Granular cell tumor of the brachial nerve in a dog: A case report.

Here, we describe the first case of a granular cell tumor (GCT) derived from the brachial nerve. Eleven-year-old neutered female Chihuahua presented to the hospital with a bulge from the left neck to the axilla. The dog had a spherical subcutaneous mass on the cervical subcutis, and cytology hinted at adenocarcinoma or neuroendocrine tumor. However, the origin of the tumor remains unknown. During resection of the mass, bleeding was difficult to control owing to the high blood flow, and tumor removal was extremely difficult. The caudal aspect of the mass was attached to the brachial nerve and had to be removed, along with parts of the nerve fibers. The patient's postoperative course was fair, but it developed paralysis of the left thoracic limb. Pathology revealed that the mass was positive for S100 and vimentin, and GCT was diagnosed. Non-oral GCTs are extremely rare. The clinical diagnosis of GCT is difficult and is often confirmed histopathologically by excision. Although most cases of GCT are benign, they must be recognized as hemorrhagic, indistinct masses that mimic malignancy. Excision carries the risk of hemorrhage and damage to the surrounding tissues to secure margins.

Descrevemos aqui o primeiro caso de um tumor de células granulares (TCG) derivado do nervo braquial. Uma chihuahua castrada de 11 anos de idade deu entrada no hospital com uma protuberância do pescoço esquerdo até a axila. A cadela apresentava uma massa subcutânea esférica no subcutâneo cervical, e a citologia indicava adenocarcinoma ou tumor neuroendócrino. Entretanto, a origem do tumor permanece desconhecida. Durante a ressecção da massa, foi difícil controlar o sangramento devido ao alto fluxo sanguíneo, e a remoção do tumor foi difícil. O aspecto caudal da massa estava ligado ao nervo braquial e teve de ser removido, juntamente com partes das fibras nervosas. A evolução pós-operatória da paciente foi regular, mas ele desenvolveu paralisia do membro torácico esquerdo. O exame anatomopatológico revelou que a massa era positiva para S100 e vimentina, e o TCG foi diagnosticado. Os TCGs não orais são extremamente raros. O diagnóstico clínico do TCG é difícil e geralmente é confirmado histopatologicamente por excisão. Embora a maioria dos casos de TCG seja benigna, eles devem ser reconhecidos como massas hemorrágicas e indistintas que simulam malignidade. A excisão acarreta o risco de hemorragia e danos aos tecidos circundantes para garantir as margens.

Effects of a Soluble Guanylate Cyclase Stimulator Riociguat on Contractility of Isolated Pulmonary Artery and Hemodynamics of U46619-Induced Pulmonary Hypertension in Dogs.

Soluble guanylate cyclase (sGC) stimulator riociguat is a relatively novel therapeutic agent for pulmonary hypertension (PH) in human medicine. Riociguat induces endothelium-independent pulmonary artery (PA) relaxation by directly activating the sGC-cyclic guanosine-3',5'-monophosphate (cGMP) pathway in muscle cells. Although riociguat may be effective in the treatment of dogs with refractory PH, basic studies on its clinical application in veterinary medicine are lacking. The present study aimed to explore the effects of riociguat on the contractility of an isolated canine PA and the hemodynamics of dogs with acute PH. In an isolated endothelium-denuded canine PA, the effects of riociguat on endothelin (ET)-1-induced contraction and cGMP levels were investigated using the Magnus method and ELISA, respectively. The effect of riociguat on the hemodynamics of the thromboxane A2 analog U46619-induced PH model dog was examined by invasive catheterization. Riociguat increased cGMP levels and reduced ET-1-induced contraction of the isolated PA. Riociguat inhibited the U46619-induced elevation of PA pressure and pulmonary vascular resistance and increased cardiac output, but it had no effect on basal systemic blood pressure. These results demonstrate for the first time that riociguat can inhibit the elevation of PA pressure through PA relaxation via an endothelium-independent increase in cGMP in dogs with PH.

A case of papillary squamous cell carcinoma in the mandible of a young French bulldog.

