Establishment and characterization of six canine hepatocellular carcinoma cell lines.

Background: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor in dogs. Although surgical resection is a major treatment option for canine HCC, there are no distinct strategies for unresectable tumor subtypes or adjuvant chemotherapy for tumors with positive margins. We aimed to establish and characterize novel HCC cell lines from canine patients. Methods: The cellular morphology, general growth features and tumorigenicity of the established cell lines were evaluated. We also examined the sensitivity of the cell lines to multi-target tyrosine kinase inhibitors (TKIs). Results: We established novel canine HCC cell lines from hepatic tumors and an additional kidney tumor of six canine patients. All cell lines showed colony forming and migratory ability. KU-cHCC-001 and KU-cHCC-001-Kidney, two cell lines exhibiting high epithelial-mesenchymal transition characteristics, showed tumorigenicity in xenografted mice. Toceranib, a veterinary TKI that targets vascular endothelial growth factor (VEGFR)/platelet-derived growth factor receptor (PDGFR)/c-kit, effectively inhibited the mitogen-activated protein kinase pathway and induced apoptosis. The established canine HCC cell lines showed greater sensitivity to toceranib than to sorafenib, a first-line treatment for human HCC targeting RAF/VEGFR/PDGFR. Sorafenib showed improved anti-tumor effects when co-treated with SCH772984, an extracellular signal-regulated kinase inhibitor. Conclusion: Our study suggests new therapeutic strategies for canine HCC, and these cell lines are valuable research materials for understanding HCC tumor biology in both humans and dogs.

Reconstructive surgery with an autologous bone graft in a dog with presumptive chronic non-bacterial osteomyelitis.

A 15-year-old Cocker Spaniel was referred to for the evaluation of left forelimb lameness. Radiographic and computed tomography examinations revealed osteolysis of the proximal left third, fourth and fifth metacarpal bones and pathological fractures of the proximal left fourth metacarpal bone. Histopathological examination via bone biopsy did not provide a definitive diagnosis, and the owner elected limb-sparing surgery. The fourth metacarpal bone and digits were amputated. Subsequently, autologous bone grafts were performed on the lytic area of the third and fifth metacarpal bones. The dog showed improvement in gait 7 weeks after reconstructive surgery. Chronic non-bacterial osteomyelitis (CNO) was diagnosed by exclusion. To the best of our knowledge, CNO has not been previously reported in dogs.

Safety evaluation of a buffer used in the lyophilization of canine platelets: type I hypersensitivity reaction in dogs caused by bovine serum albumin.

Introduction: The present study was designed to evaluate the safety of substances generally used in the preparation of lyophilized platelet products (LPPs) because the possibility of an immune response to bovine serum albumin (BSA) was considered high when using previously described technology. Methods: An intradermal skin test, followed by a drug provocation test, was conducted to observe adverse events and identify the substances responsible for an immune response. Five male beagles (2 years old) weighing 12-14 kg were used. The dogs were clinically healthy and had no history of medication use. An intradermal skin test was conducted with each substance [i.e., 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, sodium chloride, potassium chloride, sodium bicarbonate, theophylline, trehalose, and BSA] used in the conventional freeze-dry method. Results: In the intradermal skin test, three dogs tested positive at the BSA injection site and showed clinical signs after the intradermal injection, including nausea and vomiting. For the drug provocation test, all dogs received two intravenous injections of an LPP buffer solution. The initial injection was devoid of BSA, whereas the subsequent injection contained BSA. The three dogs that had reacted to BSA in the intradermal skin test exhibited adverse events such as lethargy, vomiting, and nausea immediately after intravenous injection of the LPP buffer containing BSA. All dogs recovered uneventfully after symptomatic treatment in both tests. Discussion: The high incidence and severity of type I hypersensitivity reactions observed in this study suggested that BSA is unsuitable as a component of canine LPP.

Dose-dependent effect of canine lyophilized platelet on an in vitro hemodilution model.

