IL‐4 downregulates expression of the target receptor CD30 in neoplastic canine mast cells

CD30 is a novel therapeutic target in human mast cell (MC) neoplasms. In this ‘comparative oncology’ study, we examined CD30 expression and regulation in neoplastic canine MC using a panel of immunomodulatory cytokines [interleukin‐2 (IL‐2), IL‐4, IL‐5, IL‐6, IL‐13 and stem cell factor (SCF)] and the canine mastocytoma cell lines NI‐1 and C2. Of all cytokines tested IL‐4 was found to downregulate expression of CD30 in NI‐1 and C2 cells. We also found that the CD30‐targeting antibody‐conjugate brentuximab vedotin induces growth inhibition and apoptosis in both MC lines. Next, we asked whether IL‐4‐induced downregulation of CD30 interferes with brentuximab vedotin‐effects. Indeed, pre‐incubation of NI‐1 cells with IL‐4 decreased responsiveness towards brentuximab vedotin. To overcome IL‐4‐mediated resistance, we applied drug combinations and found that brentuximab vedotin synergizes with the Kit‐targeting drugs masitinib and PKC412 in inhibiting growth of NI‐1 and C2 cells. In summary, CD30 is a new marker and IL‐4‐regulated target in neoplastic canine MC.     

FC‐33 Expression of IL‐12 receptor β2 gene in atopic dogs

Atopic dermatitis (AD) is very common in dogs, but its pathogenesis is not yet fully understood. It has been suggested that a Th2‐dominant status may be associated with the occurrence of canine AD. IL‐12 is thought to be important for the differentiation of Th1 cells. The IL‐12 receptor β2 (IL‐12Rβ2) gene is considered to play a critical role in signal transduction and is attracting attention as one of the causative genes of AD in humans. The purpose of this study was to investigate the relationship between IL‐12Rβ2 gene expression and canine AD. The canine IL‐12Rβ2 gene was cloned by RT‐PCR and its nucleotide sequences were determined. Canine IL‐12Rβ2 showed 76.8% homology at the amino acid level with human IL‐12Rβ2, and its structural motifs were well conserved. cDNA with a 91 bp deletion including the transmembrane region was also cloned, which consequently produced a frame shift and an early stop codon. The deletion region corresponded to exon 14 of the human IL‐12Rβ2 gene on chromosome 1. The expression of deleted canine IL‐12Rβ2 mRNA in phytohemagglutinin‐stimulated peripheral blood mononuclear cells was examined in seven healthy dogs and 11 AD dogs. Both deleted and intact mRNAs were expressed at constant ratios in healthy and AD dogs. The results indicate that the deletion of the transmembrane region is not associated with the occurrence of AD, and that the expression of the deleted mRNA may be constitutive and produced by alternative splicing. Funding: Self‐funded.     

PD‐1 expression by canine T cells and functional effects of PD‐1 blockade

The co‐inhibitory checkpoint molecule programmed death receptor 1 (PD‐1) can trigger T cell functional exhaustion upon binding to its ligand PD‐L1 expressed on tumour cells or macrophages. PD‐1 blocking antibodies have generated remarkable results in human cancer patients, including inducing durable responses in a number of advanced cancers. Therefore, monoclonal antibodies specific for canine PD‐1 were assessed for T cell binding and induction of functional activation. A total of 5–10% of CD4 T cells and 20–25% of CD8 T cells from healthy dogs expressed PD‐1, and PD‐1 expression was upregulated on T cells from dogs with cancer. Functionally, PD‐1 antibodies significantly enhanced T‐cell activation, as assessed by proliferation and interferon‐gamma (IFN‐γ) production. PD‐1 antibodies also reversed T‐cell suppression induced by canine soluble PD‐L1 and by tumour cells and tumour explant fragments. These findings indicate that PD‐1 antibodies have potential for use in cancer immunotherapy in dogs.     

In vitro anti‐tubulin effects of mebendazole and fenbendazole on canine glioma cells

Benzimidazole anthelmintics have reported anti‐neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC₅₀) (±SD) obtained from performing the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay after treating J3T, G06‐A, and SDT‐3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550 ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC₅₀ showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.     

