunopathogenesis of allergic dermatitis

BACKGROUND: Flea allergy dermatitis (FAD) is a common skin disease in dogs and can be induced experimentally. It often coexists with other allergic conditions. So far no studies have investigated the quantitative production of cytokine mRNA in skin biopsies and peripheral blood mononuclear cells (PBMC) in flea allergic dogs. OBJECTIVE: The aim of our study was to improve the understanding of the immunopathogenesis of allergic dermatitis as a response to fleabites. MATERIAL AND METHODS: Allergic and non-allergic dogs were exposed to fleas. Before and after 4 days of flea exposure mRNA was isolated from biopsies and PBMC. Production of chymase, tryptase, IL-4, IL-5, IL-13, TNF-alpha and IFN-gamma mRNA was measured by real-time RT-PCR. The inflammatory infiltrate in the skin was scored semi-quantitatively. The number of eosinophils, mast cells (MC) and IgE+ cells/mm2 was evaluated to complete the picture. RESULTS: FAD was associated with a higher number of MC before flea exposure and with a significant increase of eosinophils after flea exposure as compared to non-allergic dogs. The number of IgE+ cells was higher in allergic dogs before and after flea exposure. In allergic dogs mRNA for most cytokines and proteases tested was higher before flea exposure than after flea exposure. After exposure to fleas an increased mRNA production was only observed in non-allergic dogs. In vitro stimulation with flea antigen resulted in a decreased expression of most cytokines in allergic dogs before flea exposure. In contrast, in PBMC, only increased levels of IL-4 and IL-5 mRNA were observed in allergic dogs before flea exposure. However, after flea exposure and additional stimulation with flea antigen the production of mRNA for all cytokines tested was significantly increased in allergic dogs. CONCLUSION: We demonstrated that the response in biopsies and PBMC is different and that FAD is associated with a TH2 response.     

Expression of CD4 T cell cytokine genes in the colorectal mucosa of inflammatory colorectal polyps in miniature dachshunds

Inflammatory colorectal polyps (ICRPs) in miniature dachshunds are recently recognized as a major cause of large bowel diarrhea in this dog breed in Japan. ICRPs are characterized by the formation of multiple small polyps and a space-occupying large polyp in the colorectal area, and are thought to be a novel form of inflammatory bowel disease (IBD). In humans, specific cytokine patterns attributed to T helper (Th)1, Th17 and regulatory T cells have important roles in the pathogenesis of IBD. Thus, the aim of the present study was to assess the gene expression of cytokines of T cell subsets in the colorectal mucosa from dogs with ICRPs. Colorectal mucosal specimens from 10 dogs with ICRPs and 14 control dogs were used in this study. Interferon (IFN)-γ, interleukin (IL)-4, IL-17A and IL-10 mRNA expression was assessed using quantitative real-time PCR. IL-17A mRNA expression was significantly increased in large polyps compared to small polyps and controls. IFN-γ and IL-10 mRNA expression in large polyps were significantly higher than in controls. There was no significant difference in IL-4 mRNA expression among the three groups. IL-17A is thought to play important roles in the pathogenesis of ICRPs. IL-10 up-regulation could oppose the proinflammatory function of IL-17A.     

Allergen-induced production of IL-31 by canine Th2 cells and identification of immune,skin, and neuronal target cells

The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine. Peripheral blood mononuclear cells (PBMCs) were collected from naïve and house dust mite (HDM) allergen-sensitized beagle dogs and used for ex vivo characterization of cytokine production assessed using ELISpot and quantitative immunoassay. Sensitization to HDM allergen induced a T-helper type 2 (Th2) cell phenotype characterized by an increase in the production of IL-4 protein. Interestingly, repeated allergen challenge over time also resulted in an increase in IFN-γ. Further evaluation showed that co-stimulation of Th2 polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced higher levels of IL-31 compared to either stimulant alone. Production of IL-31 when PBMCs were stimulated by T cell mitogens suggests T cells as a source of IL-31. Quantitative real-time PCR was utilized to determine expression of the IL-31 receptor alpha chain in canine cell lines and tissue. Canine monocytic cells, keratinocytes, and dorsal root ganglia were shown to express the IL-31 receptor alpha chain mRNA. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus by immune cells, keratinocytes, and direct neuronal stimulation.     

