Erratum to: Evaluation of HSP70, 27 and TNF-α expression in different canine cutaneous and gland diseases

Veterinary Research Communications -     

Characterization of β-catenin expression in canine osteosarcoma

Osteosarcoma (OSA) is the most frequently occurring malignant primary bone tumour in dogs and children and arises from cells of the osteoblast lineage. Inappropriate Wnt signalling activity has been implicated in human OSA. Altered expression of β-catenin, an integral member of the Wnt signalling pathway, has been associated with numerous human cancers, including OSA. In this study, 30 of the 37 primary canine OSA tissues and 2 of the 3 metastatic OSAs were positive for β-catenin expression as determined by immunohistochemistry, whereas 2 normal bones stained negative for β-catenin. No mutations were identified in exon 3 of β-catenin in the three OSA cases in which DNA sequencing was performed. Finally, there was no relationship between β-catenin expression and overall survival time or disease-free interval. Our results indicate β-catenin is frequently expressed within the cytoplasm of neoplastic cells in canine OSA but contains no detectable mutations in exon 3, similar to human OSA.     

Hypoxia inducible factor 1α expression and effects of its inhibitors in canine lymphoma

Hypoxic conditions in various cancers are believed to relate with their malignancy, and hypoxia inducible factor-1α (HIF-1α) has been shown to be a major regulator of the response to low oxygen. In this study, we examined HIF-1α expression in canine lymphoma using cell lines and clinical samples and found that these cells expressed HIF-1α. Moreover, the HIF-1α inhibitors, echinomycin, YC-1 and 2-methoxyestradiol, suppressed the proliferation of canine lymphoma cell lines. In a xenograft model using NOD/scid mice, echinomycin treatment resulted in a dose-dependent regression of the tumor. Our results suggest that HIF-1α contributes to the proliferation and/or survival of canine lymphoma cells. Therefore, HIF-1α inhibitors may be potential agents to treat canine lymphoma.     

Cloning of Inhibin-α Gene and its mRNA Expression Pattern in the Ovary of Congjiang Xiang Pig

To reveal the relationship between inhibin-α(INHA) gene and the reproductive traits of Congjiang Xiang pig, INHA gene was cloned and sequenced taking the genomic DNA of Congjiang Xiang pigs as templates by polymerase chain reaction(PCR) method.The polymorphisms of INHA gene were tested in Congjiang Xiang pig populations with high-litter size and low-litter size using allele-specific PCR(AS-PCR) method.The expression profile of INHA gene in ovaries was detected from Congjiang Xiang pigs with high-litter yiled or low-litter yiled by Real-time PCR method.The complete coding region of INHA gene was 1095 bp in length, which coded for 364 amino acid residues.Compared with the known sequence, two candidate sites, G359A and A373G, were found out from exon 2 region of INHA gene in Congjiang Xiang pig.After investigation for the two sites in a large population, the frequency of alleles between two populations was not significant and without obvious relativity with the litter yiled of Congjiang Xiang pig.However, the INHA mRNA level in the ovary of Congjiang Xiang pig with high-litter yiled was higher than that with low-litter yiled.It suggested that INHA gene was much conserved, INHA gene expression level might be concerned for the regulation of ovary growth and follicle development in Congjiang Xiang pig breed.     

Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter. The aims of this study were to achieve high transduction efficiency with transgene expression restricted to canine breast tumor cells. Using EGFR promoter-driven AdRGD, we were able to restrict transgene expression to canine breast tumor cells with no evidence of expression in normal cells. Canine breast tumor cells transduced with EGFR promoter-driven AdRGD carrying canine caspase 3 gene showed cytotoxic activity. We constructed a second AdRGD vector that expressed oxygen-dependent degradation (ODD)-caspase 3 under the control of the EGFR promoter; the fusion protein contains a core part of the ODD domain of hypoxia inducible factor-1 alpha (HIF-1α) fused to caspase 3. Transduction of canine breast tumor cells with EGFR promoter-driven AdRGD expressing ODD-caspase 3 induced a higher rate of cell death under hypoxic conditions compared with under normoxia. The results indicate that the EGFR promoter-driven AdRGD vectors will be of value for tumor-specific transgene expression and safe cancer gene therapy in dogs.     

Abnormal functions of Leydig cells in crossbred cattle–yak showing infertility

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle–yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle–yak spermatogenesis. Testicular tissues from yaks (1–3 years old) and hybrids (F1–F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle–yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle–yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.     

