Tamara B. Wills

Correlation of ultrasound findings, liver and spleen cytology, and prognosis in the clinical staging of high metastatic risk canine mast cell tumors.

Cytologic sampling of the ultrasonographically normal spleen and liver is not implemented routinely in the clinical staging of canine cutaneous mast cell tumors and normal ultrasound findings are often accepted as sufficient evidence for ruling out splenic or liver metastasis. Our objective was to define the specificity and sensitivity of ultrasound findings for diagnosis of mast cell infiltration when verified with cytologic evaluation, and to define the prognostic role of cytologic evaluation of liver and splenic aspirates. Dogs with a diagnosis of clinically aggressive grade II, or grade III mast cell tumor treated with a combination vinblastine/CCNU chemotherapy protocol, were selected retrospectively based on availability of cytologic evaluation of spleen plus or minus liver for staging. Out of 19 dogs, 10 dogs had a grade II tumor and nine a grade III tumor. Seven dogs had mast cell infiltration of the spleen, liver, or both. The sensitivity of ultrasound for detecting mast cell infiltration was 43% for the spleen and 0% for the liver. Dogs with positive cytologic evidence of mast cell infiltration to spleen, liver, or both had significantly shorter survival (100 vs. 291 days) than dogs without evidence of mast cell infiltration (P<0.0001). Routine splenic aspiration should be performed regardless of ultrasonographic appearance in dogs with a clinically aggressive mast cell tumor.

Effects of 6% hetastarch (600/0.75) or lactated Ringer’s solution on hemostatic variables and clinical bleeding in healthy dogs anesthetized for orthopedic surgery

OBJECTIVE To evaluate and compare hemostatic variables and clinical bleeding following the administration of 6% hetastarch (600/0.75) or lactated Ringers solution (LRS) to dogs anesthetized for orthopedic surgery. STUDY DESIGN Randomized blinded prospective study. ANIMALS Fourteen, healthy adult mixed-breed hound dogs of either sex, aged 11-13 months, and weighing 20.8±1.2 kg. METHODS The dogs were randomly assigned to receive a 10 mL kg(-1) intravenous (i.v.) bolus of either 6% hetastarch (600/0.75) or LRS over 20 minutes followed by a maintenance infusion of LRS (10 mL kg(-1)  hour(-1)) during anesthesia. Before (Baseline) and at 1 and 24 hours after bolus administration, packed cell volume (PCV), total protein concentration (TP), prothrombin time (PT), activated partial thromboplastin time (APTT), von Willebrands factor antigen concentration (vWF:Ag), factor VIII coagulant activity (F VIII:C), platelet count, platelet aggregation, colloid osmotic pressure (COP) and buccal mucosal bleeding time (BMBT) were measured. In addition a surgeon who was blinded to the treatments assessed bleeding from the incision site during the procedure and at 1 and 24 hours after the bolus administration. RESULTS Following hetastarch or LRS administration, the PCV and TP decreased significantly 1-hour post-infusion. APTT did not change significantly compared to baseline in either treatment group, but the PT was significantly longer at 1-hour post-infusion than at 24 hours in both groups. No significant change was detected for vWF:Ag, FVIII:C, platelet aggregation or clinical bleeding in either group. The BMBT increased while platelet count decreased significantly at 1-hour post-infusion in both groups. The COP decreased significantly in both treatment groups 1-hour post-infusion but was significantly higher 1-hour post-infusion in the hetastarch group compared to the LRS group. CONCLUSIONS AND CLINICAL RELEVANCE At the doses administered, both hetastarch and LRS can alter hemostatic variables in healthy dogs. However, in these dogs undergoing orthopedic surgery, neither fluid was associated with increased clinical bleeding.