A 7-month-old castrated French bulldog was presented with a left-sided mandibular tumor. The initial tumor biopsy diagnosis was ameloblastoma. The owner brought this dog the Kitasato University Veterinary Teaching Hospital for more detailed examination and treatment. Computed tomography revealed a tumor on the left lateral mandibular gingiva from the caudal third of the incisor tooth to the right canine tooth, associated with severe amorphous osteolysis of the mandibular bone. The tumor was surgically excised and diagnosed as papillary squamous cell carcinoma. Currently, 2514 d (6.9 y) since the operation, the dog is healthy, without recurrence. Key clinical message: Although papillary squamous cell carcinoma is rare, many cases have been reported in the oral cavity of medium-to large-sized dogs. Based on this report, papillary squamous cell carcinoma can occur in small dogs such as young French bulldogs and a good prognosis can be achieved with proper resection.

Un cas de carcinome épidermoïde papillaire de la mandibule d'un jeune bouledogue français. Un bouledogue français castré de 7 mois a été présenté avec une tumeur mandibulaire gauche. Le diagnostic initial de biopsie tumorale était un améloblastome. Le propriétaire a amené ce chien à l'hôpital universitaire vétérinaire de Kitasato pour un examen et un traitement plus détaillés. La tomodensitométrie a révélé une tumeur de la gencive mandibulaire latérale gauche du tiers caudal de l'incisive à la canine droite, associée à une ostéolyse amorphe sévère de l'os mandibulaire. La tumeur a été excisée chirurgicalement et diagnostiquée comme un carcinome épidermoïde papillaire. Actuellement, 2514 jours (6,9 ans) depuis l'opération, le chien est en bonne santé, sans récidive.Message clinique clé :Bien que le carcinome épidermoïde papillaire soit rare, de nombreux cas ont été rapportés dans la cavité buccale de chiens de taille moyenne à grande. Sur la base de ce rapport, le carcinome épidermoïde papillaire peut survenir chez les petits chiens tels que les jeunes bouledogues français et un bon pronostic peut être obtenu avec une résection appropriée.(Traduit par Dr Serge Messier).

Timing urinary tract reconstruction in rats to avoid hydronephrosis and fibrosis in the transplanted fetal metanephros as assessed using imaging.

Chronic kidney disease leads to high morbidity rates among humans. Kidney transplantation is often necessary for severe symptoms; however, options for new curative treatments are desired because of donor shortage. For example, it has been established that the kidneys can efficiently generate urine after transplantation of the metanephros, ureter, and bladder as a group. After transplantation, the urine can indirectly flow into the recipient's bladder using a stepwise peristaltic ureter system method where the anastomosis is created via the recipient's ureter for urinary tract reconstruction. However, the growth of the regenerated metanephros varies significantly, whereas the time window for successful completion of the stepwise peristaltic ureter system that does not cause hydronephrosis of the metanephros with bladder (ureter) is quite narrow. Therefore, this study was conducted to periodically and noninvasively evaluate the growth of the transplanted metanephros, ureter, and bladder in rats through computed tomography and ultrasonography. The ultrasonographic findings highly correlated to the computed tomography findings and clearly showed the metanephros and bladder. We found that the degree of growth of the metanephros and the bladder after transplantation differed in each case. Most of the rats were ready for urinary tract reconstruction within 21 days after transplantation. Optimizing the urinary tract reconstruction using ultrasonography allowed for interventions to reduce long-term tubular dilation of the metanephros due to inhibited overdilation of the fetal bladder, thereby decreasing the fibrosis caused possibly by transforming growth factor-ß1. These results may be significantly related to the long-term maturation of the fetal metanephros and can provide new insights into the physiology of transplant regeneration of the metanephros in higher animals. Thus, this study contributes to the evidence base for the possibility of kidney regeneration in human clinical trials.

Prevention of Chronic Rejection of Marginal Kidney Graft by Using a Hydrogen Gas-Containing Preservation Solution and Adequate Immunosuppression in a Miniature Pig Model.

