2005 ACVP Young Investigator Award Abstracts

DIAGNOSTIC PATHOLOGY

First Place

CORRELATIONS BETWEEN COMPUTER-ASSISTED IMAGE ANALYSIS AND HISTOPATHOLOGY IN DETECTING EQUINE TESTICULAR LESIONS. S.M. Birch1, R.A. Pierson2, U. Blas-Machado1, G.R. Holyoak1.  1Oklahoma Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK; 2Womens Health Imaging Research Laboratory of the Royal University Hospital in Saskatoon, Canada. 

Computer-assisted image analysis (CAIA) was compared with histopathology for detection and assessment of equine testicular lesions. Testes were collected from pubertal stallions (n=65) at an abattoir in the Spring of 2003. Testes were weighed, measured, and then imaged in a waterbath using a 7.5 mHz linear-array probe (Aloka SSD-900V). All images were recorded using a DVR1000 (Sony). Tissue samples taken within 1 hr. of slaughter from both sides of cranial, caudal poles and equatorial region of each testis were fixed in modified Davidson's solution, stained (H&E) and evaluated microscopically for distribution and severity of testicular lesions. Fifty-eight of 65 stallions (89%) had evidence of seminiferous tubular degeneration, 31 of 65 (48%) had evidence of tubular atrophy and 60 of the 65 (92%) had alterations in the testicular interstitium, primarily consisting of Leydig cell hypocellularity. Ultrasound images of areas concomitant to histological sample sites were acquired at 640 Å~ 480 pixel resolution and transferred to a graphics workstation. Quantitative echotextural analyses were performed. Numerical pixel values (gray-scale values from 1 to 255) and pixel heterogeneity (standard deviation of gray-scale values) of sites were measured by CAIA (Synergyne 1©, R.A. Pierson, Saskatoon, Sask., Canada) using 4 circular areas of 20 pixels in diameter placed within the image of the parenchyma. CAIA correlated highly with seminiferous tubule degeneration (p= 0.0002, r2= 0.2036), seminiferous tubule atrophy (p< 0.0001, r2= 0.2063) and Leydig cell hypocellularity (p< 0.0001, r2= 0.2964); however, no correlations were observed with inflammation of the seminiferous tubules (p= 0.3644, r2= 0.0131). Differentiating cell types was beyond the resolution of the ultrasound instrument. CAIA shows promise as a diagnostic aid in identifying testicular degenerative lesions in stallions.

Second Place

KERNICTERUS IN AN ADULT DOG.  C. Sangster1, C. Stevenson1, B. Kidney1, D. Montgomery2, A. Allen1.1Department of Veterinary Pathology, Western College of Veterinary Medicine, Saskatoon, SK; 2Department of Veterinary Sciences, University of Wyoming, Laramie, WY.

Kernicterus is characterized by yellow discoloration of brain nuclei, accompanied by degeneration of nerve cells. This condition, which is well recognized in neonatal human infants, develops following penetration of the blood brain barrier by unconjugated bilirubin. A seven-year-old, spayed female Wheaton terrier dog was referred to the Veterinary Teaching Hospital at the Western College of Veterinary Medicine following a three day history of icterus, lethargy, anorexia and occasional vomiting. Initial biochemical analysis revealed an extreme hyperbilirubinemia of 609 µmol/L (reference interval: 1.0-4.0 µmol/L) and increased activity of hepatocellular and cholestatic liver enzymes. Following a surgical liver biopsy the dog deteriorated, seizured and died two days later. Necropsy findings included profound icterus and red and yellow mottling of the liver. Yellow discoloration of the thalamic and subthalamic nuclei was detected on subgross examination of the formalin fixed brain. Histological examination of the brain revealed neuronal necrosis within the previously mentioned nuclei in addition to Purkinje cell necrosis and the presence of Alzheimer type II astrocytes, most notably in the cerebrocortical gray matter. Histological examination of the liver revealed extensive necrosis in a periacinar to bridging pattern that often extended to portal triad areas. Kernicterus in human infants is due to a combination of factors involving bilirubin production, conjugation and transport. Rarely, adult humans are reported to acquire this condition secondary to severe hepatic disease. The few reports of this disease occurring in domestic species involve neonates, namely one foal and one kitten. We report a case of naturally occurring kernicterus in an adult dog secondary to extreme hyperbilirubinemia as a result of fulminant hepatic failure.

Third Place

ESOPHAGITIS IN CAMELIDS: REPORT OF THREE CASES.  G. Gopalakrishnan, I. Pardo, V. Vemireddi, J.A. Ramos-Vara, G.C. Johnson.  Veterinary Pathobiology, Purdue University, West Lafayette, IN; Veterinary Pathobiology, University of Missouri, Columbia, MO.

