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.

Toceranib phosphate and firocoxib-mediated partial response in a dog with advanced intranasal sarcoma.

A nine-year-old, castrated male mixed-breed dog presented with a three-month history of sneezing and stertorous breathing. Computed tomography revealed a soft tissue mass in the left nasal cavity with lysis of the cribriform plate. The mass was diagnosed as intranasal sarcoma based on histopathological analysis. The tumor cells were immunohistochemically positive for KIT and platelet-derived growth factor receptor α/ß and negative for vascular endothelial growth factor receptor 2 and cyclooxygenase-2. Treatment with toceranib phosphate (TOC) and firocoxib reduced the tumor size, which was defined as partial response (PR). After PR induction, TOC alone mediated survival for 205 days. This case report suggests that the combination of TOC and possibly firocoxib may be a therapeutic option for canine intranasal sarcoma.

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.

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.

Efficacy of Juzen-taiho-to against vincristine-induced toxicity in dogs.

Vincristine, one of the anti-cancer drugs used in veterinary practice, has adverse hematological and gastrointestinal effects in dogs. Juzen-taiho-to is a traditional Chinese medicine used for patients with anorexia in human medicine. However, the protective effects of Juzen-taiho-to against anti-cancer drug-induced toxicity in dogs have not been investigated. We therefore examined whether the administration of Juzen-taiho-to to dogs affects gastric motility, and vincristine-induced gastrointestinal and hematological toxicity. The study was composed of three trials. In the first trial, Juzen-taiho-to (450 mg/kg/day) was orally administered to five dogs. In the second and third trials, vincristine (0.75 mg/m2) was intravenously administered to each dog in the absence or presence of Juzen-taiho-to (450 mg/kg/day). During these trials, gastric motility and blood parameters were assessed. Juzen-taiho-to increased gastric motility and improved vincristine-induced gastrointestinal, but not hematological, adverse effects in dogs. This study suggested that Juzen-taiho-to may be applicable for gastrointestinal care in dogs receiving chemotherapy.

Clinical and pathological features and outcome of bilateral incidental adrenocortical carcinomas in a dog.

A 9-year-old, spayed female Chihuahua was presented with a 1-week history of lethargy and anorexia. Abdominal ultrasonography and computed tomography found bilateral adrenal masses without metastasis. Serum cortisol levels that were sampled before and after an adrenocorticotropic hormone stimulation test were within reference ranges. Lethargy and anorexia completely resolved after short-term fluid therapy; the clinical signs did not occur for approximately 8 months until her sudden death. A postmortem examination revealed bilateral adrenocortical carcinomas and liver metastasis. Primary adrenocortical carcinomas developed in the dog met the definition of bilateral incidental adrenal gland masses (IAGMs). This is the first case report to demonstrate based on histological identification that adrenocortical carcinomas cause bilateral IAGMs in dogs.

Supplementation of the fermented soy product ImmuBalance™ effectively reduces itching behavior of atopic NC/Tnd mice.

BACKGROUND: Effects of probiotics on the prevention of atopic diseases have been proposed recently. Although we have already reported the suppressive effects of the probiotic, ImmuBalance™, on a mouse model for peanuts allergy, its influence on atopic diseases remains unclear. OBJECTIVE: Potential efficacy of ImmuBalance™, which is the fermented soy product, on treatment of atopic dermatitis (AD) was investigated using a mouse model for human AD, NC/Tnd mice. METHODS: For in vivo study, ImmuBalance containing chow or a control diet were fed to NC/Tnd mice with moderate dermatitis for 2 weeks. Topical application of FK506 ointment was used as a positive control. Clinical skin severity scores, scratching behaviors, trans-epidermal water loss (TEWL), and histological features were analyzed. For in vitro study, suppressive effect of ImmuBalance™ on nerve growth factor (NGF)-activated neurite outgrowth of PC12 cells was examined. RESULTS: Clinical skin severity scores of the mice fed with ImmuBalance containing chow were gradually reduced as well as the mice treated with FK506. Feeding with ImmuBalance completely inhibited the increase in scratching behavior of NC/Tnd mice. The value of TEWL of NC/Tnd mice fed with ImmuBalance was significantly decreased. In addition, histological examination revealed that application of ImmuBalance decreased the number of PGP9.5-positive neuronal fibers in the lesional skin. When ImmuBalance extract was added to the culture, NGF-activated neurite outgrowth of PC12 cells was diminished through the inhibition of the phosphatidylinositol 3-kinase phosphorylation. CONCLUSION: ImmuBalance could exhibit favorable alterations on AD symptoms, particularly through down regulation of the itch sensation.