The transfusion of stored platelets has emerged as an efficient method for treating dogs with thrombocytopenia. However, the availability of fresh platelets is limited in veterinary medicine due to demanding storage conditions. Lyophilized platelets have long shelf lives and can be easily stored, increasing their accessibility for thrombocytopenic dogs. Due to the lack of research and information on the dose effect, canine lyophilized platelets are used at a clinical dosage without research-based evidence. This study was to evaluate the dose effect of lyophilized canine platelets on blood coagulability. Three different concentrations of lyophilized canine platelets were added to in vitro hemodilution blood model, increasing the platelet count by 25, 50, and 100 × 106/ml and coagulation profiles were analyzed. The coagulability was evaluated via the plasma fibrinogen concentration, coagulation time, thromboelastography (TEG), and platelet function analyzer (PFA). Higher concentrations of lyophilized platelets showed dose-dependent association with decreased aPTT and R-time of TEG and increased alpha angle and MA of TEG. These results showed the potential that the higher dose of canine lyophilized platelets better improve blood coagulability than the standard dose and provided the basis for further safety and clinical studies.

Correlation Between D-Dimer Concentrations and Thromboelastography in Dogs With Critical Illness: A Retrospective, Cross-Sectional Study.

A hospital-based, retrospective, cross-sectional study was performed to assess the correlation of kaolin-activated thromboelastography (TEG) with D-dimer concentrations in 59 dogs with critical illness. Dogs were included if they had one or more serious disease detected upon performing TEG and D-dimer concentration determination based on the same blood sample. According to the coagulation index (CI), the 59 dogs were divided into two groups: a hypercoagulable state, with CI > 4 (44 dogs) and a normocoagulable state, with CI < 4 (15 dogs). Moreover, the 44 dogs with CI > 4 were divided into three sub-groups according to the disease etiology, i.e., inflammatory/tumor (I/T: 25 dogs), hemodynamic compromise (H: 11 dogs), and both conditions (I/TH: 8 dogs). The median values (interquartile ranges) of the CI and D-dimer concentration were 5.46 (4.55-6.33) and 410.25 (74.63-2206.12) in the 44 dogs with CI > 4 and 3.29 (2.92-3.75) and 1164.7 (50-2250.05) in the 15 dogs with CI < 4, respectively. The D-dimer concentration was significantly increased over the reference value in the 44 dogs with a CI > 4, particularly in dogs in the I/T group. It was also significantly increased in the 15 dogs with a CI < 4. D-dimer concentrations did not correlate with any of the TEG parameters in any of the dogs. Given this lack of correlation, the D-dimer concentration may be limited as an indicator of a possible hypercoagulable state in dogs with critical illness.

Case Report: Giant Multiloculated Pseudocystic Jejunal Leiomyosarcoma in a Dog: Atypical Morphologic Features of Canine Intestinal Leiomyosarcoma.

A 10-year-old intact female Rottweiler dog weighing 29 kg presented with 2 days history of vomiting, anorexia, and lethargy to KonKuk University Teaching Hospital, Seoul, South Korea. Ultrasonography and computed tomography (CT) scannings revealed a well-demarcated, large mass (29 × 19 × 11 cm) with numerous fluid-filled cavities. Metastases to adjacent lymph nodes were also identified on CT. This large mass and the affected intestinal segments were excised for palliative purposes. Postoperatively, the dog recovered uneventfully without any complications. The cut surface of the mass showed an exophytic growth pattern of multiloculated cystic lesions filled with serosanguineous fluid, large cavities filled with necrotic exudate, and fistulous connections between the intestinal lumen and the necrotic cavity in the mass. On histopathology, the mass was a spindle cell neoplasm expanding from the jejunal muscular layer and with pseudocystic changes. Additional immunohistochemical analysis using antibodies against smooth muscle actin, desmin, and CD-117 demonstrated that the mass was consistent with a leiomyosarcoma. Six months post-operatively, plain radiography revealed an abdominal mass, suspected to be recurrence from jejunal leiomyosarcoma. The owner decided to euthanize the dog due to financial constraints. This case report describes the atypical morphology and clinical progression of a large canine jejunal leiomyosarcoma, which had similar clinical features as those of human leiomyoma and leiomyosarcoma.