Identification of neoplastic cells in blood using the Sysmex XT‐2000iV: a preliminary step in the diagnosis of canine leukemia

Background: Classification of leukemias requires specialized diagnostic techniques. Automated preliminary indicators of neoplastic cells in blood would expedite selection of appropriate tests. Objective: The objective of this study was to assess the capacity of the Sysmex XT‐2000iV hematology analyzer to identify neoplastic cells in canine blood samples. Methods: Blood samples (n=160) were grouped into 5 categories: acute leukemia (n=30), chronic leukemia (n=15), neoplasia without blood involvement (n=41), non‐neoplastic reactive conditions (n=31), and healthy dogs (n=43). WBC counts, WBC flags, scattergrams, percentages of cells with high fluorescence intensity, and percentages of cells in the lysis‐resistant region were evaluated alone or in combination to establish a “leukemic flag.” Sensitivity, specificity, negative (LR?) and positive (LR+) likelihood ratios, and the number of false‐negative (FN) and false‐positive (FP) results were calculated, and receiver operating characteristic curves were designed for numerical values. Results: Among single measurements and parameters, only the evaluation of scattergrams minimized FN and FP results (sensitivity 100%, specificity 94.8%, LR+ 19.17, and LR? 0.00), although their interpretation was subjective. The more objective approach based on the generation of a “leukemic flag” had a sensitivity of 100%, specificity of 87.0%, LR? of 0.00, and LR+ of 7.67. Conclusion: Using a novel gating strategy the Sysmex XT‐2000iV may be used effectively to screen canine blood for hematopoietic neoplasia.     

VEGFR‐2 expression in malignant tumours of the canine mammary gland: a prospective survival study

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is the main receptor activated by vascular endothelial growth factor ‐A (VEGF‐A) to promote tumour angiogenesis. Its clinical prognostic value has not been studied in canine mammary tumours (CMTs). Dogs with mammary cancer were enrolled in a survival study and the immunohistochemical expressions of VEGFR‐2 and VEGF‐A were analysed and associated with clinicopathological features. VEGFR‐2 expression was associated with VEGF immunoreactivity in cancer cells, supporting the presence of an autocrine loop that may be involved in CMTs growth and survival. VEGFR‐2 was also expressed by endothelial cells from tumour vasculature and positively associated with stromal matrix metalloproteinase‐9 (MMP‐9), suggesting the existence of a link between endothelial cells activation and up‐regulation of matrix degrading proteins. Carcinosarcomas exhibited high VEGFR‐2 expression suggesting that it may be one of the activated molecular pathways in this aggressive histological type and that VEGFR‐2 inhibitors may constitute a potential treatment to improve the prognosis of these patients. Both VEGF and VEGFR‐2 immunoreactivities were independent of patients' overall survival (OS) and disease‐free survival (DFS).     

The preliminary study on the effects of growth hormone and insulin‐like growth factor‐I on κ‐casein synthesis in bovine mammary epithelial cells in vitro

The effects of growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) on protein synthesis and gene expression of κ‐casein in bovine mammary epithelial cell in vitro were studied. The treatments were designed as follows: the growth medium without serum was set as the control group, while the treatments were medium supplemented with GH (100 ng/ml), IGF‐I (100 ng/ml), and GH (100 ng/ml) + IGF‐I (100 ng/ml). The quantity of κ‐casein protein was measured by ELISA, and the κ‐casein gene (CSN3) expression was examined by real‐time quantitative PCR (RT‐qPCR). Compared with the control group, all the experimental groups had greater (p < 0.05) expression of CSN3. The concentration of κ‐casein followed a similar response as CSN3, but the difference between the treatments and the control was not statistically significant (p > 0.05). Furthermore, no synergistic effect of GH and IGF‐I was observed for both the κ‐casein concentration and CSN3 expression. It is therefore concluded that GH or IGF‐I can independently promote the expression of CSN3 in bovine mammary epithelial cells in vitro.     

Species specificity in the adherence of staphylococci to canine and human corneocytes: a preliminary study

Staphylococcal pyoderma is rarely contagious between dogs and humans, or humans and dogs. This study investigated the hypothesis that there are species differences in adherence of Staphylococcus intermedius (the most common isolate from dogs) and Staphylococcus aureus (the most common isolate from humans) to canine and human corneocytes. Sheets of corneocytes were collected from the ventral abdomen of 10 dogs and the medial forearm of 10 humans (all healthy and without any history or physical signs of skin disease) using double-sided tape. Staphylococcus intermedius from a case of canine bacterial pyoderma and a human strain of S. aureus were prepared in phosphate buffered saline (PBS) and applied in duplicate, respectively, to the canine and human corneocyte-covered tapes using PBS as negative control. After incubation, rinsing, and staining with crystal violet, quantification of the adherent bacteria was carried out blindly by computerized image analysis. Staphylococcus intermedius was found to adhere significantly more to canine corneocytes than S. aureus (P = 0.0006), whereas S. aureus showed greater adherence to human corneocytes than S. intermedius (P < 0.0001). In addition, the pattern of adherence differed between the two organisms, with S. intermedius adhering to the entire surface and S. aureus adhering mainly to the periphery of both canine and human corneocytes. Preference for adherence to these two hosts may explain, in part, why S. intermedius and S. aureus are uncommonly isolated from human and canine skin infections, respectively.     