Kinetics of IL-6 and TNF-α changes in a canine model of sepsis induced by endotoxin

Sepsis is a major cause of death in veterinary medicine, although a better prognosis can result from an early diagnosis. To speed the diagnosis, the biomarkers TNF-α and IL-6 can provide valuable information regarding systemic inflammatory response. The purpose of this study was to investigate the changes in cytokine levels in an experimental model of sepsis using ELISA and real-time PCR. Ten adult Beagles were studied; seven received an IV bolus of high dose lipopolysaccharide solution (1mg/kg) to induce sepsis. The remaining three beagles were the control group. Blood samples were collected before and 1, 3, 6, 12, 24 and 48 h after administering LPS. Serum IL-6 level peaked at 3h (1.89 ± 0.10 ng/ml) and serum TNF-α peaked at 1h (1.11 ± 0.01 ng/ml). The expression of IL-6 mRNA in peripheral blood mononuclear cells (PBMC) increased 62-fold compared to the control group at 1h; TNF-α mRNA increased by 4.5-fold at 1h. The expressions of IL-6 and TNF-α mRNA in PBMCs changed more rapidly than serum IL-6 and TNF-α concentrations. In addition, TNF-α mRNA levels in PBMCs remained elevated longer than serum TNF-α. Our study establishes the basis for future work aimed at a better understanding of the systemic inflammatory response to infection and sepsis in canine patients.     

11-Hydroxy-β-steroid dehydrogenase gene expression in canine adipose tissue and adipocytes: stimulation by lipopolysaccharide and tumor necrosis factor α

The enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD-1) is expressed in a number of tissues in rodents and humans and is responsible for the reactivation of inert cortisone into cortisol. Its gene expression and activity are increased in white adipose tissue (WAT) from obese humans and may contribute to the adverse metabolic consequences of obesity and the metabolic syndrome. The extent to which 11β-HSD-1 contributes to adipose tissue function in dogs is unknown; the aim of the present study was to examine 11β-HSD-1 gene expression and its regulation by proinflammatory and anti-inflammatory agents in canine adipocytes. Real-time PCR was used to examine the expression of 11β-HSD-1 in canine adipose tissue and canine adipocytes differentiated in culture. The mRNA encoding 11β-HSD-1 was identified in all the major WAT depots in dogs and also in liver, kidney, and spleen. Quantification by real-time PCR showed that 11β-HSD-1 mRNA was least in perirenal and falciform depots and greatest in subcutaneous, omental, and gonadal depots. Greater expression was seen in the omental depot in female than in male dogs (P = 0.05). Gene expression for 11β-HSD-1 was also seen in adipocytes, from both subcutaneous and visceral depots, differentiated in culture; expression was evident throughout differentiation but was generally greatest in preadipocytes and during early differentiation, declining as cells progressed to maturity. The inflammatory mediators lipopolysaccharide and tumor necrosis factor α had a main stimulatory effect on 11β-HSD-1 gene expression in canine subcutaneous adipocytes, but IL-6 had no significant effect. Treatment with dexamethasone resulted in a significant time- and dose-dependent increase in 11β-HSD-1 gene expression, with greatest effects seen at 24 h (2nM: approximately 4-fold; 20nM: approximately 14-fold; P = 0.010 for both). When subcutaneous adipocytes were treated with the peroxisome proliferator activated receptor γ agonist rosiglitazone, similar dose- and time-dependent effects were noted. However, no effects were seen when adipocytes from the gonadal WAT depot were treated with rosiglitazone. The induction of 11β-HSD-1 expression, by the pro-inflammatory cytokine tumor necrosis factor α and by lipopolysaccharide may have implications for the pathogenesis of obesity and its associated diseases in the dog.     

Relationship between NF-κB expression and malignancy of canine mammary gland tumor tissues

In this study, the nuclear expression of nuclear factor kappa B (NF-κB) in 48 tissues specimens from 25 canine spontaneous mammary gland tumor (MGT) patients was assessed by immunohistochemistry to compare their levels with clinical features, histological types, prognostic outcomes and proliferative activities, including the mitotic index (MI) and cylcinD1 expression. Twelve of eighteen (66.7%) malignant tumor tissues showed greater than 10% nuclear staining, while benign tumor and hyperplastic tissues showed less than 10% nuclear staining. Higher nuclear expression of NF-κB was positively correlated with larger tumor size, lymph node metastasis and higher MI; however, no correlation was observed with distant metastasis and cyclin D1 expression. Higher NF-κB nuclear expression correlated with shorter patient survival. These findings suggest that NF-κB is a useful prognostic factor for canine MGT patients.     

Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection

Vaccines to prevent Trypanosoma cruzi infection in humans or animals are not available, and in many settings, dogs are an important source of domestic infection for the insect vector. Identification of infected canines is crucial for evaluating peridomestic transmission dynamics and parasite control strategies. As immune control of T. cruzi infection is dependent on humoral and cell-mediated immune responses, we aimed to define a serodiagnostic assay and T cell phenotypic markers for identifying infected dogs and studying the canine T. cruzi-specific immune response. Plasma samples and peripheral blood mononuclear cells (PBMCs) were obtained from forty-two dogs living in a T. cruzi-endemic region. Twenty dogs were known to be seropositive and nine seronegative by conventional serologic tests two years prior to our study. To determine canine seroreactivity, we tested sera or plasma samples in a multiplex bead array against eleven recombinant T. cruzi proteins. Ninety-four percent (17/18) of dogs positive by multiplex serology were initially positive by conventional serology. The frequency of IFNγ-producing cells in PBMCs responding to T. cruzi correlated to serological status, identifying 95% of multiplex seropositive dogs. Intracellular staining identified CD4⁺ and CD8⁺ T cell populations as the sources of T. cruzi lysate-induced IFNγ. Low expression of CCR7 and CD62L on CD4⁺ and CD8⁺ T cells suggested a predominance of effector/effector memory T cells in seropositive canines. These results are the first, to our knowledge, to correlate T. cruzi-specific antibody responses with T cell responses in naturally infected dogs and validate these methods for identifying dogs exposed to T. cruzi.     