In clinical kidney transplantation, the marginal kidney donors are known to develop chronic allograft rejection more frequently than living kidney donors. In our previous study, we have reported that the hydrogen gas-containing organ preservation solution prevented the development of acute injuries in the kidney of the donor after cardiac death by using preclinical miniature pig model. In the present study, we verified the impact of hydrogen gas treatment in transplantation with the optimal immunosuppressive protocol based on human clinical setting by using the miniature pig model. Marginal kidney processed by hydrogen gas-containing preservation solution has been engrafted for long-term (longer than 100 days). A few cases showed chronic rejection reaction; however, most were found to be free of chronic rejection such as graft tissue fibrosis or renal vasculitis. We concluded that marginal kidney graft from donor after cardiac death is an acceptable model for chronic rejection and that if the transplantation is carried out using a strict immunosuppressive protocol, chronic rejection may be alleviated even with the marginal kidney.

Pilot prescription survey of antineoplastic agents: real-world data from veterinary teaching hospitals in Japan.

The collection of real clinical records from veterinary practices and analysis of these records helps to establish evidence-based veterinary medicine and further improves animal health and welfare. Prior to the collection of nationwide clinical records, we downloaded the data from the digital accounting systems of two veterinary teaching hospitals in Japan, and the prescriptions of antineoplastic agents were surveyed for a 5-year period from 2009 to 2013. The ratio of the number of patients prescribed antineoplastic agents to the total number of prescriptions was <5% at both hospitals, and >80% of those patients were dogs. The overall number of prescriptions included more oral rather than injectable formations, whereas among antineoplastic agents, injectable formulations were prescribed more frequently at both hospitals. The most frequently prescribed agents were almost identical at both hospitals: platinum compounds, such as carboplatin and cisplatin (CDDP), vincristine and doxorubicin. The most frequently prescribed product combined with CDDP was doxorubicin at Hospital A. Antiemetic agents combined with CDDP included dexamethasone, ondansetron and metoclopramide, but these antiemetic agents were combined fewer than 10 times among 197 CDDP prescriptions. The prescription history, including the number of prescriptions, dosing intervals and combined medications, was provided by the survey. Although the present database consisted of data from two hospitals, our results indicate that a broad analysis can be conducted using integrated data from multiple hospitals and practices for further cohort studies.

Effect of Propofol Continuous-Rate Infusion on Intravenous Glucose Tolerance Test in Dogs.

Hyperglycemia causes perioperative complications and many anesthetics impair glucose metabolism and cause hyperglycemia. We evaluated the effects of propofol on blood glucose metabolism and insulin secretion during an intravenous glucose tolerance test (IVGTT) in dogs. Blood glucose, insulin, triglyceride, cholesterol, and free fatty acid (FFA) levels were measured in dogs during IVGTT in a conscious state and under the effect of 2.0% isoflurane, low-concentration propofol (0.2 mg/kg/min), and high-concentration propofol (0.4 mg/kg/min) anesthesia. Plasma glucose levels significantly increased in all of the treatment groups when compared with those in the conscious group. The prolonged half-life period of plasma glucose suggested that isoflurane and propofol attenuated glucose metabolism in dogs. Plasma insulin levels were significantly lower in the isoflurane group when compared with those in the other groups, whereas blood FFA levels were increased in the propofol groups when compared with the other groups. These results suggest that propofol itself does not directly raise plasma glucose levels, but attenuates glucose metabolism by accumulating FFA.

Association between preoperative characteristics and risk of anaesthesia-related death in dogs in small-animal referral hospitals in Japan.