Successful management of feline CD4+ CD8+ T-cell mediastinal lymphoma with pericardial effusion.

A 2-year-old, castrated, male Russian blue cat presented with acute dyspnoea, cyanosis, and lethargy. A thoracic radiography revealed a large cranial mediastinal mass; the computed tomography revealed caudal lobe atelectasis of the right lung with pericardial and pleural effusions. The cytodiagnostic tests revealed high-grade CD4+ CD8+ T-cell mediastinal lymphoma as clinical stage Vb; l-asparaginase-cyclophosphamide-doxorubicin-vincristine-prednisolone (l-CHOP)-based chemotherapy was initiated, following which the mass shrunk rapidly; 1 week after the initiation of chemotherapy, the appetite-related and respiratory symptoms improved dramatically, and the pleural and pericardial effusion resolved. The patient remains in complete remission three years after the initiation of the l-CHOP chemotherapy. Therefore, the accurate diagnosis and instantaneous initiation of chemotherapy may resolve life-threatening pleural and pericardial effusions in cats with high-grade aberrant T-cell mediastinal lymphoma.

Establishment of Canine Transitional Cell Carcinoma Cell Lines Harboring BRAF V595E Mutation as a Therapeutic Target.

Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract and tends to have a poor prognosis due to its invasive potential. Recent studies have reported that up to 80% of canine urothelial carcinoma has the BRAF V595E mutation, which is homologous to the human V600E mutation. Activating the BRAF mutation is an actionable target for developing effective therapeutic agents inhibiting the BRAF/mitogen-activated protein kinase (MAPK) pathway in canine cancer as well as human cancer. We established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring the BRAF V595E mutation. Tumor tissues highly expressed the BRAF mutant and phosphorylated extracellular signal-related kinases (ERK)1/2 proteins. The derived cell lines demonstrated activated MAPK pathways. We also evaluated the cell lines for sensitivity to BRAF inhibitors. Sorafenib, a multiple kinase inhibitor targeting RAF/vascular endothelial growth factor receptor (VEGFR), successfully inhibited the BRAF/MAPK pathway and induced apoptosis. The established canine TCC cell lines responded with greater sensitivity to sorafenib than to vemurafenib, which is known as a specific BRAF inhibitor in human cancer. Our results demonstrated that canine TCC cells showed different responses compared to human cancer with the BRAF V600E mutation. These cell lines would be valuable research materials to develop therapeutic strategies for canine TCC patients.

Case Report: Trans-Articular External Skeletal Fixation of the Hip for a Highly Comminuted Juxta-Articular Fracture of the Proximal Femur Caused by Gunshot Injury in a Cat.

This report describes a novel surgical technique for trans-articular external skeletal fixation (TA-ESF) of the hip to stabilize a rare, highly comminuted juxta-articular fracture of the proximal femoral segment involving the metaphysis and diaphysis in a cat. A 2-year-old, castrated male, Korean shorthair cat was admitted for a left femoral fracture caused by gunshot injury. Radiographs and computed tomography (CT) scans revealed a highly comminuted proximal femoral diaphyseal fracture that involved the femoral neck and the greater trochanter. The left femoral head was intact on CT. Under fluoroscopic guidance, an intramedullary Steinmann pin was placed to align the femur, and five positive-profile, end-threaded pins were placed in the left hemipelvis and distal femur. A triangulated frame of connecting bars was constructed using Steinmann pins and epoxy resin for TA-ESF. This secured the pins placed in the pelvis and femur as well as the intramedullary pin, providing proximal femoral stabilization by trans-articular fixation of the hip. The cat began placing weight on the left pelvic limb 4 days postoperatively, and progressively obtained near-normal limb function by day 112. The TA-ESF was partially dismantled to a linear tie-in construct on day 64 and was completely removed on day 161. Final radiographs on day 161 revealed lack of bone healing in the proximal segment, especially femoral head and neck, despite functional recovery of the injured leg. At one year postoperatively, the cat had normal limb function without any noticeable complications. On follow-up 29 months after surgery, the owner reported that the cat had normal limb function without any noticeable complications. Despite insufficient bone healing in the cat, TA-ESF of the hip allowed for satisfactory functional recovery of this challenging juxta-articular fracture of the proximal femur, which was not amenable to stabilization with a traditional non-load-sharing fixation system.

