Kv 11.1 Expression Is Associated With Malignancy of Canine Mammary Gland Tumors.

BACKGROUND/AIM: The expression level of the voltage-dependent potassium channel Kv 11.1 was shown to be associated with the clinicopathological features, aggressiveness, and prognosis of human breast cancer. Canine mammary gland tumor (cMGT) is the most common tumor type in intact female dogs; however, the significance of Kv 11.1 in cMGT is unknown. The aim of this study was to identify Kv 11.1 expression in 57 benign and malignant cMGT tissues from dogs and to investigate the correlation of Kv 11.1 expression with the clinicopathological parameters and prognosis of cMGT. MATERIALS AND METHODS: A total of 57 samples were collected from cMGTs surgically resected at the Veterinary Medical Teaching Hospital, Seoul National University and subjected to immunohistochemistry assay using rabbit anti-Kv 11.1 polyclonal antibody. Immunohistochemical staining results were evaluated as the sum of intensity and percentage scores. The correlation between immunohistochemistry scores and clinicopathological parameters was investigated. RESULTS: Immunohistochemical analysis revealed that Kv 11.1 immunoreactivity was higher in benign cMGTs than in malignant cMGTs. Kv 11.1 expression was significantly associated with tumor malignancy (p<0.001), tumor size (p<0.001), histological grade (p<0.05), and age at the time of mastectomy (p<0.05). CONCLUSION: This study presents the first evidence of Kv 11.1 expression in cMGTs and indicates an inverse correlation between Kv 11.1 expression and tumor malignancy. Kv 11.1 expression can be used as a prognostic biomarker and a tool for the management of cMGTs.

Therapeutic effects of platelet derived growth factor overexpressed-mesenchymal stromal cells and sheets in canine skin wound healing model.

Adipose-derived mesenchymal stromal cells (Ad-MSCs) have excellent potential for skin wound repair. Moreover, platelet-derived growth factor (PDGF) has strong wound healing properties. The purpose of the present study was to compare the healing effects of PDGF-overexpressing canine allogeneic Ad-MSCs (PDGF-MSCs) and their cell sheets (PDGF-CSs) as compared to unexpressed Ad-MSCs (U-MSCs) and their cell sheets (UCSs) in a cutaneous wound healing model induced upon dogs. In in vitro study, the expression of immunomodulatory and growth factors was assessed by qRT-PCR. In in vivo study, cells and sheets were transplanted into a square-shaped full-thickness (1.5×1.5 cm) skin defect model created in 12 dogs. After 5 and 10 days, wounds were harvested and evaluated macroscopically and histopathologically. The qRT-PCR results showed that the PDGF-B gene was significantly upregulated (p<0.05) in PDGF-CS and PDGF-MSCs groups. Upon gross analysis of the wound, all stromal cells and their sheet groups showed accelerated (p<0.05) cutaneous wound healing compared to the negative control groups. As compared to U-MSCs and UCSs, the PDGF-MSCs showed significant epithelization on days 5 and 10 of healing, whereas PDGF-CSs showed improved epithelization only on day 10. In the granulation tissue analysis, PDGF-CSs and UCSs promoted more formation (p<0.05) of upper granulation tissue, collagen, and activated fibroblasts than PDGF-MSCs, and U-MSCs. Especially, the PDGF-CSs presented the highest formation and maturation of granulation tissue among all groups. All considered, PDGF overexpressed stromal cells or cells sheets can improve cutaneous wound healing in a canine model.

Author Correction: Methylation of LINE-1 in cell-free DNA serves as a liquid biopsy biomarker for human breast cancers and dog mammary tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Methylation of LINE-1 in cell-free DNA serves as a liquid biopsy biomarker for human breast cancers and dog mammary tumors.