Interactions between TGFβ1 and cyclic strain in modulation of myofibroblastic differentiation of canine mitral valve interstitial cells in 3D culture

ObjectivesThe mechanisms of myxomatous valve degeneration (MVD) are poorly understood. Transforming growth factor-beta1 (TGFβ1) induces myofibroblastic activation in mitral valve interstitial cells (MVIC) in static 2D culture, but the roles of more physiological 3D matrix and cyclic mechanical strain are unclear. In this paper, we test the hypothesis that cyclic strain and TGFβ1 interact to modify MVIC phenotype in 3D culture.Animals, materials and methodsMVIC were isolated from dogs with and without MVD and cultured for 7 days in type 1 collagen hydrogels with and without 5 ng/ml TGFβ1. MVIC with MVD were subjected to 15% cyclic equibiaxial strain with static cultures serving as controls. Myofibroblastic phenotype was assessed via 3D matrix compaction, cell morphology, and expression of myofibroblastic (TGFβ3, alpha-smooth muscle actin – αSMA) and fibroblastic (vimentin) markers.ResultsExogenous TGFβ1 increased matrix compaction by canine MVIC with and without MVD, which correlated with increased cell spreading and elongation. TGFβ1 increased αSMA and TGFβ3 gene expression, but not vimentin expression, in 15% cyclically stretched MVIC. Conversely, 15% cyclic strain significantly increased vimentin protein and gene expression, but not αSMA or TGFβ3. 15% cyclic strain however was unable to counteract the effects of TGFβ1 stimulation on MVIC.ConclusionsThese results suggest that TGFβ1 induces myofibroblastic differentiation (MVD phenotype) of canine MVIC in 3D culture, while 15% cyclic strain promotes a more fibroblastic phenotype. Mechanical and biochemical interactions likely regulate MVIC phenotype with dose dependence. 3D culture systems can systematically investigate these phenomena and identify their underlying molecular mechanisms.     

Breed-related expression patterns of Ki67, γH2AX,and p21 during ageing in the canine liver

Veterinary Research Communications - Cellular senescence is a molecular hallmark of ageing that is associated with multiple pathologies, and DNA damage marker γH2AX, together with cell cycle...     

A novel NF-κB inhibitor improves glucocorticoid sensitivity of canine neoplastic lymphoid cells by up-regulating expression of glucocorticoid receptors

Lymphoid neoplasms including lymphoma and leukemia are one of the most life-threatening disorders in dogs. Many lymphoid malignancies are well-treated with glucocorticoid (GC); however, GC resistance sometimes develops and its mechanism remains uncertain. Since constitutive activation of nuclear factor-κB (NF-κB) has been reported to play roles in lymphoid malignancies, we examined whether inhibition of NF-κB activity with a synthetic inhibitor IMD-0354 affected GC sensitivity of canine neoplastic lymphoid cells, CL-1 and GL-1. Dexamethasone failed to inhibit proliferation of these cells, in which low expression of glucocorticoid receptors (GR) was identified. In the presence of IMD-0354, GR expressions in CL-1 and GL-1 were increased, consequently dexamethasone inhibited their proliferation. These results indicated that GR expression might be down-regulated by spontaneous activation of NF-κB, resulting in GC resistance. Taken together, interference of NF-κB activity may have the synergistic effect in combination chemotherapy with GC for treatment against lymphoid malignancies.     

Pro-inflammatory and pro-apoptotic responses of TNF-α stimulated bovine mammary endothelial cells

Coliform mastitis may be severe in periparturient cows due to enhanced expression of pro-inflammatory cytokines that contribute to disease pathogenesis. Tumor necrosis factor (TNF)-α is implicated with the severity of coliform mastitis by provoking inflammatory responses in affected tissues. The endothelium is an integral organ in regulating inflammatory responses and loss of endothelial integrity may be fatal. Studies in humans suggest that endothelial cell apoptosis may be a consequence of TNF-α exposure and contributes to the development of sepsis, however, its impact on bovine mammary endothelial cells (BMEC) is unknown. We sought to determine the inflammatory and apoptotic responses of primary BMEC exposed to TNF-α in vitro. Stimulation of endothelial monolayers with TNF-α resulted in significant increase of toll-like receptor 4, interleukin-6 and -8, and intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 gene expression in a time-dependent manner. Caspase-8 and caspase-3 mRNA expression, as well as caspase enzyme activity, also increased significantly following TNF-α stimulation. Cell viability assessed by ATP activity and BMEC apoptosis determined by flow cytometry revealed no significant changes across time with TNF-α stimulation. Results suggest that TNF-α stimulation, at the dose used in this study, can elicit a pro-inflammatory response in BMEC, but not induce apoptosis. The impact of TNF-α on mammary vascular function and the subsequent impact on the pathophysiology of severe coliform mastitis warrant further investigation.