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Annual Meeting Information

ACVP Young Investigator Awards

Diagnostic Pathology

First Place:
Duncan Russell, BVMS, Cornell University

IMMUNOHISTOCHEMICAL DETECTION OF VITAMIN D RECEPTOR IN CANINE MAST CELL TUMORS. D. Russell1, K. Rassnick1, H. Erb1, M. Vaughan2, S. McDonough1. 1College of Veterinary Medicine, Cornell University, Ithaca, NY, and 2Roswell Park Cancer Inst., Buffalo, NY.
The active form of vitamin D (1-alpha, 25-dihydroxycholecalciferol; calcitriol) has potent anti-tumor activity against a number of human malignancies. Despite promising data to suggest that calcitriol could be an effective adjunct to current chemotherapy modalities, the role of calcitriol in veterinary neoplasia is poorly understood. It was recently shown that vitamin D inhibits growth of canine mast cell tumors (MCTs) in vitro, presumably due to ligand-mediated activation of the vitamin D receptor (VDR). The aim of the current study was to investigate VDR expression in non-neoplastic cutaneous mast cells and MCTs by means of immunohistochemistry, using a monoclonal antibody specific for VDR. Target sample size is 8 non-neoplastic samples and 75 MCTs. To date, VDR expression has been evaluated in 2 canine dermatopathies containing non-neoplastic mast cells, and 18 cutaneous MCTs. Nuclear VDR immunostaining was identified in both samples of non-neoplastic cutaneous mast cell infiltrates. Five grade I MCTs, 6 grade II MCTs, and 7 grade III MCTs were examined and nuclear immunostaining was detected in all samples. Non-neoplastic mast cell samples had weak and moderate VDR immunostaining. In MCTs, VDR immunostaining was weak to moderate in 9 samples and intense in 9 samples. This data supports previous in vitro findings, and might indicate a role of VDR in canine mast cell neoplasia. In the future, immunohistochemical demonstration of VDR in biopsy specimens could be used to select appropriate patients for vitamin D therapy.

Second Place:
Marcia Ilha, DVM, MS, University of Tennessee

REPRODUCTIVE PATHOLOGY OF 32 FEMALE VIETNAMESE POTBELLIED PIGS. M. R. S. Ilha1, S. J. Newman1, S. van Amstel2, K. Fecteau3, B. Rohrbach3. 1Department of Pathobiology, 2Department of Large Animal Clinical Sciences, 3Department of Comparative Medicine, University of Tennessee, College of Veterinary Medicine, Knoxville, TN.
Thirty-two female Vietnamese potbellied pigs from local rescues were spayed and the reproductive tracts submitted for gross and histological examination. The pig’s ages varied from four months to 19 years. Abdominal enlargement was the only clinical sign noted in four pigs. Eight pigs with normal uterine histology were used as controls. The 24 remaining pigs had diffuse cystic endometrial hyperplasia (CEH). Additionally, 14 pigs between the ages of 5-19 years had single or multiple spindle cell tumors (SCT) weighing up to 24.4 kg in the uterine horns or broad ligament. These SCTs included fibroleiomyomas (1), leiomyomas (11), and leiomyosarcomas (7). Endometrial mucosal masses were present in ten pigs, three of which had concurrent SCTs, and were classified as adenocarcinomas (2), adenomas (12), and adenomyosis (9). Pyometra and endometritis were present in three pigs. Additional findings included multiple ovarian follicles, corpora lutea, ovarian cysts, parovarian cysts and cysts within the oviduct wall. Immunohistochemistry for estrogen (ER) and progesterone receptors (PR) was performed in all uterine sections, and SCTs were stained for smooth muscle actin (SMA). In CEH, adenomyosis, and adenomas, about 70% of epithelial nuclei expressed ER and PR; in adenocarcinomas, the expression was 20%. Six adenomas had multifocal areas of squamous differentiation that did not stain for ER or PR. All SCTs were strongly SMA positive, and greater than 50% of nuclei expressed ER and PR. Endogenous estrogen and progesterone hormones play an important role in the development of uterine pathology in intact potbellied pigs. ER and PR nuclear expression is reduced with malignant epithelial transformation.