Breast cancer (BC) is one of the most common cancers in both women and female dogs. Methylation changes of LINE-1 have been reported in human cancers. The aim of this study was to determine the hypomethylation of canine LINE-1 in liquid biopsies for canine mammary tumors (CMT) and to assess its diagnostic performance in human plasma. BC associated LINE-1 methylation was measured by methylation sensitive (HpaII) and insensitive (MspI) restriction enzyme digestion followed by real-time PCR using the cfDNA isolated from 300 µl of plasma. The relative level of methylated canine LINE-1 was less than 0.4 in the benign and malignant CMTs (0.29 ± 0.061 and 0.39 ± 0.066, respectively) when it was 0.92 ± 0.067 in the healthy controls. The area under the ROC curve (AUC) was significantly high in both benign and malignant tumors (0.97 and 0.93). Furthermore, this approach was also successfully implemented in a set of 26 human BCs with 10 healthy controls (AUC = 0.78). Altogether, our data suggest that the comparative approach using a dog model might be helpful to rapidly develop a new diagnostic biomarker and that the methylation of LINE-1 in cfDNA may be a good target as a diagnostic marker of both human BC and CMT.

Intra-abdominal necrotic lipoma diagnosed by computed tomography as a paraprostatic cyst.

A dog with a history of diarrhea and dyschezia exhibited an oval-shaped, soft-tissue opacity mass in the abdomen on radiographs. CT examination revealed a large fluid-filled structure displacing the urinary bladder, prostate, and colon. The mass had continuity with the prostate; therefore, it was tentatively diagnosed as a paraprostatic cyst. Cytologic examination was performed and the mass was considered a non-inflammatory cyst. However, after surgery, histopathologic examination revealed a necrotic, inflamed cystic lipoma. This case shows that unusual intra-abdominal lipomas may have a cystic appearance.

Effect of Gelatin on Osteogenic Cell Sheet Formation Using Canine Adipose-Derived Mesenchymal Stem Cells.

Osteogenically differentiated cell sheet techniques using mesenchymal stem cells (MSCs) are available to stimulate bone regeneration. The advantage of the cell sheet technique is delivering live cells effectively into the focal region. We developed a novel osteogenic cell sheet technique by adding gelatin to osteogenic cell medium. Gelatin-induced osteogenic cell sheets (GCSs) were compared to conventional osteogenic cell sheets (OCSs). Undifferentiated MSCs (UCs) were used as a control. The morphology of these cell sheets was evaluated microscopically and histologically. The time-dependent cell proliferation rate was estimated by DNA quantification. The expression of osteogenic gene markers and the number of calcium depositions were assessed by quantitative real-time polymerase chain reaction and Alizarin red S (ARS) staining, respectively. GCSs were thicker and stronger than OCSs. GCSs showed a significantly higher cell proliferation rate compared to OCSs (p < 0.05). GCSs exhibited significantly higher upregulation of BMP-7 mRNA compared to OCSs (p < 0.05). Both GCSs and OCSs showed negative ARS reactivity on day 10, but only GCSs showed positive ARS reactivity on day 21. With this technique, we observed active cell proliferation with abundant ECM and upregulation of osteogenic bone markers, and our results suggest that GCSs could be promising for therapeutic applications in bone regeneration.

Comparison of viability and antioxidant capacity between canine adipose-derived mesenchymal stem cells and heme oxygenase-1-overexpressed cells after freeze-thawing.

Allogenic adipose-derived mesenchymal stem cells (Ad-MSCs) are an alternative source for cytotherapy owing to their antioxidant and anti-inflammatory effects. Frozen-thawed allogenic Ad-MSCs can be used instantly for this purpose. However, the viability and function of frozen-thawed Ad-MSCs have not been clearly evaluated. The purpose of this study was to compare the viability and function of Ad-MSCs and heme oxygenase-1 (HO-1)-overexpressed Ad-MSCs in vitro after freeze-thawing. The viability, proliferation, antioxidant capacity and mRNA gene expression of growth factors were evaluated. Frozen-thawed cells showed significantly lower viability than fresh cells (77% for Ad-MSCs and 71% for HO-1 Ad-MSCs, P<0.01). However, the proliferation rate of frozen-thawed Ad-MSCs increased and did not differ from that of fresh Ad-MSCs after 3 days of culture. In contrast, the proliferation rate of HO-1-overexpressed Ad-MSCs was lower than that of Ad-MSCs. The mRNA expression levels of TGF-ß, HGF and VEGF did not differ between fresh and frozen-thawed Ad-MSCs, but COX-2 and IL-6 had significantly higher mRNA expression in frozen cells than fresh cells (P<0.05). Fresh Ad-MSCs exhibited higher HO-1 mRNA expression than frozen-thawed Ad-MSCs, and fresh HO-1-overexpressed Ad-MSCs exhibited higher than fresh Ad-MSCs (P<0.05). However, there was no significant difference between fresh and frozen HO-1-overexpressed Ad-MSCs. The antioxidant capacity of HO-1-overexpressed Ad-MSCs was significantly higher than that of Ad-MSCs. Cryopreservation of Ad-MSCs negatively affects viability and antioxidant capacity, and HO-1-overexpressed Ad-MSCs might be useful to maximize the effect of Ad-MSCs for cytotherapy.