Small Bowel Obstruction Induced by Concurrent Postoperative Intra-Abdominal Adhesions and Small Bowel Fecal Materials in a Young Dog.

A 7-month-old neutered male poodle dog presented with general deterioration and gastrointestinal symptoms after two separate operations: a jejunotomy for small-intestinal foreign body removal and an exploratory laparotomy for diagnosis and treatment of the gastrointestinal symptoms that occurred 1 month after the first surgery. The dog was diagnosed as having small-bowel obstruction (SBO) due to intra-abdominal adhesions and small-bowel fecal material (SBFM) by using abdominal radiography, ultrasonography, computed tomography, and laparotomy. We removed the obstructive adhesive lesion and SBFM through enterotomies and applied an autologous peritoneal graft to the released jejunum to prevent re-adhesion. After the surgical intervention, the dog recovered quickly and was healthy at 1 year after the surgery without gastrointestinal signs. To our knowledge, this study is the first report of a successful treatment of SBO induced by postoperative intra-abdominal adhesions and SBFM after laparotomies in a dog.

Longitudinal assessment of B-RAF V595E levels in the peripheral cell-free tumor DNA of a 10-year-old spayed female Korean Jindo dog with unresectable metastatic urethral transitional cell carcinoma for monitoring the treatment response to a RAF inhibitor (sorafenib).

Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract. In this case study, a dog with metastatic urethral TCC was treated with sorafenib. The tumor expression levels of receptor tyrosine kinase genes, including VEGFR-1, VEGFR-2, PDGFR-α, PDGFR-ß, ALK, EGFR, ErbB2, and B-RAF, were analyzed. VEGFR was overexpressed in tumor tissues compared to the normal tissues. Considering the high frequency of B-RAF mutation in canine urological tumors, the B-RAF gene was examined, and the B-RAF V595E mutation was detected in the tumor tissue. Therefore, the antitumor effect of sorafenib, a multi-tyrosine kinase inhibitor, on unresectable metastatic urethral TCC characterized by B-RAF V595E was evaluated and circulating cell-free tumor DNA (ctDNA) was assessed for monitoring the treatment response. After the initiation of oral sorafenib therapy (4 mg/kg/day escalated to 10 mg/kg/day), the dysuria was alleviated gradually, and the patient remained stable for 3 months. During that treatment period, the patient showed various levels of changes associated with B-RAF V595E mutation in ctDNA as evident from longitudinal plasma samples after initiation of sorafenib therapy. The findings of this study suggest that ctDNA may serve as a useful non-invasive tool for monitoring the treatment response to anticancer drugs.

Case Report: Surgical Correction of a Cystic Duct Stump Leakage Following Cholecystectomy Using an Autologous Rectus Sheath Graft in a Dog.

A 2.7 kg, 13-year-old, castrated male Yorkshire Terrier was presented with bile peritonitis after cholecystectomy. Exploratory coeliotomy to identify and correct bile leakage revealed that the transected end of the cystic duct was open with no in-situ ligatures or vascular clips. The residual cystic duct stump was too short to ligate or seal directly. An autologous rectus sheath graft, harvested from the internal leaf of the rectus sheath, was used to patch the cystic duct stump. The graft was secured over the open duct using several simple interrupted sutures and covered with an omentalization. The clinical signs resolved after surgery, except for a transient increase in hepatobiliary enzyme levels and intrahepatic bile duct dilatation. The enzyme levels returned to near normal on day 25 after surgery. No intrahepatic bile duct dilatation was detected on day 55 after surgery. The owner was contacted for 3 years post-operatively and reported that the dog remained healthy without any long-term complications. Grafting using autologous rectus sheath can be used to treat cystic duct stump leakage that cannot be managed with direct closure using traditional modalities due to spatial constraints.