Third Place:
Gillian Shaw, DVM, MS

CHITINOLYTIC MYCOTIC SHELL DISEASE AND BRANCHITIS IN CAPTIVE AMERICAN HORSESHOE CRABS. G. Shawa1, K. Brennan1, T. Southard1, S. Trembley1, D. Huso1, C. Morrell1, K. Tuxbury3, R. Montali1, N. Kwiatkowski2, W. Merz2, J. Mankowski1. 1 Department of Molecular and Comparative Pathobiology, and 2Department of Pathology, Johns Hopkins University, Baltimore, MD, and 3National Aquarium, Baltimore, MD.
Tissues from 22 American horseshoe crabs (Limulus polyphemus) from the National Aquarium in Baltimore were submitted after the crabs died or were euthanized because of shell and book gill lesions. Carapace lesions were present on both the dorsal and ventral prosoma of all animals, and consisted of variably sized, circular, tan lesions, which occasionally had associated softening of the shell. The book gill leaflets and occasionally the opercula also contained multifocal, variably sized, yellow to off-white, circular lesions, which were occasionally proliferative. Histologically, the carapace lesions contained variably pigmented, septate, irregularly branching fungal hyphae located on the surface and within the cuticle of the carapace and extending into the sub-cuticular regions in some areas. Fungal hyphae were associated with cuticular necrosis and large numbers of viable and degenerate amebocytes. Mixed bacterial populations were variably present in association with the lesions. Individual book gill leaflets were infiltrated by fungal hyphae (morphologically similar to those in the carapace) accompanied by viable and degenerate amebocytes and necrotic debris. Cultures of gill lesions of two individuals yielded Fusarium solani isolates. Other findings include encysted metazoan parasites, necrotizing myositis and cellulitis, hepatopancreatitis, and erosive lesions of appendages. These findings demonstrate that concurrent mycotic branchitis and dermatitis are serious health issues for captive horseshoe crabs.

Experimental Disease

First Place:
Chelsea Martin, DVM, Ohio State University

EFFECT OF ZOLEDRONIC ACID THERAPY IN AN IN VIVO MODEL OF BONE INVASIVE FELINE ORAL SQUAMOUS CELL CARCINOMA. C. K. Martin, J. L. Werbeck, N. K. Thudi, L. M. Gooding, T. D. Wolfe, R. E. Toribio, T. J. Rosol, Departments of Veterinary Biosciences and Veterinary Clinical Sciences, Ohio State University, Columbus, OH.
The hypothesis of this study is that zoledronic acid will inhibit tumor-induced osteolysis and reduce tumor growth and invasion in a xenograft mouse model of bone invasive feline oral squamous cell carcinoma (FOSCC). Squamous cell carcinoma is the most common malignancy of the feline oral cavity and is frequently associated with osteoclastic bone resorption. Zoledronic acid (ZOL) is a nitrogen-containing bisphosphonate which inhibits osteoclastic bone resorption. A luciferase-expressing FOSCC cell line (SCCF2Luc) was injected into perimaxillary submucosa of 30 athymic nude mice. Mice were treated with 100Âµg/kg zoledronic acid, or vehicle, twice weekly for 4 weeks. ZOL treatment mildly reduced tumor growth (bioluminescence), prevented loss of bone volume and surface area (ÂµCT), and was associated with reduced osteolysis and increased periosteal new bone formation (ÂµCT, faxitron radiography and histomorphometry). ZOL had no effect on tumor invasion around the incisor or into the nasal cavity. Numerous large tartrate-resistant acid phosphatase-positive osteoclasts were observed at the tumor-bone interface in both vehicle and ZOL-treated mice, however osteoclasts in ZOL-treated mice had evidence of degeneration characterized by increased cytoplasmic vacuolation. The results demonstrate that ZOL-mediated inhibition of osteolysis was independent of osteoclast activation and maturation. ZOL may have reduced the rate of osteoclast-mediated dissolution of bone matrix, or shortened osteoclast life span. ZOL reduced FOSCC-induced osteolysis and bioluminescence, but invasive behavior was unchanged. ZOL will be valuable as an adjuvant therapy for FOSCC for maintaining mandibular and maxillary bone volume and function.