Functional recovery after spinal cord injury in dogs treated with a combination of Matrigel and neural-induced adipose-derived mesenchymal Stem cells.

BACKGROUND AIMS: Previous studies have reported that scaffold or cell-based transplantation may improve functional recovery following spinal cord injury (SCI), but these results were based on neuronal regeneration and cell replacement. In this study, we investigated whether a combination of Matrigel and neural-induced mesenchymal stem cells (NMSC) improved hindlimb function in dogs with SCI, and what mechanisms were involved. METHODS: We pre-differentiated canine adipose-derived mesenchymal stem cells into NMSC. A total of 12 dogs subjected to SCI procedures were assigned to one of the following three transplantation treatment groups: phosphate-buffered saline (PBS); Matrigel; or Matrigel seeded with NMSC. Treatment occurred 1 week after SCI. Basso, Beattie and Bresnahan (B.B.B.) and Tarlov scores, histopathology, immunofluorescence staining and Western blot analysis were used to evaluate the treatment effects. RESULTS: Compared with dogs administered PBS or Matrigel alone, dogs treated with Matrigel + NMSC showed significantly better functional recovery 8 weeks after transplantation. Histology and immunochemical analysis revealed that the combination of Matrigel + NMSC reduced fibrosis from secondary injury processes and improved neuronal regeneration more than the other treatments. In addition, the combination of Matrigel + NMSC decreased the expression of inflammation and/or astrogliosis markers. Increased expressions of intracellular molecules related to neuronal extension, neuronal markers and neurotrophic factors were also found in the Matrigel + NMSC group. However, the expression of nestin as a neural stem cell marker was increased with Matrigel alone. CONCLUSIONS: The combination of Matrigel + NMSC produced beneficial effects in dogs with regard to functional recovery following SCI through enhancement of anti-inflammation, anti-astrogliosis, neuronal extension and neuronal regeneration effects.

Comparison of canine umbilical cord blood-derived mesenchymal stem cell transplantation times: involvement of astrogliosis, inflammation, intracellular actin cytoskeleton pathways, and neurotrophin-3.

Canine mesenchymal stem cells (cMSCs) derived from umbilical cord blood represent a potentially useful source of stem cells for therapy. The aim of this study was to compare the effects of different transplantation times of cMSCs after spinal cord injury (SCI). A total of 21 dogs were subjected to SCI by balloon-induced compression of the first lumbar vertebrae for 12 h. Of the 21 dogs, 12 were divided into four groups of three according to the time of stem cell (1 × 10(6)) transplantation at the injury site: control no treatment, 12 h, 1 week, and 2 weeks. The remaining 9 animals were negative harvest (HA) time controls for each treatment group (n = 3). Olby and Tarlov scores were used to evaluate functional recovery of the hindlimbs. Markers for neuronal regeneration (Tuj-1, nestin, MAP2, and NF-M), astrogliosis (GALC, GFAP, and pSTAT3), signal molecules for actin cytoskeleton (RhoA, Cdc42, and Rac1), inflammation (COX-2), and neurotrophins (NT-3) were evaluated by Western blot analysis. Scores of the 1-week transplantation group showed significant improvement compared to controls. Hematoxylin and eosin (H&E) staining revealed less fibrosis at the injury site in the 1-week transplantation group compared to other groups and immunohistochemistry showed increased expression of neuronal markers. Furthermore, in both 1-week and 2-week transplantation groups, Tuj-1, nestin, MAP2, NF-M, NT-3, and GFAP increased, but pSTAT3, GALC, and COX2 decreased. RhoA decreased and Rac1 and Cdc42 increased in the 1-week transplantation group. In conclusion, transplantation of cMSCs 1 week after SCI was more effective in improving clinical signs and neuronal regeneration and reducing fibrosis formation compared to the other transplantation times evaluated. Subsequently, these data may contribute to the optimization of timing for MSC transplantation used as a therapeutic modality.