The effect of a topical antioxidant formulation including N-acetyl carnosine on canine cataract: a preliminary study

OBJECTIVE: To determine the efficacy of a topical antioxidant formulation including N-acetyl carnosine in the treatment of canine cataract in a preliminary nonplacebo, controlled, unmasked study. ANIMALS STUDIED: Thirty dogs of varying breeds and ages with a spectrum of lens opacities ranging from nuclear sclerosis to total mature cataract. METHODS: Dogs were treated three times daily with topical 2% N-acetyl carnosine in a buffered vehicle containing the antioxidants glutathione, cysteine ascorbate, L-taurine and riboflavin (Ocluvet, Practivet, Phoenix, AZ, USA). Dogs were examined prior to treatment and at 2, 4 and 8 weeks during treatment, by direct and indirect ophthalmoscopy and slit-lamp biomicroscopy after pharmacologic pupil dilation. Photographic documentation of lens opacity was achieved by retroillumination photography, with three photographs taken at each examination time-point. A lens opacification index (LOI), determined by integration of the grayscale level of each pixel across the image, was evaluated by computerized image analysis of digitized images. Alteration in mean LOI was determined for each animal, having normalized the initial LOI. RESULTS: Fifty-eight eyes of 30 dogs were evaluated, 22 with mature cataract, 13 with immature cataract, 9 with cataract associated with other intraocular disease such as uveitis and 14 with nuclear sclerosis alone. One dog was unilaterally anophthalmic after previous enucleation and one had a phthytic eye after previous uveitis-induced glaucoma. Image analysis showed a reduction in mean LOI in all cataract groups (mean resolution in opacity of 2.3 +/- 0.33% for all cataracts), although this was only statistically significant in those eyes with immature cataract (mean resolution of opacity 4.5 +/- 0.33%) or nuclear sclerosis (mean decrease in opacity 5 +/- 0.37%). Reduction in lens opacity was seen in eyes with mature cataract (0.5 +/- 0.4%) and in miscellaneous cataract associated with intraocular inflammation (1.3 +/- 0.4%), but these changes were not statistically significant. Owner evaluation of visual capability, however, suggested improvement in vision in 80% of cases by the end of the study. CONCLUSIONS: This study demonstrates some marginal reduction in lens opacification in a substantial number of cases of canine cataract with the use of a topical nutritional antioxidant formulation including N-acetyl carnosine. Lens opacification was improved with treatment in eyes with immature cataract or nuclear sclerosis while in eyes with mature cataract or cataract with associated intraocular inflammatory pathology less reduction was seen.     

Comparative morphometric analysis of recurrent and nonrecurrent canine basal cell carcinomas: a preliminary report

Background: Most reports of canine basal cell carcinomas (BCCs) focus on morphologic appearance rather than biologic behavior. Rare recurrences and no metastases have been reported in dogs. Quantitative nuclear morphometry may be useful in predicting tumor recurrence. Objective: The aim of the present study was to compare quantitative nuclear parameters of canine BCCs that did not recur within 60 weeks of excision with those that recurred. Methods: Cytologic specimens obtained from spontaneous BCCs from 11 dogs were analyzed by computerized nuclear morphometry. The dogs were monitored by their owners over a period of 60 weeks to detect local recurrence of the tumor; recurrent tumors were confirmed histologically. For each initial tumor specimen, nuclei of at least 100 neoplastic cells were measured by 2 independent observers, and mean nuclear area (MNA), mean nuclear perimeter (MNP), and mean nuclear diameter (MND) were calculated. Results: Six nonrecurrent and 5 recurrent tumors were analyzed. Neoplastic cells from BCCs that subsequently recurred had higher MNA (102.41 ± 4.57 μm²), MNP (36.27 ± 0.61 μm), and MND (11.21 ± 0.27 μm) than cells from nonrecurrent BCCs (MNA 87.66 ± 4.79 μm², MNP 33.51 ± 0.78 μm, MND 10.36 ± 0.29 μm) (P<.001; Mann–Whitney U‐test). Conclusion: Based on these preliminary results, nuclear morphometry may be a useful tool to predict local recurrence of BCCs in dogs.     

