Tumour necrosis factor-α induces C-C motif chemokine ligand 5 production in canine keratinocytes.

BACKGROUND: C-C motif chemokine ligand (CCL)5 induces skin inflammation in healthy dogs. In addition, CCL5 is overexpressed in the skin of experimental models of canine atopic dermatitis (cAD). Tumour necrosis factor (TNF)-α has been shown to be upregulated in cAD. However, it remains unclear whether TNF-α induces CCL5 production in canine keratinocytes. HYPOTHESIS/OBJECTIVES: To determine the effect of TNF-α on CCL5 production in canine keratinocyte culture and investigate possible synergy with interferon (IFN)-γ and interleukin (IL)-4. MATERIALS AND METHODS: CCL5 protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatant of a cell line of canine progenitor epidermal keratinocyte (CPEK) cells stimulated with TNF-α with or without inhibitors of the TNF receptor signalling pathway. CCL5 protein concentrations also were measured in CPEK cells stimulated with TNF-α in the absence or presence of IFN-γ, a T-helper (Th)1-type cytokine, and/or IL-4, a Th2-type cytokine. RESULTS: TNF-α increased CCL5 production in CPEK cells in time- and dose-dependent manners. Inhibitors of the TNF receptor signalling pathway diminished CCL5 production. Although neither IFN-γ nor IL-4 alone induced CCL5 production in CPEK cells, the combination of TNF-α and IFN-γ, and not IL-4, synergistically enhanced CCL5 production in these cells. CONCLUSIONS AND CLINICAL RELEVANCE: TNF-α may be involved in skin inflammation in dogs by promoting CCL5 production in keratinocytes. Furthermore, the synergistic effect of TNF-α and IFN-γ suggests that the local Th1-type milieu may aggravate skin inflammation. Further studies are required to elucidate the role of TNF-α-induced CCL5 production of keratinocytes in the pathogenesis of cAD.

Pilot evaluation of a single oral fecal microbiota transplantation for canine atopic dermatitis.

The gut microbiota has been suggested to be involved in the pathogenesis of canine atopic dermatitis (cAD). However, the gut microbiota has not been well characterized in dogs with atopic dermatitis (AD). In addition, the efficacy of fecal microbiota transplantation (FMT) in dogs with AD remains unclear. This research, therefore, aimed to characterize the gut microbiota of dogs with AD and conduct pilot evaluation of the efficacy of a single oral FMT on clinical signs and the gut microbiota of dogs with AD. For these purposes, we used 12 dogs with AD and 20 healthy dogs. The 16S rRNA analysis of the fecal microbiota revealed significant differences between 12 dogs with AD and 20 healthy dogs. Next, a single oral FMT was performed in 12 dogs with AD as a single-arm, open-label clinical trial for 56 days. A single oral FMT significantly decreased Canine Atopic Dermatitis Extent and Severity Index (CADESI)-04 scores from day 0 (median score, 16.5) to day 56 (8) and Pruritus Visual Analog Scale (PVAS) scores from days 0 (median score, 3) to day 56 (1). Furthermore, a single oral FMT changed the composition of the fecal microbiota of dogs with AD at the phylum and genus levels. The number of common amplicon sequence variants in the fecal microbiota between donor dogs and dogs with AD was positively correlated with CADESI-04 and PVAS reduction ratios 56 days after FMT. Our findings suggest that the gut microbiota plays a pivotal role in the pathogenesis of cAD, and that oral FMT could be a new therapeutic approach targeting the gut microbiota in cAD.

The immunoreceptor SLAMF8 promotes the differentiation of follicular dendritic cell-dependent monocytic cells with B cell-activating ability.

Follicular dendritic cells (FDCs) play a crucial role in generating high-affinity antibody-producing B cells during the germinal center (GC) reaction. Herein, we analysed the altered gene expression profile of a mouse FDC line, FL-Y, following lymphotoxin ß receptor stimulation, and observed increased Slam-family member 8 (Slamf8) mRNA expression. Forced Slamf8 expression and SLAMF8-Fc addition enhanced the ability of FL-Y cells to induce FDC-induced monocytic cell (FDMC) differentiation. FDMCs accelerated GC-phenotype proliferation in cultured B cells, suggesting that they are capable of promoting GC responses. Furthermore, a pulldown assay showed that SLAMF8-Fc could bind to SLAMF8-His. Overall, the homophilic interaction of SLAMF8 promotes FDMC differentiation and SLAMF8 might act as a novel regulator of GC responses by regulating FDMC differentiation.

Expression of genes encoding interleukin 15 and its receptor subunits in the duodenal and colonic mucosae of dogs with chronic enteropathy.