Esophagitis is not well documented in camelids. We report three cases of this condition. All three animals were adults. Animal # 1 was a llama with a history of acute recumbency and good appetite. Necropsy findings included emaciation, moderate gastrointestinal parasitism and ulcerative esophagitis, more severe in the cranial third of the esophagus. Histologically, there was extensive ulceration and bacterial colonization. Animal # 2 was a camel with chronic weight loss and intermittent diarrhea. Grossly, the middle and caudal portions of the esophagus had multiple ulcers covered by fibrin. Microscopically, lesions in the esophagus were similar to animal # 1. Animal # 3 was a llama and had severe esophagitis involving the caudal 2/3 of the esophagus. Microscopically, numerous bacteria were associated with the ulcerated mucosa. This animal was diagnosed with dysautonomia. Bacterial isolation from the esophagus of animal # 1 included alpha-hemolytic Streptococcus and Clostridium subterminale; animal # 2 had heavy growth of Arcanobacterium pyogenes; Salmonella spp. was isolated from the esophagus of animal # 3. Viral isolation (esophagus, lung, intestine) and FA for multiple respiratory and enteric viruses were negative in all three animals. Immunohistochemistry for BVD in the esophageal lesions was also negative. Camelids are capable of regurgitating gastric contents and also emesis. Emesis can be the result of overloading of C-1, diaphragmatic hernia, esophageal obstruction, and ingestion of poisonous plants. Ingestion of caustic substances was suspected in one case, but the lack of oral lesions argued against it. Gastric reflux/emesis, releasing gastric acid into the esophagus was likely in the animal with dysautonomia (#3) and possibly in another case (# 2).

EXPERIMENTAL DISEASE

First Place

SLUG-MEDIATED EFFECTS OF CUTANEOUS UVR EXPOSURE. K. Newkirk, A. Parent, C. Choi, F. Duncan, D. Kusewitt. The Ohio State University, Columbus, OH.

Slug, a member of the Snail family of zinc finger transcription factors, modulates epithelial-mesenchymal transformation (EMT), the transformation of anchored epithelial cells into motile mesenchymal cells. EMT plays an important role in development, in wound healing, and in carcinoma progression and metastasis. Slug has also been found to have anti-apoptotic functions. We are characterizing the role of Slug in the development of cutaneous squamous cell carcinomas by determining the susceptibility of homozygous and heterozyous Slug knockout mice and their wild type counterparts to skin cancer induced by chronic UVR exposure. Following only two UVR exposures, however, a marked difference in the UVR response of Slug knockout and wild type mice was seen. Slug knockout mice failed to exhibit the skin erythema and peeling seen in wild type mice. This resistance to sunburn was unexpected and prompted an additional study to investigate the role of Slug in the acute response to UVR. Our findings have demonstrated a role for Slug in this process. Although the extent of UVR-induced DNA damage (cyclobutane pyrimidine dimers) was similar in all genotypes, homozygous Slug knockout mice did not show the increase in skin thickness (dermal edema) seen in the wild type mice. Both homozygous and heterozygous Slug knockout mice had markedly reduced dermal neutrophil infiltrates at 48h post-UVR compared to wild type mice. Both homozygous and heterozygous animals also had more cleaved caspase-3-positive epidermal cells than wild type animals, suggesting increased UVR-induced apoptosis. Additionally, the homozygous knockout mice demonstrated delayed expression of keratin 6 and reduced epidermal hyperplasia in response to UVR. These findings indicate an important role for Slug in the apoptotic and inflammatory response to UVR.

Second Place

14-3-3 PROTEIN REGULATES VIRAL-INDUCED NEURODEGENERATION. K.L. Helke1,2, N.J. Haughey3, J.L. Mankowski1,2. Depts of Comparative Medicine1, Pathology2 and Neurology3, Johns Hopkins University, Baltimore, MD.