Parotid Duct Ectasia in a Dog.

A 3yr old spayed female Brittany was referred with a cylindrical mass on the right side of her face. Three months earlier, a small stone embedded in the right parotid duct was noted and removed through a ductal incision by the referring veterinarian. At referral, the dog's general physical condition was normal except for a cylindrical mass on the right cheek. Skull radiographs showed a possibly retained sialolith or dystrophic mineralization within the previous surgical site. Aspiration of the mass yielded a thick, yellow/tan, mucopurulent fluid. Cytology of the fluid demonstrated degenerative neutrophils without bacteria. The cylindrical mass was excised with a carbon dioxide laser. The caudal end of the mass was connected to the parotid salivary gland and the rostral one-third of the mass tapered to a point. The caudal end of the mass was ligated with 3-0 polydioxanone and excised immediately rostral to the ligation. Histopathology revealed that the mass was parotid duct ectasia. The dog completely recovered with some mild temporary facial paresis and had no recurrence of parotid duct ectasia at 4 mo follow-up. The case report describes a dog with an unusual parotid salivary duct ectasia caused by parotid duct sialolith removal.

Suspected anaphylactic shock associated with administration of tranexamic acid in a dog.

A 10-year-old male castrated Maltese was referred with clinical signs of hematuria, stranguria, and pollakiuria. The dog was diagnosed with sterile hemorrhagic cystitis with urethroliths and cystoliths. To remove the uroliths, the dog underwent retrograde urohydropropulsion followed by a cystotomy. The following day, persistent bleeding in the urinary bladder was identified with large hematoma, hematuria and anemia. In order to reduce bleeding, the dog received 10 mg/kg of tranexamic acid (TXA) intravenously. Immediately after TXA administration, the dog developed anaphylactic shock manifested by hypotension, hypothermia, tachycardia and a dull mentation. Thus, an emergency treatment including bolus injection of crystalloid, administration of dexamethasone and diphenhydramine, and oxygen supplementation was given, after which the dog quickly recovered within a few minutes.

Concurrent pulmonary hypoplasia and congenital lobar emphysema in a young dog with tension pneumothorax: a rare congenital pulmonary anomaly.

BACKGROUND: Pulmonary hypoplasia (PH) and congenital lobar emphysema (CLE) are very rare congenital pulmonary anomalies in veterinary medicine. PH refers to the incomplete pulmonary development due to embryologic imbalance of bronchial development between the lung buds, while CLE is defined as alveolar hyperinflation due to bronchial collapse during expiration caused by bronchial cartilage dysplasia, external bronchial compression, and idiopathic etiology. CLE may develop into pulmonary blebs or bullae that may rupture and induce a spontaneous pneumothorax. There are no reports on concurrent PH and CLE in animals. CASE PRESENTATION: A 7-month-old castrated male Italian Greyhound weighing 5.5 kg presented with vomiting and acute onset of severe dyspnea without any previous history of disease. After emergency treatment including oxygen supplementation and thoracocentesis, plain radiology and computed tomography scanning were performed and lobar emphysema with multiple bullae in the left cranial lung lobe associated with tension pneumothorax was identified. Since the pneumothorax was not resolved despite continuous suction of intrathoracic air for 3 days, a complete lobectomy of the left cranial lung lobe was performed. The excised lobe was not grossly divided into cranial and caudal parts, but a tissue mass less than 1 cm in size was present at the hilum and cranial to the excised lobe. Postoperatively, the dog recovered rapidly without air retention in the thoracic cavity. Histopathologically, the mass was identified as a hypoplastic lung tissue with collapsed alveoli, bronchial dysplasia, and pulmonary arterial hypertrophy. Additionally, the excised lung lobe presented CLE with marked ectasia of alveoli, various blebs and bullae, and general bronchial cartilage dysplasia. According to gross and histopathologic findings, the dog was diagnosed with concurrent PH and CLE in the left cranial lung lobe. During 16 months of follow-up, the dog was well and without any respiratory problems. CONCLUSIONS: This case report confirmed the clinical and histologic features of two different types of rare congenital pulmonary anomalies, PH and CLE, which occurred concurrently in a single lung lobe of a young dog. The condition was successfully managed with lobectomy.