A pro-inflammatory role of interleukin (IL)-15 and IL-15 receptor (R) in chronic intestinal inflammation, such as inflammatory bowel disease, has been reported in humans. However, the contribution of IL-15 signaling in the pathogenesis of canine chronic enteropathy (CE) remains unclear. Therefore, as a first step in elucidating the importance of IL-15 signaling in canine CE, we measured the mRNA expression of IL-15 and IL-15R subunits, including IL-15Rα, IL-15Rß, and IL-15Rγ, in the duodenal and colonic mucosae of healthy dogs and those with CE, including food-responsive enteropathy (FRE), antibiotic-responsive enteropathy (ARE), and immunosuppressant-responsive enteropathy (IRE). Real-time PCR analysis revealed significantly lower IL-15Rα mRNA expression levels in the duodenal mucosa of dogs with IRE compared to healthy dogs. In contrast, the mRNA expression levels of IL-15, IL-15Rß, and IL-15Rγ in the duodenal mucosa and IL-15, IL-15Rα, IL-15Rß, and IL-15Rγ in the colonic mucosa did not differ among healthy dogs and those with FRE, ARE, or IRE. These findings suggest that decreased mRNA expression of IL-15Rα might be involved in the pathogenesis of duodenitis in dogs with IRE. Moreover, even in canine CE, IL-15 signaling appears to play different roles in duodenitis and colitis in dogs with FRE, ARE, and IRE. However, there were no correlations between the gene expression levels of IL-15Rα and clinical severity or histopathological scores in the duodenum of dogs with IRE. Further studies are necessary to investigate the IL-15Rα protein localization and to determine how impaired IL-15Rα expression contributes to the development of duodenitis in dogs with IRE.

The independent association between salivary alpha-amylase activity and arterial stiffness in Japanese men and women: the Toon Health Study.

Psychological stress is considered to be a potential contributor in the development of arterial stiffness. However, an independent association between arterial stiffness and biological markers of stress has not yet been established. We examined the independent association between salivary alpha-amylase (sAA) activity and arterial stiffness, not mediated by cardiometabolic disease associated with arterial stiffness, in a sample of healthy Japanese men and women. Participants (992 in total, 296 men and 696 women aged 30-79 years) had neither previous cardiovascular events or stroke, nor coexisting hypertension, diabetes, or dyslipidemia. Arterial stiffness was measured by the cardio-ankle vascular index (CAVI), and increased CAVI was defined as a CAVI value of 9 or higher. A saliva sample was collected in the morning and sAA was measured with a commercial assay kit. Higher sAA activity was positively associated with greater arterial stiffness particularly among women (ß = 0.070; 95% CI = 0.014-0.126; p = 0.01), and not across all participants (ß = 0.042; 95% CI = -0.005-0.089; p = 0.08) and in men (ß = -0.005; 95% CI = -0.097-0.087; p = 0.91). The association was strongest in the group of women aged 60 years and older (ß = 0.121; 95% CI = 0.018-0.224; p = 0.02). Although the association between sAA and increased CAVI (CAVI ≥ 9) was not significant in all and sex subgroups, odds ratios (OR) for CAVI ≥ 7 were significantly high in all participants (OR = 1.25; 95% CI = 1.03-1.53) and women (OR = 1.43; 95% CI = 1.12-1.82). Elevation of sAA was associated with an increase in arterial stiffness, particularly for women aged 60 years or older.

Clinical characteristics of dogs presenting with vomiting as a gastrointestinal sign of chronic enteropathy.

Vomiting is a major gastrointestinal (GI) sign of chronic enteropathy (CE) in dogs. Previous studies have reported clinical characteristics of dogs with CE, who developed diarrhea with or without vomiting as GI signs. However, to characterize clinical features of dogs with CE appropriately, dogs presenting with vomiting without diarrhea should be included in the analysis. Thus, this study aimed to characterize clinical features and outcomes of dogs that presented with vomiting without diarrhea. Based on their presenting GI signs, we retrospectively classified 66 dogs with CE into "Vomiting", "Diarrhea", or "Vomiting and diarrhea" groups and compared clinical and histological characteristics of each group. We found that 18 of the 66 dogs with CE (27%) presented with vomiting without diarrhea as a GI sign. Compared to the other 2 groups, the Vomiting group was significantly associated with food-responsive enteropathy (FRE), Beagle, lower clinical severity scores, higher plasma albumin levels, and higher histological scores for eosinophils in the duodenal lamina propria according to the univariate analysis. The multivariate analysis revealed that FRE and higher histological scores for eosinophils in the duodenal lamina propria were significant variables in the Vomiting group. Moreover, the survival time was the longest in the Vomiting group among dogs with CE. These findings are of clinical significance as they indicate that presenting with vomiting without diarrhea may not only be helpful in differentiating FRE from the other types of CE, but also in predicting the prognosis.