Second Place:
Janildo Reis, Jr., DVM, University of Georgia

DETECTION OF VESICULAR STOMATITIS NEW JERSEY VIRUS IN EXPERIMENTALLY INFECTED CATTLE USING SITU HYBRIDIZATION AND IMMUNOHISTOCHEMISTRY. J. Reis1, L. Rodriguez1, D. Mead2, G. Smoliga2 C. Brown1, 1University of Georgia; 2Plum Island Animal Disease Center, ARS-USDA.
Vesicular stomatitis in cattle can be produced experimentally by inoculation at the coronary band, via scarification with virus culture or through feeding of infected black flies. The inoculating dose required to produce infection is approximately 1,000 times lower when using flies as compared to scarification. In this study, tissues taken from cattle experimentally infected with each method were compared, using histopathology, immunohistochemistry, and riboprobe in situ hybridization to detect pathologic damage, presence of viral protein, and viral replication. Cattle were euthanized 24 hours after infection and tissues collected into formalin. Epithelial disruption of the coronary bands was similar in both - with the development of vesicles seen histopathologically. These vesicles contained abundant replicating virus as detected by in situ hybridization and viral protein, as detected by immunohistochemistry. However, there were marked differences in the draining lymph nodes, where those cattle infected by fly bite had many more infected cells. These cells were positive using in situ hybridization with a negative sense riboprobe indicating active viral replication. Cells were morphologically consistent with antigen presenting cells. This preliminary study shows for the first time active viral replication in lymph nodes and provide further evidence that black flies have a facilitating component for viral replication in host cells.

Third Place:
Jennifer McCleese, DVM, Ohio State University

A NOVEL HSP90 INHIBITOR, STA-12-1474, EXHIBITS BIOLOGIC ACTIVITY AGAINST OSTEOSARCOMA. J. K. McCleese1, W. Ying2, J. Barsoum2, C. A. London1. 1Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH and 2Synta Pharmaceuticals Corp., Lexington, MA
Abstract Osteosarcoma (OSA), the most common malignant bone tumor in dogs and children occurs with a higher frequency in the canine (10,000 cases/yr) versus the human (1,000 cases/yr) population. In both species, no significant improvements in treatment have been made for over 15 yrs. Heat shock protein 90 (Hsp90), a molecular chaperone responsible for ensuring several kinases, hormone receptors, and transcription factors maintain an active functional state, represents an attractive therapeutic target for cancer since many of its client proteins contribute to tumor growth and survival. We hypothesized that Hsp90 exists in a multi-chaperone active complex in OSA cells, allowing selective targeting of malignant cells, promoting client protein down-regulation and subsequent cell death. To test this, we used a novel Hsp90 inhibitor, STA-12-1474, to block ATP-dependent Hsp90 activity. Our data demonstrate dose and time dependent inhibition of canine and human OSA cell viability ultimately resulting in apoptosis, with enhanced sensitivity for malignant compared to normal canine osteoblasts. Hsp90 was associated with co-chaperones p23 and Hop in canine OSA cells but not normal canine osteoblasts, indicating formation of an active complex in the malignant cells. Inducible Hsp70 levels increased with STA-12-1474 treatment, consistent with induction of cellular stress following Hsp90 inhibition. STA-12-1474 promoted down-regulation of rhHGF induced p-Met and Met as well as, p-Stat3, p-Akt and Akt, while total Stat3 levels remained unchanged. Finally, no additive or synergistic activity was identified when STA-12-1474 and doxorubicin were combined. In summary, these data indicate that Hsp90 represents a relevant target for therapeutic intervention in OSA, although strategies combining chemotherapy with Hsp90 inhibition require further evaluation.

Natural Disease

First Place:
Chelsea Himsworth, DVM, University of Saskatchewan

DESTRUCTIVE POLYARTHRITIS IN ABORTED BOVINE FETUSES: A POSSIBLE ASSOCIATION WITH UREAPLASMA DIVERSUM INFECTION? C. Himsworth1, J. Hill2, Y. Huang1, E. Waters3, G. Wobeser1. 1Department of Veterinary Pathology, 2Department of Veterinary Microbiology, Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada, 3Prairie Diagnostic Services Inc., Saskatoon, Saskatchewan, Canada.
Irregularly distributed, severe, erosive polyarthritis was diagnosed in five aborted, bovine, near-term fetuses. Ureaplasma spp. was identified in four cases by PCR or culture, and genotyping of PCR product from one case confirmed the presence of Ureaplasma diversum specifically. Fetal arthritis and Ureaplasma diversum-associated joint pathology have not been previously reported in the veterinary literature; however, other Ureaplasma spp. are known to cause reproductive disease and arthritis in humans. We suggest that U. diversum infection may be associated with the arthritis seen in these fetuses, and that thorough examination of multiple fetal joints should be part of the routine pathological examination of the bovine fetus.