Acute hemorrhagic diarrhea syndrome associated with contaminated foreign bodies (used feminine hygiene products) in a Golden Retriever dog.

A one-year-old male Golden Retriever presented with acute onset of vomiting and hemorrhagic diarrhea since 2 days. The dog was depressed, showing abdominal pain, 12% dehydration, tachycardia, and a bounding pulse. Diagnostic imaging showed severe dilatation and fluid retention of the entire gastrointestinal tract with decreased motility. A foreign body was found in the gastroduodenal region, but there was no obstruction or plication. The dog was tentatively diagnosed with acute hemorrhagic diarrhea syndrome and rapidly recovered after supportive treatment. However, on the morning of day 4, anorexia and vomiting recurred, and diagnostic imaging revealed intestinal plication with free peritoneal fluid, not found on the previous image. An emergency laparotomy revealed the foreign body to be two used feminine hygiene products. These contaminated products were suspected to induce acute hemorrhagic diarrhea syndrome, and led to subsequent complication in this large dog.

Pyothorax induced by an intrathoracic foreign body in a miniature dachshund: Migration of a popsicle stick from the stomach.

A four-year-old dachshund presented with a two-week history of pyrexia, depression, and cough. Four months earlier, the owner observed the dog swallow a whole popsicle stick, but the animal showed no clinical signs at that time. Radiography, ultrasonography, and computed tomography confirmed an intrathoracic linear foreign body and pleural effusion in the right thorax. The pleural fluid was bloody and purulent, and contained inflammatory cells and Escherichia coli. The dog was diagnosed with pyothorax induced by a foreign body, and was treated successfully by surgical removal of the foreign body, partial lung lobectomy, thoracic lavage, and antibiotics. The foreign body was identified as a popsicle stick that the dog had eaten.

Canine Sialolithiasis: Two Case Reports with Breed, Gender, and Age Distribution of 29 Cases (1964-2010).

This study was conducted to investigate the clinical data of two cases of canine sialolithiasis and to analyze 29 cases identified in the Veterinary Medical Database by year of admission, breed, gender, and age. Medical records from the University of Missouri Veterinary Medical Teaching Hospital were reviewed and two dogs diagnosed with sialolithiasis (calcium oxalate) were identified between 1990 and 2010. The two dogs had cervical or pharyngeal sialocele and were successfully treated by sialolith removal and concurrent sialoadenectomy. Signalments of dogs with sialolithiasis between 1964 and 2010 were collected from the Veterinary Medical Database and evaluated. Several breeds of dogs were represented and the 10 to <15 yr old age group was shown to have significant association with sialolithiasis. Sialolithiasis is a rare veterinary condition. In this study, older dogs were at higher risk. In dogs, concurrent sialocele was common and good outcome could be expected after surgical removal of sialoliths with concurrent sialoadenectomy.

Primo vessel as a novel cancer cell migration path from testis with nanoparticle-labeled and GFP expressing cancer cells.

BACKGROUND/AIM: Recently, a novel circulatory system, the primo vascular system (PVS), was found to be a potent metastatic route of cancer cells. The aim of the current work is to demonstrate that cancer cells injected into the testis migrate through the primo vessel (PV). MATERIALS AND METHODS: NCI-H460 cells labeled with fluorescent nanoparticles (FNP) or green fluorescent protein (GFP) gene transfection were injected into testicular parenchyma in 24 rats. After 24 hours of injection, the abdominal cavity was investigated via a stereomicroscope, to detect the PVS, and the samples were analyzed histologically with 4',6-diamidino-2-phenylindole (DAPI) and hematoxylin and eosin. RESULTS: Injected cancer cells were detected inside the PVS distributed on the abdominal organs. Some were detected inside intestinal parenchyma into which the attached primo vessels (PVs) entered. CONCLUSION: The results supported the fact that the PVS may be a novel migration path of cancer cells, in addition to the lymphatic and hematogenous routes.