Case Report: Surgical Treatment of Type IV Spinal Dermoid Sinus in a Shiba Inu.

A 2-year-old spayed female Shiba Inu was presented with progressive non-ambulatory bilateral paraparesis, back pain, and urinary incontinence. CT and MRI revealed multiple vertebral malformations and type IV dermoid sinus. Hemilaminectomy was performed in T1-T5 to remove the dermoid sinus and granulomatous lesion that infiltrated into the spinal cord parenchyma. Histopathological examination of the excised tissue revealed type IV dermoid sinus with granulomatous meningomyelitis. After surgery, back pain was resolved, and the dog recovered ambulation and voluntary urination at the time of follow-up 4 months after surgery.

House dust mite-derived serine protease upregulates gene expression of interleukin-33 in canine keratinocytes via protease-activated receptor-2.

BACKGROUND: The involvement of interleukin (IL)-33 produced by keratinocytes has been suggested in the pathogenesis of canine atopic dermatitis (cAD). House dust mite (HDM)-derived proteases induce the production of various cytokines and chemokines in keratinocytes via protease-activated receptor-2 (PAR-2); however, their effects on IL-33 mRNA expression in canine keratinocytes have not been determined. HYPOTHESIS/OBJECTIVE: To clarify whether HDM-derived proteases induce IL-33 mRNA expression in canine keratinocytes via PAR-2. METHODS AND MATERIALS: Expression of IL-33 mRNA was quantified by real-time PCR in a cell line of canine progenitor epidermal keratinocytes (CPEK) stimulated with Dermatophagoides farinae (Der f) whole body extract, Der f pre-treated with cysteine protease and serine protease inhibitors, and trypsin. Trypsin and Der f-mediated IL-33 mRNA expression also was measured in CPEK cells treated with a PAR-2 antagonist. RESULTS: Der f enhanced IL-33 mRNA expression in CPEK cells in incubation time- and dose-dependent manners. Der f pre-treated with a serine protease inhibitor, and not a cysteine protease inhibitor, abrogated an increase in IL-33 mRNA expression in CPEK cells. Trypsin also enhanced IL-33 mRNA expression in CPEK cells. Trypsin-mediated IL-33 mRNA expression was completely abolished by a PAR-2 antagonist, while Der f-mediated IL-33 mRNA expression was partially and significantly diminished by it. CONCLUSIONS AND CLINICAL RELEVANCE: Der f-derived serine protease upregulated IL-33 mRNA expression in CPEK cells at least in part via PAR-2. These findings suggest that HDM may be involved in the development of C AD by increasing IL-33 mRNA expression in keratinocytes.

Long-term management of a cat with nasopharyngeal lymphoma by chlorambucil.

Background: Lymphoma in the nasal cavity is the most common tumor of cats' upper respiratory tract. However, the effect of single-agent chlorambucil on nasal or nasopharyngeal lymphoma has not been evaluated in cats. Case Description: An 8-year-old, castrated male Scottish Fold weighing 3.5 kg presented with an 8-month history of nasal discharge, sneezing, and mild epistaxis. CT and rhinoscopy revealed nasal discharge and slight swelling of the nasopharyngeal mucosa, but no masses and local invasions were detected. Histopathological and immunohistochemical analyses of the nasopharyngeal mucosa demonstrated B-cell lymphoma in the cat. The treatment with chlorambucil led to long-term management of the cat without any side effects. No recurrences of clinical signs have been observed for 754 days. Conclusion: The present case report suggests that chlorambucil can be a therapeutic option for feline localized nasopharyngeal B-cell lymphoma without masses and local invasions.

Expression of genes encoding inflammasome sensor subunits in the duodenal and colonic mucosae of dogs with chronic enteropathy.

Inflammasomes play a pivotal role in gastrointestinal homeostasis and inflammation. However, it remains elusive whether the nucleotide-binding oligomerization domain-like receptor (NLR) family inflammasomes, such as NLR family pyrin domain-containing (NLRP) 3, NLRP6, and NLRP12, are involved in the pathogenesis of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE), immunosuppressant-responsive enteropathy (IRE), and non-responsive enteropathy (NRE). Thus, we measured mRNA expression of NLRP3, NLRP6, and NLRP12 in the intestinal mucosa of 35 dogs with CE (ARE, four dogs; FRE, 11 dogs; IRE and NRE, 20 dogs) and seven healthy dogs. As per real-time PCR analysis, significant increases in mRNA expression of NLRP3 and NLRP12 were noted in the colonic but not in the duodenal mucosa of dogs with FRE compared to healthy dogs. These findings suggested that the NLRP3 and NLRP12 inflammasomes might contribute to the development of colitis in dogs with FRE.

Successful outcome after a single endoscopic fecal microbiota transplantation in a Shiba dog with non-responsive enteropathy during the treatment with chlorambucil.