OBJECTIVE: To explore the major risk factors linking preoperative characteristics and anaesthesia-related death in dogs in referral hospitals in Japan. STUDY DESIGN: Observational cohort study. ANIMALS: From April 1, 2010 to March 31, 2011, 4323 dogs anaesthetized in 18 referral hospitals in Japan. METHODS: Questionnaire forms were collated anonymously. Death occurring within 48 hours after extubation was considered as an anaesthesia-related death. Patient outcome (alive or dead) was set as the outcome variable. Preoperative general physical characteristics, complete blood cell counts, serum biochemical examinations and intraoperative complications were set as explanatory variables. The risk factors for anaesthesia-related death were evaluated using chi-square test or Fisher's exact test, followed by multivariable logistic regression analysis of the data. Significance was set at p < 0.05. RESULTS: Thirteen dogs that died from surgical error or euthanasia were excluded from statistical analysis. The total mortality rate in this study was 0.65% [28/4310 dogs; 95% confidence interval (CI), 0.41-0.89]. Furthermore, 75% (95% CI, 55.1-89.3) of anaesthesia-related deaths occurred in dogs with pre-existing diseases. Most of the deaths occurred postoperatively (23/28; 82.1%; 95% CI, 63.1-93.9). Preoperative serum glucose concentration <77 mg dL-1 (6/46; 13.0%; 95% CI, 4.9-26.3), disturbance of consciousness (6/50; 12.0%; 95% CI, 4.5-24.3), white cell count >15,200 µL-1 (16/499; 3.4%; 95% CI, 1.9-5.5) and American Society of Anesthesiologists grade III-V (19/1092; 1.7%; 95% CI, 1.1-2.7) were identified as risk factors for anaesthesia-related death. Intraoperative hypoxaemia (8/34; 23.5%; 95% CI, 10.7-41.2) and tachycardia (4/148; 2.7%; 95% CI, 0.7-6.8) were also risk factors for anaesthesia-related death. CONCLUSIONS AND CLINICAL RELEVANCE: The results revealed that certain preoperative characteristics were associated with increased odds of anaesthesia-related death, specifically low serum glucose concentration and disturbances of consciousness. Greater attention to correcting preanaesthetic patient abnormalities may reduce the risk of anaesthesia-related death.

Transcatheter arterial embolization for treatment of hepatocellular carcinoma in a cat.

CASE DESCRIPTION: A 16-year-old 6.8-kg (15-lb) castrated male domestic shorthair cat was evaluated because of a 3 × 6-cm mass in the right medial lobe of the liver. CLINICAL FINDINGS: The cat had a history of frequent vomiting and anorexia along with 10% weight loss over the past year. TREATMENT AND OUTCOME: Transcatheter arterial embolization was selected because surgery (standard first-line treatment) was declined and only 1 vessel feeding the tumor was apparent on contrast-enhanced CT. A 4F sheath was placed in the left carotid artery, and a 3.3F guide catheter was advanced into the celiac artery. A 0.014-inch guidewire and 1.7F microcatheter were inserted into the hepatic artery through the guiding catheter and advanced into the feeding vessel. A mixture of polyvinyl alcohol particles and contrast agent was injected for embolization. A hypoechoic area in the tumor was identified on ultrasonography on posttreatment day 6, and necrotic and degenerated cells in the area were identified cytologically. By posttreatment day 71, vomiting had resolved and CT revealed decreased tumor size, but altered attenuation suggested a more solid mass on day 205. No feeding vessel for embolization was found on contrast-enhanced CT, so ultrasonic emulsification to remove the tumor was performed on day 231. No recurrence was seen on contrast-enhanced CT on day 420 or day 721. CLINICAL RELEVANCE: Findings suggested that transcatheter arterial embolization may be suitable for treating hepatic tumors in cats, but alternative approaches are needed in cats, compared with dogs, owing to anatomic differences.

Influence of pneumoperitoneum and postural change on the cardiovascular and respiratory systems in dogs.

We investigated the influence of pneumoperitoneum#(PP) and postural change under inhalation anesthesia with isoflurane, which is routinely used in dogs, on the cardiovascular and respiratory systems. As test animals, 6 adult beagles were used. To induce anesthesia, atropine, butorphanol and propofol were intravenously injected. Anesthesia was maintained with 1.3 MAC (1.7%) isoflurane. The following were the experiment conditions: I:E ratio, 1:1.9; tidal air exchange, 20 ml/kg; and ventilation frequency, 14 times/min. Respiration was regulated so that the PaCO2 was approximately 35 to 40 mmHg before the start of the experiment. PP with CO2 (intraperitoneal pressure 15 mmHg) and a postural change (15°C) was performed during the experiment. As parameters of circulatory kinetics, heart rate (HR), mean aortic pressure (MAP), mean pulmonary arterial pressure (MPAP), central venous pressure (CVP), femoral venous pressure (FVP) and cardiac output (CO) were measured. As parameters of respiratory kinetics, airway pressure (PAW) and blood gas (BG) were measured. There were significant increases in HR, MAP, MPAP, CVP, FVP, CO, PAW and PaCO2 after PP in the horizontal position. There were significant increases in CVP, FVP, PAW and PaCO2 after PP in the Trendelenburg position. There were significant increases in the MPAP, CVP, FVP, PAW and PaCO2 after PP in the inverse Trendelenburg position. There was a significant difference in FVP after PP between the Trendelenburg position and inverse Trendelenburg position. The results of this experiment suggest that appropriate anesthesia control, such as changing the ventilation conditions after PP, is required for laparoscopic surgery under inhalation anesthesia with isoflurane.