Activation of the Aryl Hydrocarbon Receptor by TCDD Inhibits Mammary Tumor Metastasis in a Syngeneic Mouse Model of Breast Cancer

Treatment with aryl hydrocarbon receptor (AhR) agonists can slow or reverse the growth of primary mammary tumors in rodents, which has fostered interest in developing selective AhR modulators for treatment of breast cancer. However, the major goal of breast cancer therapy is to inhibit metastasis, the primary cause of mortality in women with this disease. Studies conducted using breast cancer cell lines have demonstrated that AhR agonists suppress proliferation, invasiveness, and colony formation in vitro; however, further exploration using in vivo models of metastasis is warranted. To test the effect of AhR activation on metastasis, 4T1.2 mammary tumor cells were injected into the mammary gland fat pad of syngeneic Balb/c mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Primary tumor growth was monitored for 4 weeks, at which time metastasis was determined. TCDD treatment suppressed metastasis by approximately 50%, as measured both in the lung and in mammary glands at sites distant from the primary tumor. Primary tumor growth was not suppressed by TCDD exposure nor was proliferation of 4T1.2 cells affected by TCDD treatment in vitro. Taken together, these results suggest that the protective effect of AhR activation was selective for the metastatic process and not simply the result of a direct decrease in tumor cell proliferation or survival at the primary site. These observations in immunologically intact animals warrant further investigation into the mechanism of the protective effects of AhR activation and support the promise for use of AhR modulators to treat breast cancer.

Mechanistic exploration of AhR-mediated host protection against Streptococcus pneumoniae infection

Streptococcus pneumoniae is a primary cause of invasive bacterial infection and pneumonia and is one of the leading causes of death worldwide. In prior studies we showed that pre-treating mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent agonist of the aryl hydrocarbon receptor (AhR), protects against S. pneumoniae-induced mortality and reduces pulmonary bacterial burden. The current studies were conducted to help elucidate the mechanism for this protective effect, and to characterize the response in the lung during the first 10h following infection. C57Bl/6 mice were treated with TCDD one day prior to intranasal infection with serotype 3 S. pneumoniae. Monitoring of bacteria in the lung airways revealed that bacterial growth was inhibited in the TCDD-treated animals within 10h of infection. To address the mechanism of this rapid protective response, macrophages, neutrophils, and invariant Natural Killer T (iNKT) cells were quantified, and levels of natural antibodies produced by B-1 B cells were evaluated. Functional assays addressed whether AhR activation reduced the capacity of lung epithelial cells to bind bacteria, and whether TCDD treatment enhanced production of antimicrobial agents in the lung or blood. None of the hypothesized mechanisms was able to explain the protective effect. Finally, the exposure paradigm was manipulated to test whether administration of TCDD after instillation of the bacteria was also protective. Results showed that TCDD must be administered in advance of exposure to bacteria, suggesting that the lung environment is rendered inhospitable to the pathogens.

Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells.

BACKGROUND Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells. OBJECTIVES The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood. METHODS Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti-CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146-positive cells were stained with fluorescein-conjugated Ulex europaeus agglutinin 1 (UEA-1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA-anticoagulated whole blood samples from 10 healthy client-owned dogs. RESULTS The anti-CD146-coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA-1-positive cells were obtained from whole blood, while >85-90% of cultured canine aortic endothelial cells were UEA-1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10-40 microm in size. Using immunomagnetic isolation, 43.4 +/- 15.6 CECs/mL (range 24-70/mL) were isolated from canine whole blood samples. CONCLUSIONS Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.

Presumptive increase in protein-bound serum calcium in a dog with multiple myeloma

An 11-year-old male castrated Australian Shepherd was presented with a history of lethargy, panting, and weight loss for 1 month. Physical examination revealed a moderately enlarged spleen. Laboratory abnormalities included thrombocytopenia and marked hypercalcemia, with hyperglobulinemia, hypoalbuminemia, and a monoclonal spike in the beta-globulin region on serum protein electrophoresis. Serum total calcium concentration was markedly increased (16.5 mg/dL, reference interval 8.9-11.4 mg/dL) but ionized calcium concentration (1.39 mmol/L) was within the reference interval (1.25-1.45 mmol/L). Isosthenuria was noted, but the dog was not polyuric or polydipsic. Serum parathyroid hormone concentration was within reference limits and parathyroid hormone-related peptide concentration was 0 pmol/L. Radiographic findings were largely unremarkable. Results of cytologic evaluation of a fine-needle aspirate specimen from the spleen indicated plasma cell neoplasia. Based on the results of the electrophoresis, splenic aspirates, radiographs, and hypercalcemia, a diagnosis of splenic multiple myeloma was made. The marked hypercalcemia, normal ionized calcium and parathyroid hormone concentrations, and lack of osteolytic lesions indicated a presumptive increase in protein-bound serum calcium, likely due to binding to molecules of the paraprotein (M protein). Protein binding of calcium in dogs with multiple myeloma should be considered as a potential mechanism of elevated total serum calcium concentration.