Double‐blinded cross‐over study on the efficacy of pentoxifylline for canine atopy

The pathogenesis of canine atopy has not been established completely. Recent studies have shown that tumour necrosis factor alpha and Interleukin‐6 play a role in allergic reactions in humans and mice. Pentoxifylline (PTX) suppresses synthesis of these cytokines and may be a useful therapy for modulating the symptoms of canine atopy. The objectives of this study were to investigate the effects of PTX (10mg kg^?1 twice daily for 4 weeks) on clinical signs (erythema and pruritus) and intradermal skin test reactivity in atopic dogs (n = 10). The study was a double‐blinded, placebo controlled, crossover clinical trial with a washout period of 2 weeks between treatments. Clinical signs were evaluated and scored by the investigator and owners. During PTX treatment, scores of pruritus and erythema decreased significantly. PTX did not affect intradermal skin test reactivity to house dust mite at 15 min (allergen of reference for this study).     

Effect of desmopressin on immune-mediated haemorrhagic disorders due to canine monocytic ehrlichiosis: a preliminary study

To evaluate the possible use of desmopressin acetate (DDAVP) in haemorrhagic disorders consequent to canine monocytic ehrlichiosis (CME), three dogs infected by Ehrlichia canis, with a history of thrombocytopenia and recent bleeding, were studied. The dogs were administered desmopressin (1 μg/kg b.w. s.c.) every 24 h on three occasions. Blood samples were collected immediately before, and after 2 and 48 h the first DDAVP administration, to assess haematological, clinical chemistry and clotting time parameters. Spontaneous bleeding stopped within 1 h after the first DDAVP injection. Buccal mucosa bleeding time (BMBT) was shortened from 9.6 to 2.3 min within 2 h after the treatment. A statistically significant increase in platelet PLT count and fibrinogen, and a statistically significant decrease of PT and aPTT were observed after DDAVP administration. The haemorrhagic disorders caused by CME appear to be quickly corrected by DDAVP administration, giving the clinician the time necessary to administer appropriate chemotherapy.     

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild‐type p53

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.     

Expression and functionality of TRPV1 receptor in human MCF‐7 and canine CF.41 cells

As canine mammary tumours (CMT) and human breast cancer share clinical and prognostic features, the former have been proposed as a model to study carcinogenesis and improved therapeutic treatment in human breast cancer. In recent years, it has been shown that transient receptor potential vanilloid 1 (TRPV1) is expressed in different neoplastic tissues and its activation has been associated with regulation of cancer growth and progression. The aim of the present research was to demonstrate the presence of TRPV1 in human and canine mammary cancer cells, MCF‐7 and CF.41, respectively, and to study the role of TRPV1 in regulating cell proliferation. The images obtained by Western blot showed a signal at 100 kDa corresponding to the molecular weight of TRPV1 receptor. All tested TRPV1 agonists and antagonists caused a significant decrease (P < 0.05) of cell growth rate in MCF‐7 cells. By contrast, in CF.41 cells capsaicin and capsazepine induced a significant increase (P < 0.05) in cell proliferation, whereas resiniferatoxin (RTX) and 5‐iodo‐resiniferatoxin (5‐I‐RTX) had no influence on CF.41 cell proliferation. Further studies are needed to elucidate the underlying molecular mechanism responsible for the different effects evoked by TRPV1 activation in MCF‐7 and CF.41 cells.     

Alpha-chloralose poisoning in dogs and cats: a retrospective study of 33 canine and 13 feline confirmed cases

Alpha-chloralose (AC) is an anaesthetic compound also used as a rodenticide, and has dose-dependent central nervous system mixed effects of excitation and depression. The objectives of this study were to detail the clinical and clinicopathological characteristics, as well as the treatment and prognosis, of AC toxicosis in dogs and cats. Medical records were retrospectively reviewed for AC poisoning between the years 1989 and 2004, and 33 dogs and 13 cats were included in the study. The most common clinical signs were seizures, muscle tremor, hyperaesthesia, hypothermia, salivation, myosis, stupor, coma and ataxia. Coma was more common, while salivation and ataxia were less common in cats compared to dogs. Although hypothermia was very common, especially in cats (90.9%), hyperthermia was frequently observed in dogs (21%). Treatment in all patients was supportive and symptomatic, and the most commonly used anticonvulsants were diazepam and barbiturates; however, severe unresponsive seizures in three dogs had to be controlled with inhalant gas anaesthesia. The hospitalisation period was 1-3 days, and the overall mortality rate was 6.5%. Alpha-chloralose poisoning seems to have a favourable prognosis in dogs and cats.