Effect of the phosphodiesterase type 5 inhibitor tadalafil on pulmonary hemodynamics in a canine model of pulmonary hypertension.

Phosphodiesterase type 5 (PDE5) inhibitors are used for treating pulmonary arterial hypertension (PAH) in dogs. The long-acting PDE5 inhibitor tadalafil was recently approved for treatment of PAH in humans. Basic information related to the pharmacological and hemodynamic effects of tadalafil in dogs is scarce. In this study, the hemodynamic effects of tadalafil after intravenous (IV) and oral administration were investigated in a healthy vasoconstrictive PAH Beagle dog model induced by U46619, a thromboxane A2 mimetic. Six healthy Beagle dogs were anesthetized with propofol and maintained with isoflurane. Fluid-filled catheters were placed into the descending aorta to measure systemic arterial pressure and in the pulmonary artery to measure pulmonary arterial pressure (PAP). U46619 was infused via the cephalic vein to induce PAH. IV infusion of U46619 significantly elevated PAP from baseline in a dose-dependent manner. U46619-elevated PAP and pulmonary vascular resistance was significantly attenuated by the simultaneous infusion of tadalafil at 100 and 200 µg/kg/h. Likewise, oral administration of tadalafil at 1.0, 2.0, and 4.0 mg/kg significantly attenuated U46619-elevated PAP in a dose-dependent manner. U46619-elevated systolic and mean PAP decreased significantly 1 h after oral tadalafil administration at 4.0 mg/kg, and this effect was maintained for 6 h. In conclusion, tadalafil had a pharmacological effect in dogs and IV infusion of tadalafil induced pulmonary arterial relaxation, while oral administration of tadalafil decreased PAP. These results suggest that tadalafil may offer a new therapeutic option for treating dogs with PAH.

Magnetic resonance imaging assessment of pituitary posterior lobe displacement in dogs with pituitary-dependent hyperadrenocorticism.

The displacement and signal intensity (SI) of the pituitary posterior lobe were evaluated on T1 weighted magnetic resonance (MR) images in 28 dogs with pituitary dependent hyperadrenocorticism (PDH). Compared with normal dogs, the posterior lobe was displaced dorsally in the pituitary of the PDH dogs. Correlation between the pituitary height/brain area (P/B) ratio and the displacement of the posterior lobe in the PDH dogs suggests that dorsal displacement of the posterior lobe increases in accordance with enlargement of the pituitary. As to the SI of the posterior lobe, the PDH dogs showed significantly lower SI in comparison to the normal dogs. Taken together, these results suggest that assessment of the displacement and SI of the posterior lobe of the pituitary on T1-weighted MR images is useful for the diagnosis of pituitary adenoma. In pituitary microadenoma that presents no apparent tumorigenesis on MRI, evaluation of these values may be useful for diagnosis and selection of an appropriate therapy.

Evaluation of the function of polymorphonuclear neutrophilic leukocytes in healthy dogs given a high dose of methylprednisolone sodium succinate.