A 7-year 6-month-old, castrated male Shiba dog presented with a 1-month history of lethargy, anorexia, vomiting, and frequent watery diarrhea. Weight loss, hypoalbuminemia, anemia, and leukocytosis were detected at the first visit. The dog was diagnosed with non-responsive enteropathy (NRE) based on clinical and histopathological examinations. Since the dog did not respond to the immunosuppressive drugs, fecal microbiota transplantation (FMT) was performed during the treatment with chlorambucil. A single endoscopic FMT into the cecum and colon drastically recovered clinical signs and clinicopathological abnormalities and corrected dysbiosis in the dog. No recurrence or adverse events were observed. The present case report suggests that FMT, possibly together with chlorambucil, might be a treatment option for NRE in Shiba dogs that have poorer prognosis compared with other dog breeds.

Presence of the house dust mite allergen in the gastrointestinal tract of dogs with chronic enteropathy: A potential inducer of interleukin-1ß.

House dust mite (HDM) is an environmental allergen ubiquitously present indoors, causing allergic inflammation in dogs. However, it is unclear whether HDM allergens can be detected in the gastrointestinal (GI) tract of dogs. In addition, although expression of interleukin (IL)-1ß is increased in the intestinal mucosa of dogs with chronic enteropathy (CE), the role of HDM allergens in the production of IL-1ß has not been evaluated. The objectives of this study were to determine the presence of HDM allergens in the GI tract of dogs and to elucidate the effect of HDM on IL-1ß expression in canine macrophages. HDM allergen, Dermatophagoides pteronyssinus (Der p) 1, was quantified in the gastric and duodenal fluids and the duodenal and colonic mucosae of dogs with CE and healthy laboratory dogs, and faeces of dogs with CE, healthy laboratory dogs and healthy client-owned dogs. Gene expression and protein levels of IL-1ß were measured in HDM-stimulated canine peripheral macrophages from healthy laboratory dogs. Der p 1 was detected in the gastric and duodenal fluids of dogs with CE and healthy laboratory dogs, and faeces of all dogs examined. Der p 1 levels in the duodenal and colonic mucosae were significantly higher in dogs with CE than in healthy laboratory dogs. HDM increased both gene expression and protein levels of IL-1ß in canine macrophages. These findings demonstrate the presence of HDM allergens in the GI tract of dogs and the possible involvement of HDM allergens in the pathogenesis of CE by promoting IL-1ß expression in macrophages.

Effects of a selective casein kinase 1δ and ε inhibitor on FcεRI expression and IgE-mediated immediate-type cutaneous reactions in dogs.

The molecular clock network in mast cells has been shown to be a factor responsible for circadian regulation of allergic inflammation. PF670462 is a selective inhibitor of casein kinase 1δ and ε (CK1δ/ε) that control the posttranslational modification of clock proteins. The aims of this study were to evaluate the effects of PF670462 on gene and protein expression of FcεRI, the high-affinity IgE receptor, in canine mast cells and on IgE-mediated immediate-type cutaneous reactions in dogs. PF670462 decreased mRNA expression of FcεRIα and ß, but not γ, and protein expression of FcεRI in a canine mast cell line. Furthermore, PF670462 suppressed IgE-mediated immediate-type cutaneous erythema in dogs. These findings indicate that CK1δ/ε function as regulators for FcεRI expression and IgE-mediated cutaneous reactions in dogs.

Oral faecal microbiota transplantation for the treatment of Clostridium difficile-associated diarrhoea in a dog: a case report.

BACKGROUND: Successful clinical outcomes of faecal microbiota transplantation (FMT) for recurrent Clostridium difficile infection have been reported in humans and a marmoset. However, it has been unclear whether oral FMT was effective for the treatment of C. difficile-associated diarrhoea in dogs. CASE PRESENTATION: An 8-month-old, intact male French bulldog was presented with a 4-month history of intermittent large bowel diarrhoea. Physical and clinical examinations did not identify any specific causes for diarrhoea. Real-time PCR analysis and immunochromatography detected C. difficile antigen and toxin A&B genes and proteins in a faecal sample. Based on these findings, diarrhoea in the dog was considered to be induced by C. difficile-associated colitis. The dog was treated with oral FMT, in which a faecal solution obtained from a healthy beagle was orally administered to the subject. Stool consistency and frequency and faecal blood and mucus became normal 2-3 days after oral FMT, and real-time PCR analysis and immunochromatography was negative for C. difficile antigen and toxin A&B genes and proteins. No adverse events were observed. CONCLUSION: The present case report demonstrated that oral FMT was an effective treatment for C. difficile-associated diarrhoea in a dog. The findings in this report provide a rationale to evaluate clinical efficacy of oral FMT for other gastrointestinal diseases in dogs.