Gingival overgrowth in dogs associated with clinically relevant cyclosporine blood levels: observations in a canine renal transplantation model.

OBJECTIVE: To investigate the development of gingival hyperplasia in dogs after renal transplantation and administration of microemulsified cyclosporine A (MCsA). STUDY DESIGN: Experimental study. ANIMALS: Healthy adult mongrel dogs (n=5). METHODS: As part of study on renal transplantation, dogs administered MCsA (20 mg/kg/day), azathioprine, and prednisolone to prevent graft rejection were monitored for development of gingival changes. Prednisolone was discontinued after 3 months. MCsA dose was adjusted to maintain whole blood trough concentration of 400-700 ng/mL. Gingival change was evaluated by weekly examination and photodocumentation, and gingival biopsy for histopathology was performed at 28 weeks. RESULTS: One dog was lost because of acute graft rejection. Gingival hyperplasia developed in 3 of 4 dogs. The earliest gingival changes occurred in the interdental papillae at 20 weeks after transplantation. On histopathology, the underlying connective tissue was thickened and contained increase numbers of fibroblasts and inflammatory infiltrates. CONCLUSIONS: Long-term immunosuppression with an MCsA-based treatment likely induces substantial gingival hyperplasia when therapeutic, immunosuppressive blood levels of MCsA were maintained for 32 weeks. CLINICAL RELEVANCE: MCsA is used for immune-mediated diseases and preventing rejection after transplant in dogs. MCsA blood levels, and gingival hyperplasia should be monitored by routine examination of the interdental papilla in dogs administered MCsA for long periods.

Palliative intravenous cisplatin treatment for concurrent peritoneal and pleural mesothelioma in a dog.

A 10-year-old Maltese dog was presented with abdominal distention and dyspnea. Cytological examination of pleural and peritoneal effusion was suggestive of malignant effusion of glandular origin. Numerous, multifocal, tan to white nodules were disseminated throughout the surface of the abdominal organs and peritoneum at biopsy. Histologically, the tumors were revealed to be an epithelial type of mesothelioma. Neoplastic cells co-expressed cytokeratin and vimentin. Intravenous administration of cisplatin was chosen as the treatment. During treatment, the dog's overall body condition improved and the clinical signs were relieved without significant side effects. The survival time from diagnosis to sudden death by unknown cause was 153 days.

Mutation and overexpression of p53 as a prognostic factor in canine mammary tumors.

We concentrated ourselves to evaluate the prognostic significance of the p53 gene mutations, its protein expression and MIB-1 index as a proliferative marker in canine mammary tumors. In the present study, a total of 20 cases were examined, among which there were 5 malignant mixed tumors, 4 mammary gland adenocarcinomas, 1 papillary adenocarcinoma, 8 benign mixed tumors and 2 mammary gland adenomas. Positive immunostaining for p53 with PAb240 antibody was found in 2 benign (20%) and 3 malignant (30%) tumors. However, PAb421 antibody did not give positive result at all. In Western blot analysis, the p53 expression in benign and malignant tumors was detected in 4 and 3 cases, respectively. p53 mutations were found in 6 cases out of the cases with detected p53 protein expression. The MIB-1 index in benign and malignant tumors were 17.6+/-20.8% and 29.0+/-27.2%, respectively and there was no significant difference between tumor types. There was a significant correlation between p53 mutations and p53 overexpression (correlation coefficient = 0.5, p < 0.05). In Kaplan-Meier survival analysis, the p53 index was associated with significantly shortened survival time (p < 0.01). In multivariate analysis, p53 overexpression was only an independent factor for indicator of worse prognosis in canine mammary tumors (p = 0.01). These results demonstrated that p53 gene mutations and protein overexpression using the PAb240 anti-p53 antibody were useful predictors of increased malignant potential and poor prognosis in canine mammary tumors.