Glucocorticoid sensitivity depends on expression levels of glucocorticoid receptors in canine neoplastic mast cells.

Glucocorticoid (GC) administration with or without other chemotherapeutic reagents is a commonly used option in the treatment of mast cell malignancies. However, the responsiveness of mast cell tumors to GC treatment varies in individuals, and the regulatory mechanisms determining the GC sensitivity of malignant mast cells remain unclear. Since the expression of the GC receptor (GR) has been reported to be associated with GC sensitivity in human neoplastic lymphocytes, we attempted to investigate the relationship between GR levels and GC sensitivity by using neoplastic mast cells derived from canine mast cell tumors (MCTs). To elucidate the regulatory mechanisms involved in GC responsiveness, we analyzed various canine MCT cell lines and tissue samples from dogs with MCT. While the proliferation of canine MCT cells was suppressed by the addition of GC to the culture, we found that MCT cells derived from humans and rodents, as well as canine lymphoma cells, responded poorly to GC. However, there were also some variations in responsiveness to GC treatment among canine MCT cell lines used in this study. Using real-time polymerase chain reaction and Western blot analysis, we elucidated the relationship between GR expression and responsiveness to GC in canine MCT cells. Furthermore, to assess the involvement of GR expression in GC sensitivity in vivo, clinical investigations were conducted on dogs with cutaneous MCT. Written informed consent was obtained from owners, and the affected dogs were treated with prednisolone (0.5-2.0 mg kg(-1)day(-1), administered orally) 1 or 2 weeks prior to the surgical removal of the tumors. Tumor volume was measured according to WHO criteria both before and after prednisolone treatment, and the GC sensitivity of each MCT was determined on the basis of the reduction in tumor volume. Of the 15 dogs with MCT, 11 responded to treatment with prednisolone completely or partially, whereas 4 dogs showed no response. Examination of clinical samples obtained by surgical removal revealed that GR expression levels were significantly lower in GC-resistant MCT tissues than in GC-sensitive MCT tissues. Thus, these results strongly indicate that GR expression may contribute to GC sensitivity in canine MCT.

Silencing of int6 gene restores function of the ischaemic hindlimb in a rat model of peripheral arterial disease.

AIMS: Intermittent claudication (IC) is one of the serious symptoms of peripheral arterial disease (PAD) and is characterized by pain in the legs or buttocks that worsens with exercise and subsides with rest. The concept of 'therapeutic angiogenesis' for PAD has been widely proposed; however, the methodology, including cell transplantation, is still unclear. In this study, we examined the clinical efficacy of silencing the int6 gene, which encodes a protein that stabilizes hypoxia-inducible factor (HIF)-2α, on angiogenesis in PAD. METHODS AND RESULTS: An animal model for IC was established in Sprague-Dawley rats by external iliac artery ligation and evaluated by quantitative analysis of gait disturbance. Next, we explored the therapeutic effects of int6 siRNA injected into the adductor magnus muscle on IC. Recovery of hindlimb function occurred in the early stages after int6 siRNA injection. The number of blood vessels showed an obvious increase in the int6 siRNA-treated muscles. Angiography revealed the recovery of peripheral circulation at the affected sites. Early up-regulation of HIF-2α and other angiogenic factors, including basic fibroblast growth factor and hepatocyte growth factor, was also apparent in the int6 siRNA-treated sites. We also confirmed the up-regulation of HIF-2α and its translocation to the nucleus in the int6 siRNA-injected muscle. CONCLUSION: A single injection of int6 siRNA promoted angiogenesis via up-regulation of HIF-2α-related angiogenic factors in the muscles of the affected hindlimb and reduced gait disturbance. The int6 gene may be a novel therapeutic target for the treatment of IC in patients with PAD.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is critical for interleukin-4 expression in response to FcepsilonRI receptor cross-linking.

Basophils mediate many of their biological functions by producing IL-4. However, it is unknown how the Il4 gene is regulated in basophils. Here, we report that CCAAT/enhancer-binding protein α (C/EBPα), a major myeloid transcription factor, was highly expressed in basophils. We show that C/EBPα selectively activated Il4 promoter-luciferase reporter gene transcription in response to IgE cross-linking, but C/EBPα did not activate other known Th2 or mast cell enhancers. We found that the PI3K pathway and calcineurin were essential in C/EBPα-driven Il4 promoter-luciferase gene transcription. Our mutation analyses revealed that C/EBPα drove Il4 promoter-luciferase activity depending on its DNA binding domain. Mutation of the C/EBPα-binding site in the Il4 promoter region abolished C/EBPα-driven Il4 promoter-luciferase activity. Our results further showed that a mutation in nuclear factor of activated T cells (NFAT)-binding sites in the Il4 promoter also negated C/EBPα-driven Il4 promoter-luciferase activity. Our study demonstrates that C/EBPα, in cooperation with NFAT, directly regulates Il4 gene transcription.