Glanzmann thrombasthenia in a 17-year-old Peruvian Paso mare

A 17-year-old Peruvian Paso mare was evaluated for bilateral epistaxis that had been present for at least 3 years. The mare had mild anemia, platelet count within the reference interval, unremarkable coagulation times, and a negative Coggins test. On endoscopic examination, structural abnormalities were not observed in the nasal cavities, pharynx, larynx, trachea, or either guttural pouch, but petechiation was noted in the nasal mucosa. Additional tests revealed prolonged cutaneous bleeding time, normal concentration of von Willebrand factor antigen, an abnormal clot retraction test, and failure of plalelet aggregation in response to agonists, suggesting a functional disorder of platelets. Genetic analysis indicated the horse was homozygous for a 10-base-pair deletion that included the last 3 base pairs of exon 11 and the first 7 base pairs of intron 11 of the gene encoding glycoprotein IIb. The diagnosis was Glanzmann thrombasthenia (GT) caused by a structural defect in glycoprotein IIb. GT is an autosomal recessive disorder caused by a defect in the glycoprotein IIb-IIIa complex on platelet surfaces. Separate genes encode each glycoprotein, and mutations in either gene can result in GT. This case of GT is unique given the age of the mare at the time of diagnosis. We conclude that GT, although an inherited disorder, should be considered in horses with suspected dysfunctional platelets, regardless of age.

What is your diagnosis? Vertebral mass in a dog

A 1-year-old, castrated male, mixed-breed dog was presented for sporadic episodes of kyphosis, tremors, and vocalizing. On neurologic examination, the lesion was localized to spinal cord segments T3-L3. Magnetic resonance imaging of the spine showed an expansile mass occupying most of the ventral aspect of the spinous process of T6. Fine-needle aspirates of the mass were examined cytologically. A moderately cellular population of pleomorphic spindle cells and abundant mucinous matrix were observed. The cytologic diagnosis was spindle cell neoplasia, with myxosarcoma and fibrosarcoma as the primary differential diagnoses. The dog was euthanized. Histopathologic evaluation of the mass and surrounding tissue confirmed a low-grade spindle cell sarcoma, with severe compressive myelopathy and mild neutrophilic inflammation. The neoplastic cells stained positive for mucopolysaccharides with Alcian blue, resulting in a final diagnosis of low-grade (grade 1) myxosarcoma. Fine-needle aspiration was useful in making a preliminary diagnosis of myxosarcoma in this case. Myxosarcoma should be included in the differential diagnosis for a vertebral mass in a young dog.

What is your diagnosis? Intracranial mass in a cat

A 14-year-old female spayed cat was presented with a 3-4-month history of circling to the left and intermittent head pressing. Neurologic examination findings localized the lesion to the left supratentorial region. Using magnetic resonance imaging, an extra-axial mass was found on the dorsal aspect of the brain at the level of the frontal and parietal lobes, compressing and displacing the brain ventrally and caudally. Craniectomy was performed and the mass was submitted for cytologic and histopathologic evaluation. Impression smears revealed abundant cholesterol crystals and loose clusters of mildly pleomorphic spindle cells, compatible with a meningioma. The histopathologic diagnosis was meningioma with psammoma bodies and numerous cholesterol clefts. Abundant cholesterol crystals within meningiomas in cats may present a diagnostic challenge when nucleated cells are scant. Other differential diagnoses for abundant cholesterol crystals in an intracranial mass include cholesterol granulomas and keratinizing cysts.