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2004 ACVP Young Investigator Award WInners

2004 ACVP Young Investigator Award Abstracts

DIAGNOSTIC PATHOLOGY

First Place:

LANGERHANS CELL HISTIOCYTOSIS IN A CAT.
M.D.M. Busch1 and P.F. Moore2. 1Department of Pathology, Wildlife Conservation Society, Bronx, NY; 2University of California, School of Veterinary Medicine: Pathology, Microbiology and Immunology, Davis, CA.

A 9-year old, female spayed, domestic shorthaired cat that presented with a chronic history of clinical airway disease was euthanized after the development of progressive respiratory signs and anorexia despite symptomatic treatment. At necropsy, the lungs were effaced by myriad, small, 0.5 to 1.5 cm diameter, irregularly shaped, tan masses. Throughout the renal cortices there were similar multifocal, variably sized, tan masses. Histologically, the lungs contained multifocal to coalescing, intraalveolar and intrabronchiolar sheets of oval to pleomorphic cells that resembled histiocytes, and which were surrounded by bands of fibrous connective tissue. Rare mitotic figures and few foci of necrosis were evident. Sheets of similar cells multifocally infiltrated the renal cortices. The proliferative cells in the lungs and kidneys expressed vimentin and CD18; they were negative for cytokeratin, CD45R, CD79a, and CD3. The cells in the lung, but not in the kidney, also expressed E-cadherin. This pattern of CD expression in combination with ultrastructural demonstration of Birbecks granules in some of the proliferating cells in the lungs and kidneys demonstrates their Langerhans cell origin and supports a diagnosis of Langerhans cell histiocytosis (LCH). The presence of large aggregates of lesional cells distinguishes LCH from reactive dendritic cell proliferative diseases. To the authors’ knowledge, this report describes the first conclusive case of Langerhans cell histiocytosis in a cat.

Second Place:

DABSKA-LIKE TUMOR IN A CHIMPANZEE (Pan Troglodytes).
D.A. Alves1, M.F. Stidworthy2, J.M. Hamilton3, J.C.M. Lewis2, D.A. Belote1, and M.G. Mense1. 1Armed Forces Institute of Pathology, Washington, D.C.; 2International Zoo Veterinary Group, Keighley, West Yorkshire, UK; and 3IDEXX Laboratories, Wetherby, West Yorkshire, UK.

In humans, the Dabska tumor (malignant endovascular papillary angioendothelioma, papillary intralymphatic angioendothelioma) is considered an extremely rare, low-grade malignancy that most often affects infants and children. It shows a wide anatomic distribution, with predilection for the skin. The medical literature suggests that these lesions recur locally and rarely spread to lymph nodes without systemic metastasis. Close clinical monitoring for regrowth or metastasis is recommended. This 4 year-old male chimpanzee (Pan troglodytes) had a rapidly appearing, approximately 75 x 50 mm in diameter, raised, cutaneous, sparsely haired, plaque-like dermal nodule in the mid-lumbar region of the back. Microscopically, within the dermis, neoplastic cells variably occlude dilated lymphatics and extend as papillary structures from the lymphatic lining. When not visibly intravascular, the cells form nodules and nests, and infiltrate the adjacent dermis. Neoplastic cells are spindled to polygonal and are supported by a moderate fibrovascular stroma that is often centrally hyalinized. Cells have variably distinct borders, scant to small amounts of eosinophilic fibrillar to amorphous cytoplasm, and oval nuclei with finely stippled chromatin and 1-2 distinct nucleoli. There is mild anisokaryosis and mitotic figures average 1-2 per high power field. Immunohistochemically, neoplastic cells are positive for vimentin, CD31, CD34, Factor VIII related antigen, and S100; and negative for kermix, keratin 903, cytokeratin 7, cytokeratin 20, epithelial membrane antigen (EMA), Ber EP4 epithelial antigen, and Melan A. To date, this lesion has not recurred after removal. To our knowledge, there are no published reports of a similar primary lymphatic neoplasm in a non-human primate.

Third Place:

DIAGNOSIS OF NECROTIZING MYOCARDITIS AND MYOCARDIAL INFARCTION DUE TO NEOSPORA CANINUM IN AN ADULT MASTIFF DOG BY MULTIPLEX PCR AND IMMUNOHISTOCHEMISTRY.
E.K. Meseck1, B.L. Njaa1, N.J. Haley3, E.H. Park4 and S.C. Barr2. Department of Biomedical Sciences4, Section of Pathology1 and Department of Clinical Sciences2, College of Veterinary Medicine, Cornell University, Ithaca, NY; Department of Microbiology, Immunology and Pathology3, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO.

A three-year-old male castrated Mastiff dog died suddenly with locally extensive, severe, acute necrotizing myocarditis and myocardial infarct. A novel multiplex polymerase chain reaction (PCR) using fresh myocardial tissue provided rapid preliminary results positive for Neospora caninum and negative for Toxoplasma gondii. PCR results were confirmed by identification of tachyzoite organisms on hematoxylin and eosin stained histologic sections and by immunohistochemistry with a murine monoclonal antibody. Immunohistochemistry for Toxoplasma gondii was negative. Myocarditis is one of several presentations of neosporosis in adult dogs, but sudden death secondary to myocardial infarct has only been previously published once. As in this case, the previously reported case involved a young adult Mastiff, which may suggest an innate susceptibility to Neospora caninum myocarditis in this breed.

EXPERIMENTAL DISEASE

First Place:

PERIPHERAL NEUROPATHY IN SIV/HIV INFECTION.
V.A. Laast, S.E. Queen, P.M. Tarwater, C. Pardo, M.C. Zink, and J.L. Mankowski. Department of Comparative Medicine, Department of Neurology, School of Medicine, Johns Hopkins University, Baltimore, MD.