OBJECTIVE: To evaluate effects of a high dose of methylprednisolone sodium succinate (MPSS) on function of polymorphonuclear neutrophilic leukocytes (PMNs) in dogs. ANIMALS: 7 healthy male Beagles (body weight, 10.5 to 15 kg; age, 2 to 4 years). PROCEDURES: All dogs were treated by IV administration of a high dose of MPSS (30 mg/kg). Additional doses of MPSS (15 mg/kg) were administered IV at 2 and 6 hours and then at 6-hour intervals until 48 hours after the initial dose. Blood samples were collected before and 1, 2, 4, 7, and 14 days after completion of the MPSS administrations and used for evaluation of PMN functions. Isolated PMNs were used for assessment of functions, such as adhesion, migration, phagocytosis, and oxidative burst. RESULTS: On days 1, 2, and 4 after completion of MPSS administration, there was a decrease in PMN expression of adhesion markers such as CD11b and CD18. There was a decrease in the phagocytotic ability of PMNs on days 1, 2, and 7 after completion of MPSS administration, with a reduction in the oxidative burst of PMNs detected on day 7. No significant changes were identified for migration. All functional changes returned to their pretreatment values by 14 days after completion of MPSS treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with a high dose of MPSS suppressed PMN functions in dogs. Analysis of these results suggested that treatment with a high dose of MPSS can suppress some of the major functions of PMNs for at least 7 days.

Priming effects of lipopolysaccharide and inflammatory cytokines on canine granulocytes.

Granulocytes play a pivotal role in natural immunity. Under inflammatory conditions, granulocytes are universally primed by several agents, such as endotoxins and inflammatory cytokines. Primed granulocytes exert potent adhesiveness, chemotaxis, phagocytosis and reactive oxygen species (ROS) production, effectively eliminating invading agents. Reactivity against priming agents is known to vary with species; however, there have been few reports on the effects of priming agents on canine granulocytes. In the present study, we assayed the priming effects of lipopolysaccharide (LPS), recombinant canine tumor necrosis factor-alpha (rcTNF-alpha) and recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) on canine granulocyte function in vitro. Isolated recombinant canine were primed with various concentrations of LPS, rcTNF-alpha and rcGM-CSF, and CD11b expression was assayed. Furthermore, actin polymerization, phagocytosis and ROS production were then assayed at primer concentrations where enhancement of CD11b expression was observed. LPS did not enhance canine granulocyte function. Phagocytosis and actin polymerization were not enhanced by priming agents; however, rcTNF-alpha and rcGM-CSF enhanced CD11b expression and ROS production in canine granulocytes. These results suggest that priming effects are mainly reflected in CD11b expression and ROS production, with rcGM-CSF and rcTNF-alpha having a priming effect similar to that observed in humans.

Effects of lidocaine hydrochloride on canine granulocytes, granulocyte CD11b expression and reactive oxygen species production.

Lidocaine hydrochloride (Lido) is widely used for analgesia in veterinary medicine; however, in humans, it has been suggested that Lido attenuates granulocyte functions, such as adhesion and reactive oxygen species (ROS) production. Thus, Lido may affect canine granulocyte function; however, there have been no reports on the effects of Lido on canine granulocyte function. Thus, we studied the effects of Lido on canine granulocyte CD11b expression and ROS production. We further studied the effects of Lido on the priming of canine granulocyte CD11b expression and ROS production by recombinant canine granulocyte macrophage colony stimulating factor (rcGM-CSF). Isolated granulocytes were incubated with 3, 30 or 300 microg/ml Lido, or with Lido followed by priming with 5 ng/ml rcGM-CSF. CD11b was detected by the immune fluorescent antibody method, and the mean fluorescence intensity (MFI) was assayed by flow cytometry. ROS production was assessed by the peak time (PT) of ROS production and area under the luminol reaction curve (AUC), which represents total ROS production quantity against opsonized zymosan stimuli. Only 300 microg/ml Lido (tissue level observed by regional block) significantly attenuated both the MFI of CD11b and its enhancement by rcGM-CSF. Moreover, at this concentration, the AUC and its enhancement by rcGM-CSF were significantly attenuated by Lido; in contrast, Lido did not affect PT. In conclusion, Lido suppressed granulocyte adhesion to the endothelium and antiseptic capability by suppressing CD11b expression and/or ROS production. Particular care should thus be exercised when performing regional anesthesia block using Lido.