Although the presence of 14-3-3 proteins in cerebrospinal fluid has been documented in several neurodegenerative diseases, the underlying pathophysiologic role of 14-3-3 in mediating neuronal damage in HIV-dementia, as well as other chronic neurodegenerative conditions, remains unknown. 14-3-3 is a ubiquitously expressed adaptor protein that is especially abundant in neurons. By binding and sequestering pro-apoptotic mediators such as Bad, 14-3-3 proteins inhibit downstream mitochondrial death pathways. Conversely, when 14-3-3 dissociates from pro-apoptotic mediators, apoptotic pathways are activated, leading to cell death. To determine whether 14-3-3 plays a crucial role in the regulation of apoptosis in neurons during chronic SIV/HIV infection of the CNS, we examined whether 14-3-3 binding to pro-apoptotic mediators decreased with development of SIV CNS disease when neuronal damage and loss occur. By co-immunoprecipitating 14-3-3- and Bad from brain homogenates and then measuring Bad levels by quantitative immunoblotting, dissociation of 14-3-3 from pro-apoptotic Bad was demonstrated in SIV-infected macaques with CNS disease versus uninfected control animals. To determine whether specific neurotoxic viral proteins disrupted binding of 14-3-3 to pro-apoptotic mediators in neurons, thereby triggering neuronal damage and apoptosis, cultured primary rat neurons were treated with recombinant HIV Tat. Viral induced neuronal damage was measured by MTS assay and Live/ Dead fluorescent assay and then compared with extent of 14-3-3 binding to pro-apoptotic mediators, including Bad measured by co-immunoprecipitation and quantitative immunoblotting. With increasing doses of Tat, neuronal damage correlated with loss of 14-3-3-Bad complex formation, indicating a central role for 14-3-3 proteins in regulating viral-induced neurodegeneration.

Third Place

SODIUM/IODIDE SYMPORTER REGULATION BY tRA, FORSKOLIN, RAS AND PI3K IN MCF-7 HUMAN BREAST CANCER CELLS. K.A.B. Knostman1, Z. Zhang2, C. C. Capen1, S.M. Jhiang3 1. Department of Veterinary Biosciences, 2 Ohio State Biochemistry Program, and 3 Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH.

NATURAL DISEASE

First Place

PROGNOSTIC SIGNIFICANCE OF INTRATUMORAL MICROVESSEL DENSITY IN CANINE SOFT-TISSUE SARCOMAS.  R. Luong, D.M. Craft, K.E. Baer, S.N. Ettinger, T.J. Scase, P.J. Bergman. Department of Pathology, The Animal Medical Center, New York NY;  Flaherty Comparative Oncology Laboratory, The Animal Medical Center, New York NY; South Bay Veterinary Specialists, San Jose CA; and Animal Health Trust, Newmarket, Suffolk, UK.

Canine soft-tissue sarcomas (STS) are a heterogenous group of neoplasms that are classified collectively, since they have similar histologic features and clinical behavior. Prognosis has been traditionally based on histologic grading, but the prognostic value of certain cellular proliferation markers has recently been demonstrated. Another method of predicting the behavior of neoplasms is intratumoral microvessel density (IMD), which is a measure of the tumor angiogenesis (intratumoral new vessel growth). The general consensus regarding IMD and tumor prognosis is that higher IMD values correlate with faster growing tumors and increased tumor metastases.  The prognostic significance of IMD has been documented in many human neoplasms and in a limited number of canine and feline neoplasms. To evaluate the prognostic value of IMD in canine STS, we studied 57 STS and compared IMD with histologic grade. Using immunohistochemistry, the STS were labeled with anti-Factor VIII-related antigen (FVIII-RA) antibodies to determine their IMD. There is positive association between increasing mean and highest microvessel counts based on FVIII-RA expression with increasing histologic grade. Mean microvessel counts were 112 vessels/mm2 (grade I), 163/mm2 (grade II), and 186/mm2 (grade III). Highest vessel counts were 134 vessels/mm2 (grade I), 201/mm2 (grade II), and 230/mm2 (grade III). Determination of IMD in canine STS using FVIII-RA immunohistochemistry is positively associated with histologic grade. Additional studies are ongoing, investigating relationships between IMD and clinical parameters, and the use of anti-CD31 antibodies to determine IMD.

Second Place

MARE REPRODUCTIVE LOSS SYNDROME: IRRADIATED EASTERN TENT CATERPILLARS INDUCE ABORTION IN MARES.  M.M. Sebastian, L.R. Harrison, W. Bernard, K. Newman, J.M. Donahue, M.L. Vickers, T.D. Fitzgerald, B. Lynn, T. Tobin.  VDIL,University of Georgia,Tifton,GA; LDDC,University of Kentucky,Lexington,KY; Rood and Riddle Equine Hospital, Lexington, KY; Venture Laboratories, Lexington, KY; Department of Biology, State University of New York, Cortland, NY; Department of Chemistry, University of Kentucky, Lexington, KY; and Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington KY.