Second Place:
Teresa Southard, DVM, PhD

SPONTANEOUS CUTANEOUS AND SUBCUTANEOUS TUMORS IN AGED MALE LONG-EVANS RATS. T. Southard, B. Simons, C. Brayton. Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD.
Skin and subcutaneous masses were the most common reason for pathology submissions from a colony of aged Long-Evans (L/E) rats used in behavioral studies. Masses that were amenable to surgical removal were submitted as biopsies. If the mass could not be surgically removed, the rat was euthanized and submitted for necropsy. The rats were retired male breeders and ranged in age from 14 months to 2 years. More than 100 cutaneous and subcutaneous masses from this colony were examined from 2003 to 2008, and histopathological diagnoses showed a predominance of mesenchymal lesions over nonmammary epithelial lesions. The most common diagnosis was fibroma (32 rats). Other benign tumors of mesenchymal origin included peripheral nerve sheath tumors/neurofibromas, lipomas and a single fibrolipoma. Malignant mesenchymal tumors included fibrosarcomas, myxosarcomas, undifferentiated sarcomas and one histiocytic sarcoma. The most common epithelial lesions were epidermal inclusion cysts. Benign epithelial neoplasms included pilomatricoma, sebaceous adenomas and infundibular keratinizing acanthomas. Malignant epithelial tumors were rare and consisted of one squamous cell carcinoma and one sebaceous carcinoma. Because small tumors may go unnoticed by investigators and animal care staff, this is not a true incidence study; however, the high proportion of mesenchymal tumors in these rats contrasts with a previous report of a high incidence of epithelial tumors in this strain.

Third Place:
Jennifer Hughes Hanks, DVM, MS

HEPATITIS AND ENTERITIS CAUSED BY A NOVEL HERPES VIRUS IN TWO MONITOR LIZARDS (VARANUS SPP.). J. Hughes-Hanks, S. Schommer, S. Younger, W. J. Mitchell, D. Shaw. Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, MO.
Reported cases of herpesviral-induced disease are uncommon in reptiles, with the exception of the chelonians. Associated lesions include stomatitis, hepatitis, and papillomatosis. Two monitor lizards presented for postmortem examination with a history of rapid death. Tan, 1-2 mm foci were disseminated on the serosal surface of the intestine. The small and large intestines contained viscous white material. Multifocal, tan pinpoint foci were present on the surface of the liver. Histologically, there were multifocal areas of necrosis in the lamina propria of the small intestine and in the liver. Many degenerate cells surrounding the necrotic foci contained large amphophilic intranuclear inclusion bodies which peripheralized the chromatin. Enveloped icosahedral virions consistent with herpesvirus were detected by electron microscopy. A 180bp fragment was amplified from samples of small intestine and liver using conserved herpesvirus primers targeting the DNA polymerase. The sequence of the fragment was determined to be most closely related to Varanid Herpesvirus 2 (80% nucleotide identity, 86% amino acid identity). Based on histological and molecular findings, a novel pathogenic herpesvirus of Varanid lizards is proposed.

Toxicologic Pathology

First Place:
Kimberly Maratea, DVM, MS, Purdue University

SHORT-TERM INTERFERENCE WITH TGF-BETA SIGNALING CAUSES DELAYED DEVELOPMENT OF HEART VALVE DISEASE IN SPRAGUE-DAWLEY RATS. K. A. Maratea1, H. HogenEsch1, K. B. Donnelly2, R. Y. Reams2, J. F. Van Vleet1, P. W. Snyder1. 1Department of Comparative Pathobiology, Purdue University, West Lafayette,IN, 2Lilly Research Laboratories, Greenfield, IN.
Coordinated signaling of multiple transforming growth factor-beta (TGF-β) superfamily members is known to have a critical role in heart valve development. A novel, rat model of valve disease induced by a small molecule TGF-β type I receptor kinase inhibitor (LY2109761) supports the hypothesis that dysregulation of TGF-β signaling is a mechanism underlying the pathogenesis of heart valve disease. Young Sprague-Dawley rats were treated with LY2109761 for 4 consecutive days; tissues were collected on days 2, 5, 14, and 28. Microscopic valve lesions characterized by leaflet thickening and myxomatous degeneration were observed on days 14 and 28. Valve lesions were accompanied by accumulation of α-SMA-positive, activated valve interstitial cells (VIC) in the 14-day group. The Ki-67 proliferation index was increased in treated valves on days 2, 5, and 14. Changes in gene expression were consistent with upregulated TGF-β signaling on days 5 and 14, and increased bone morphogenic protein (BMP) signaling on day 14. Additionally, expression of multiple genes associated with extracellular regulation of TGF-β superfamily signaling was significantly decreased on day 2. The effect of treatment on cell proliferation, VIC activation, and expression of TGF-β-related genes appears to be temporary, since these parameters were comparable to control valves at day 28. Microscopic valve lesions, however, did not regress by day 28, indicating they may be irreversible. These findings demonstrate that short-term disruption of TGF-β signaling with LY2109761 predisposes heart valves to pathological remodeling, and suggest that precise regulation of multiple TGF-β superfamily members and downstream effectors is required to maintain postnatal valve architecture.