Management of perianal fistulae in five dogs using azathioprine and metronidazole prior to surgery

OBJECTIVE: To evaluate combination therapy with azathioprine and metronidazole in German Shepherd Dogs with perianal fistulae. DESIGN: Prospective study. PROCEDURE: Five dogs (31.5 to 36.0 kg) with perianal fistulae were treated with azathioprine (50 mg per dog orally every 24 h) and metronidazole (400 mg per dog orally every 24 h). Patients were re-evaluated at 2 week intervals by inspection, palpation, photographs of the perineal region and assessment of white blood cell counts where possible. Treatment was continued until improvement in lesions reached a plateau. Surgical excision of residual fistulae and anal sac remnants was then performed, with medical therapy continued for an additional 3 to 6 weeks. RESULT: Signs attributable to anal irritation were reduced or eliminated in all dogs within 2 weeks, although visible healing of lesions progressed more slowly. Ulcerated lesions reduced in surface area and depth, and some fistulae healed completely. Non-healing areas were usually associated with anal sac rupture or chronic fibrosis. Visible improvement typically reached a plateau 4 to 6 weeks after commencing treatment. Immunosuppressive therapy continued for 5 to 24 weeks before surgical intervention to remove anal sacs (four dogs) and/or residual fistulae (five dogs). All dogs remain disease free 7 to 10 months postoperatively. No important complications of treatment were encountered. CONCLUSION: Azathioprine with metronidazole effectively reduced perianal irritation, and the severity and extent of lesions prior to surgery. Treatment was economical even in large dogs and associated with few untoward sequelae. The combined use of immunosuppressive and antimicrobial therapy followed by surgery minimised potential morbidity associated with aggressive use of either medical of surgical treatment alone.     

Gene expression of selected signature cytokines of T cell subsets in duodenal tissues of dogs with and without inflammatory bowel disease

Inflammatory bowel disease (IBD) is a common cause of chronic diarrhoea in dogs. In people, specific cytokine patterns attributed to T cell subsets, especially T helper cell [Th]1, Th17 and regulatory T(reg) cells have emerged in IBD. In contrast, no specific involvement of a distinct T cell subset has been described so far in canine IBD. Thus, the aim of the present study was to assess gene expression of signature cytokines in duodenal tissues from 18 German shepherd dogs with IBD (group 1), 33 dogs of other breeds with IBD (group 2) and 15 control dogs (group 3). Relative quantification of IL-17A, IL-22, IL-10, IFNy and TGFβ was performed. Expression of IL-17A was significantly lower in groups 1 and 2 compared to group 3 (p=0.014), but no difference in the expression of IL-22 (p=0.839), IFNγ (p=0.359), IL-10 (p=0.085) or TGFβ (p=0.551) across groups was detected. Thus, no clear evidence for the involvement of Th-17 signature cytokines in canine IBD at the mRNA level could be shown. The contribution of specific T cell subsets to the pathogenesis of canine IBD warrants further investigation.     

Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs

The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.     

Partial divergence of cytokine mRNA expression in bronchial tissues compared to bronchoalveolar lavage cells in horses with recurrent airway obstruction

The aim of this study was to investigate mRNA levels of cytokines in bronchial epithelium in horses with recurrent airway obstruction (RAO) during acute crisis and remission. Additionally, cytokine mRNA levels in endobronchial biopsies and bronchoalveolar lavage (BAL) cells were compared. Seven RAO horses were examined while in respiratory crisis following provocation and again while in remission after 2 months on pasture, during which time six healthy horses on pasture were also examined. Quantitative real-time PCR (RT-PCR) was used to assess mRNA expression for cytokines IL-5, IL-6, IL-8, IL-10, IL-17 and transforming growth factor beta1 (TGF-beta1) in endobronchial biopsies and bronchoalveolar lavage. Expression of IL-8 mRNA was significantly upregulated during crisis in both endobronchial biopsies and BAL cells (p=0.036), while there was a similar trend for upregulation of IL-10 mRNA only in BAL cells that approached significance (p=0.059). Moreover, during crisis the expression of IL-8 mRNA in BAL cells was positively correlated to relative IL-6 mRNA expression (r(s)=0.971, p=0.001) and bronchial epithelial expression of IL-10 and TGF-beta1 mRNA were positively correlated (r(s)=0.943, p=0.005). In comparing the relationship of mRNA expression in BAL to biopsy in individual RAO horses, there was a positive correlation with IL-6 to IL-8 mRNA expression in BAL during respiratory crisis (r(s)=0.971, p=0.001) that also correlated positively with IL-8 expression in biopsies on pasture (r(s)=0.986, p<0.0001 for both). Regarding RAO horses at pasture versus controls neither the cytokine mRNA levels in endobronchial biopsy nor in BAL cells differed significantly. These results further support previous findings that IL-8 mRNA in both BAL cells and bronchial epithelium is upregulated in RAO horses during crisis. However, apart from IL-8, it appears that expression of other cytokines, including IL-5, IL-6, IL-10, IL-17 and TGF-beta1 in bronchial epithelium does not necessarily mirror cytokine expression in BAL cells in individual horses with RAO. Accordingly, examination of markers of inflammation in endobronchial tissue provides complementary but not necessarily identical information to that obtained in BAL cells. Given the potential for repeated sampling over time bronchial biopsy can serve as an invaluable additional tool for investigation of time-dependent changes in inflammatory process in this animal model of asthma.