Cultivation and characterization of canine skin-derived mast cells.

It is essential to develop a technique to culture purified skin-derived mast cells (SMCs) to facilitate immunological research on allergic diseases in dogs. This study was performed to develop an efficient culture system for canine SMCs and to characterize the cells in comparison to canine bone marrow-derived mast cells (BMMCs). Enzymatically digested skin biopsy samples were cultivated in serum-free AIM-V medium supplemented with recombinant canine stem cell factor. Three to five weeks after the initiation of culture, mast cells were collected by a magnetic activated cell separation system using anti-c-Kit antibody. The collected cells were composed of a uniform population showing morphological characteristics of mast cells with a round or oval nucleus and abundant toluidine blue-positive metachromatic granules in the cytoplasm. The results of flow cytometric analysis for the presence of cell membrane c-Kit and Fc epsilon receptor I (FcepsilonRI) indicated that approximately 90% of the cells were mast cells. The cytoplasmic granules were positive for both tryptase and chymase. Apparent dose-dependent degranulation was induced by antibody-mediated cross-linking of immunoglobulin E (IgE) bound to the cells. These cytological and immunological characteristics observed in SMCs were mostly similar to those observed in BMMCs; however, IgE-mediated degranulation was significantly lower in SMCs than BMMCs. The culture system for canine SMCs developed in this study would be useful in understanding the pathophysiology and developing anti-allergic therapeutics in canine allergic dermatitis.

IL-3 induces basophil expansion in vivo by directing granulocyte-monocyte progenitors to differentiate into basophil lineage-restricted progenitors in the bone marrow and by increasing the number of basophil/mast cell progenitors in the spleen.

Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in approximately 4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3R alpha-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen.

Identification of c-kit mutations-independent neoplastic cell proliferation of canine mast cells.

Gain-of-function mutations in the proto-oncogene c-kit have been considered the molecular mechanism of neoplastic proliferation of mast cells. However, the importance of c-kit gene mutations is not well evaluated in canine mast cell tumors (MCTs). In the present study, we established and characterized a mast cell line, HRMC, derived from a dog with MCT. We also examined c-kit mutations in HRMC cells and assessed an inhibitory effect of a tyrosine kinase inhibitor, STI571, on HRMC cells. HRMC cells had cytoplasmic metachromatic granules, chymase and tryptase, and expressed both KIT and FcepsilonRI on the cell surface. HRMC cells contained histamine and released beta-hexosaminidase through FcepsilonRI cross-linking and calcium ionophore stimulation. Nucleotide sequence analysis demonstrated no mutations in an open reading frame of c-kit cDNA and genomic DNA of the juxtamembrane domain of c-kit in HRMC cells. STI571 did not show any inhibitory effects on the proliferation of HRMC cells. These findings clearly demonstrated the existence of c-kit mutations-independent neoplastic canine mast cell proliferation. The growth factor-independent mast cell line established in this study might be valuable to explore novel mechanisms of c-kit mutations-independent neoplastic proliferation of mast cells in dogs.

Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors.

Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (FcepsilonRI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with FcepsilonRI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders.

The initial response of CD4+ IL-4-producing cells.

Naive CD4(+) T cells were reported to produce small amounts of IL-4 in vitro, which are implicated to be sufficient to initiate T(h)2 response in vivo. However, IL-4-producing naive CD4(+) T cells are difficult to study in vivo because they are present in low numbers shortly after the first antigen exposure. Here, we used IL-4/green fluorescence protein (GFP) reporter mice (G4 mice) to track the initial response of CD4(+) IL-4-producing cells. We first established a flow cytometry method to estimate the number of GFP(+) cells. We demonstrated the effectiveness of this method by showing that the responding CD4(+)GFP(+) cells exhibited an activated phenotype, possessed the capacity to express IL-5 and IL-13, but not IFN-gamma mRNA, and showed enhanced levels of GATA3 and c-maf mRNA expression. More importantly, we showed that the cell lines derived from FACS-sorted CD4(+)GFP(+) cells were antigen specific. By using this newly established method, we showed that the majority of responding GFP(+) cells were CD4(+) T cells. Our study provides direct ex vivo evidence to show that a small percent of CD4(+) T cells that have no previous experience of antigenic stimulation might produce IL-4 to initiate T(h)2 response.