Late instar Central Kentucky Eastern tent caterpillar (ETC, Malacosoma americanum) larvae were irradiated at 25, 30 and 40 kilo Grays to evaluate the ability of radiation to destroy viruses and bacteria but to conserve enzymatic activity. Based on the results of this initial study,  ETC larvae were irradiated in bulk at 30 kiloGrays and administered at 100g/day by oral gavage to six late term pregnant mares until they aborted or for 10 days. Non-irradiated larvae and normal saline were administered to late term pregnant mares and served as positive (n =6) and negative (n =6) controls, respectively. Three of the six mares receiving irradiated ETC aborted on experimental days 12,14, and 24, respectively. Mares receiving non-irradiated ETC (positive controls) aborted within a period of 1.2 -5 days. No negative control mare aborted. Umbilical cords from two of the mares receiving irradiated ETC had funisitis (inflammation of umbilical cord) similar to that observed in naturally occurring Late Fetal Loss / Mare Reproductive Loss Syndrome (LFL/MRLS). Actinobacillus spp and Streptococcus spp, the same bacteria which were isolated in naturally occurring MRLS abortions, were isolated from multiple organs and placenta of these fetuses. Irradiation of ETC substantially reduced the abortifacient potential of ETC larvae in pregnant mares. Abortions induced by irradiated ETC closely resembled field LFL/MRLS and the data suggest that bacterial or viral agents associated with ETC are not the primary cause of MRLS. This is also the first experimentally induced funisitis in any animal species.

Third Place

EPITHELIOID VARIANT OF HEMANGIOMA AND HEMANGIOSARCOMA IN THE DOG, HORSE AND COW.  A.L. Warren, B.A. Summers. College of Veterinary Medicine, Cornell University, Ithaca, NY.

Epithelioid hemangiomas, hemangioendotheliomas and angiosarcomas are well recognized histologic variants of human endothelial tumors that in the past have been confused with neoplasms of epithelial or histiocytic origin. We describe 11 epithelioid endothelial vascular tumors in dogs, horses and a cow that show microscopic features that are similar to these tumors in humans. Nine tumors were located within the dermis and subcutis, one in the gastrocnemius tendon and one in the skeletal muscle of the thigh. Key histologic features included the epithelioid appearance of neoplastic endothelial cells, papillary and gland-like acinar arrangements, and infrequent, though striking, cytoplasmic vacuolation of neoplastic cells forming signet ring formations that rarely contained a single erythrocyte. All tumors were positive for von Willebrand factor with variable intensity and distribution and uniformly negative for cytokeratin. Based on conventional morphologic criteria, growth pattern (invasive or not) and metastasis (known in one case at the time of biopsy), we subdivided these tumors into epithelioid hemangiomas (n = 3) and hemangiosarcomas (n = 8); none qualified as epithelioid hemangioendotheliomas. Additional follow up information obtained by written questionnaire was available for 6/11 animals and revealed local recurrence in one epithelioid hemangioma and epithelioid hemangiosarcoma and regional metastasis in one epithelioid hemangiosarcoma. This series represent a novel group of vascular endothelial tumors in domestic animals of which there are only two previous cases reported in the veterinary literature.

TOXICOLOGIC PATHOLOGY

First Place

PATHWAY OF P53-DEPENDENT TROPHOBLAST CELL APOPTOSIS IN CYTOSINE ARABINOSIDE-TREATED PLACENTA. H. Yamauchi, K. Katayama, M. Ueno, K. Uetsuka, H. Nakayama, K. Doi. Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.

Increased placental apoptosis is thought to be related with intrauterine growth retardation or other abnormal pregnancies in human. We previously reported that cytosine arabinoside (Ara-C), a teratogenic DNA damaging agent, induced trophoblast cell apoptosis in rat placenta. In the present study, we injected Ara-C into pregnant rats and mice on day 13 and 12 of gestation, respectively, to investigate the mechanisms of Ara-C-induced placental apoptosis in relation to p53, a tumor suppressor gene which is involved in the onset of apoptosis after DNA damage. In Ara-C-treated placenta, p53 protein expression was elevated and phosphorylation of p53 at serine 15 indicating activation of p53 protein was also increased. Increased expression of pro-apoptotic p53 target genes, noxa and puma, was detected with a reverse transcription-polymerase chain reaction. In p53-deficient mice, induction of apoptosis was almost completely abrogated. In addition, cDNA microarray analysis detected up-regulation of other p53 target genes such as p21 and reactive oxygen species related genes such as heat shock protein. These results indicate that Ara-C-induced placental apoptosis requires p53 activation, and they also suggest that oxidative stress mediates induction of DNA damage in Ara-C-treated placenta. Because oxidative stress is thought to affect the pathogenesis of abnormal human pregnancy, it is possible that p53 would contribute to placental dysfunction through induction of trophoblast cell apoptosis in response to DNA damage.

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