Effect of fetal bovine serum and heat-inactivated fetal bovine serum on microbial cell wall-induced expression of procoagulant activity by equine and canine mononuclear cells in vitro.

OBJECTIVE: To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. SAMPLE POPULATION: Mononuclear cells from 18 horses and 3 dogs. PROCEDURES: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. RESULTS: Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPS-binding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosan-induced procoagulant activity of canine cells. CONCLUSION AND CLINICAL RELEVANCE: Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPS and zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays.

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.

Purification and characterization of canine serum ferritin-binding proteins.

Ferritin-binding protein (FBP) is known to interact with circulating ferritins in mammals. Canine FBPs were purified from canine serum by affinity chromatography and were identified as IgM, IgG, and IgA by immunoblotting with alkaline phosphatase-labeled antibodies to canine IgM, IgG, and IgA heavy chains. Following further purification by application to a Sephacryl S-300 column, canine FBPs were separated into 81.3- and 27.7-kDa bands by sodium dodecyl sulfate-polyacryamide gel electrophoresis, and the 81.3-kDa band reacted with the anti-canine IgM heavy chain antibody. Purified canine FBP bound to canine liver ferritin, but not to canine albumin and transferrin. FBP showed greater binding to the expressed bovine ferritin H-chain homopolymer than to the expressed bovine ferritin L-chain homopolymer. The binding of FBP with canine liver ferritin was dose-dependently inhibited by anti-rat liver ferritin antibody, and the anti-ferritin antibody dissociated the bound FBP in a dose-dependent manner, even after binding FBP with liver ferritin. The canine ferritin H subunit peptide fragment with amino acid residues 148-155 (NH(2)-GDHVTNLR-COOH) in its C-terminal region was recognized by FBP. These results indicate that canine serum FBPs are autoantibodies to ferritin (IgM, IgG, and IgA) and that anti-ferritin autoantibody (IgM) recognizes the C-terminal region of ferritin H subunit.

Short report: cloning of the Babesia gibsoni cytochrome B gene and isolation of three single nucleotide polymorphisms from parasites present after atovaquone treatment.

We determined the nucleotide sequence of the Babesia gibsoni cytochrome b (cytb) gene. DNA was extracted from B. gibsoni isolated from Aomori Prefecture, Japan, and 1,288 basepairs of the cytb gene, including 1,071 basepairs of the open reading frame, were sequenced. The cytb gene of B. gibsoni obtained from three dogs that had been experimentally infected with B. gibsoni and treated with atovaquone was also sequenced. The B. gibsoni cytb gene obtained from all three atovaquone-treated dogs contained a single polymorphism resulting in an amino acid change in one of the putative ubiquinone-binding sites of Plasmodium falciparum. This polymorphism was homologous to mutations in other apicomplexan protozoa that exhibit resistance to atovaquone. Two other single polymorphisms were identified in parasites isolated from two of the dogs. These results indicate that single nucleotide polymorphisms in the sequence for mitochondrial cytb gene may be associated with decreased susceptibility of Babesia species to atovaquone.

Development of a SYBR green real-time polymerase chain reaction assay for quantitative detection of Babesia gibsoni (Asian genotype) DNA.

A real-time fluorogenic polymerase chain reaction (PCR) assay based on SYBR green that allows for sensitive, reproducible, and accurate quantification of Babesia gibsoni (Asian genotype). DNA from peripheral blood of infected dogs was developed. Standard curves were created by plotting the input amount of a standard template, constructed with plasmid DNA containing 182 base pairs (bp) of the p18 gene, against threshold cycle numbers. The curves showed a wide dynamic range (1,000,000-fold input) and high correlation values (>0.99). The PCR amplification efficacy of the standard template was similar to that of intact genomic DNA obtained from peripheral blood with B. gibsoni infection. The detection limit of the assay was 9 parasites/microl of blood with B. gibsoni infection. The intra-assay and interassay coefficients of variation of the threshold cycles ranged from 0.70% to 1.89% and from 1.18% to 1.92%, respectively. This assay system was found to be reproducible and accurate for the quantification of parasite DNA in experimentally infected dogs and far more sensitive than traditional microscopic examination.