Second Place:
Angela Brice, DVM, Johns Hopkins University School of Medicine

MINOCYCLINE ARRESTS CD4+ T CELLS IN THE G0/G1 PHASE OF THE CELL CYCLE AND INCREASES THE ACTIVATION THRESHHOLD. A. K. Brice, D. R. Graham, M. C. Zink. Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD.
Proteins such as cMyc, cyclin D1, CDK4 and pRb are essential for progression through the G0/G1 and S phases of the cell cycle. Minocycline, a semisynthetic tetracycline derivative, decreases CD4+ T cell proliferation and IL2 responsiveness. The mechanism for this immunosuppression has not been clearly elucidated. We demonstrated that minocycline-treated CD4+ T cells were arrested in the G0/G1 phase and had significantly lower expression of proteins necessary for G0/G1 phase progression including cMyc, cyclin D1, CDK4 and pRb. We further showed that minocycline-treated CD4+ T cells had significantly lower surface CD25 expression following stimulation by anti-CD3 and anti-CD28 antibodies as compared to untreated controls. High levels of exogenous IL-2 followed by higher concentrations of stimulating antibodies significantly increased surface CD25 expression in treated cultures. These data suggest that minocycline targets the G0/G1 phase of the cell cycle through the reduced expression of key cell cycle regulators and that minocycline-treated T cells have a heightened threshold for activation. Since HIV relies on host cell cycle progression for its life cycle, this may explain our and others finding that minocycline suppresses HIV replication.

Third Place:
Tzu-Yin Lin, DVM

OSUHDAC-42, A NOVEL HDAC INHIBITOR, EXHIBITS BIOLOGIC ACTIVITY AGAINST HUMAN AND CANINE MALIGNANT MAST CELLS THROUGH BOTH HISTONE DEPENDENT AND HSP90 DEPENDENT PATHWAYS. T-Y. Lin1, M. Sridhar2, S. Fossey1, C. A. London1,2. 1Department of Veterinary Biosciences, 2Department of Veterinary Clinical Sciences. Ohio State University, Columbus, OH.
Epigenetic changes including DNA hypermethylation and histone hypoacetylation are found in many cancers. Evidence suggests that inhibition of histone deacetylation is a promising approach to modulating some of these epigenetic changes in both solid tumors and hematologic malignancies. Preliminary data indicate that histone deacetylase inhibitors (HDACis) may have biologic activity against malignant mast cell disease. The purpose of the following studies was to investigate the histone-dependent and histone-independent mechanisms of HDAC inhibition in malignant mast cells. Briefly, human and canine malignant mast cell lines were treated with OSUHDAC-42 (a novel phenylbutyrate-based HDACi) and 17-AAG (an HSP90 inhibitor), and cell viability, cell cycling, cell invasion, and the effects on several signaling pathways were evaluated. Treatment with OSUHDAC-42, but not 17-AAG, induced hyperacetylation of H3, H4 and alpha-tubulin along with upregulation of p21. Both OSUHDAC-42 and 17-AAG downregulated the expression of pKit, total Kit, pAkt, total Akt, and total HER2/Neu in a time-dependent and reversible manner. 17-AAG downregulated both pSTAT3/STAT5 and total STAT3/STAT5, but OSUHDAC-42 exhibited variable effects on these targets. OSUHDAC-42 and 17-AAG induced dissociation between HSP90 and Kit and subsequent upregulation of HSP70 expression following loss of HSP90 activity. Additionally, treatment of the malignant mast cells with OSUHDAC-42 induced loss of cell viability, cell cycle arrest and apoptosis via activation of caspase3/7. Lastly, OSUHDAC-42 and 17-AAG blocked malignant mast cell invasion and downregulated MMP2 expression. In conclusion, the novel HDACi OSUHDAC-42 exhibits biologic activity against malignant mast cells through both histone-dependent and HSP90-dependent pathways, representing a promising new therapeutic approach for mast cell disease.

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