Peripheral neuropathy is the most frequent neurological complication associated with HIV-1 infection, yet its pathogenesis remains undefined. Distal Symmetrical Polyneuropathy (DSP) is the most common form of peripheral neuropathy in HIV infection, affecting approximately 30-35% of HIV-infected individuals. We have developed an accelerated, consistent SIV/macaque model of HIV CNS disease with strong parallels to HIV infection in which SIV-infected macaques also develop moderate to marked multifocal ganglionitis characterized by focal and diffuse mononuclear infiltrates, neuronophagia and neuronal loss. To determine whether this SIV/macaque model paralleled HIV associated DSP, hematoxylin and eosin sections of dorsal root ganglia (DRG) and trigeminal ganglia (TG) from SIV-infected macaques euthanized during terminal infection were evaluated to identify infiltrating inflammatory cells and to assess the level of neurodegeneration or neuronal loss. To further characterize the inflammatory cell population, tissue microarrays containing DRG and TG from SIV-infected and control uninfected macaques were immunostained for SIV gp41 protein (as a measure of viral burden), and a macrophage activation marker (CD68) followed by quantitative digital image analysis. Neuronal loss was measured by unbiased stereological methods (fractional area). There was a marked increase in the number of CD68-positive macrophages in perineuronal regions in the DRG and TG in SIV-infected animals compared to controls. Further there was marked loss of neurons associated with the presence of infiltrating/activated macrophages. Macrophages are hosts for virus replication and secrete neurotoxic viral gene products as well as pro-inflammatory cytokines and chemokines that promote neurodegeneration. These studies confirm that alterations in the PNS develop in SIV-infected macaques that closely parallel those observed in HIV-infected individuals.

Second Place:

HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 MITOCHONDRIAL LOCALIZING PROTEIN p13 SENSITIZES JURKAT T CELLS TO RAS-MEDIATED APOPTOSIS.
H. Hiraragi(1,2), B. Michael(1,2), A. Nair(1,2), M. Silic-Bennussi(4), V. Ciminale(4), and M.D. Lairmore(1,2,3). (1) Center for Retrovirus Research, (2) Department of Veterinary Biosciences, (3) Comprehensive Cancer Center, The Ohio State University, Columbus, OH and (4) Department of Oncology and Surgical Services, University of Padova, Padova, Italy.

Human T Lymphotropic Virus-1 (HTLV-1), a deltaretrovirus similar to bovine leukemia virus (BLV), is the etiological agent of adult T-cell lymphoma/leukemia (ATL) and other lymphocyte-mediated inflammatory diseases. HTLV-1 is a complex retrovirus that encodes typical retroviral structural and enzymatic gene products, but also unique regulatory and accessory proteins. Using alternative splicing of its pX region, the virus encodes a series of viral regulatory and accessory proteins, including a singly-spliced open reading frame (ORF) II product p13. We have shown that selective mutations of infectious viral clones that ablate the expression of ORFII products (p30 and p13) resulted in reduced virus loads and host humoral responses in rabbits. p13 has also been shown to localize to mitochondria, and suppress cell growth and tumorgenicity in a mouse model, but its function in human lymphocytes remains undetermined. Herein, we analyzed functional properties of human lymphocytes expressing p13, using both transient expression plasmids, and lentiviral vectors stably expressing p13. Our data revealed that the p13-expressing Jurkat T cells were markedly sensitive to ceramide- and FasL-induced apoptosis in a dose-dependent manner, as measured by percentage of apoptotic cells by Annexin V staining assay. Furthermore, pre-incubation of the p13 expressing cell with farnesyl transferase inhibitor, a down-regulator of Ras, protected against cell death, providing evidence for the participation of Ras-mediated signaling in the molecular alterations induced by p13 in human lymphocytes.

Third Place:

THE ROLE OF APOPTOSIS DURING THE RECOVERY PROCESS OF THE GLOMERULAR STRUCTURE IN RAT THY-1 NEPHRITIS.
A. Haishima1, K. Inoue1, M, Murakami2 and K. Shirota1. Research Institute of Biosciences, Azabu University1, Laboratory of Molecular Biology, School of Veterinary Medicine, Azabu University2, Sagamihara, Kanagawa, Japan.

Apoptosis might play an important role in the glomerulonephritis. The purpose of this study was to investigate whether the number of apoptotic cells and the levels of gene expression of apoptosis-relating proteins, Bax and Bcl-2, alter in the renal glomeruli during the course of rat Thy-1 nephritis. We dissected kidneys at 2,3,5,7,14,28 and 56 days post-injection of rabbit anti-Thy-1 antibody. Severe mesangiolysis and influx of monocytes into the glomeruli were seen at day 2. The total cell number and PCNA-positive cells in the glomeruli significantly increased and peaked at day 7. Thereafter glomerular cells decreased and the glomerular structure returned to normal by day 56. Both Bcl-2 and Bax were localized in the mesangial area of glomeruli by immunostaining, moreover bcl-2 and bax mRNA was localized in the same area as their proteins by in situ hybridization at day 7. TUNEL-positive cells appeared at day 7 and their number increased at day 14. For quantitative analysis of the gene expression of bax and bcl-2, 200 glomeruli were dissected from the frozen sections of each kidney with a laser microdissection system. The level of gene expression of bax in the glomeruli was highest at day 14, whereas that of bcl-2 did not significantly change during the course of the nephritis. These results suggest that apoptosis might have an important role in excluding glomerular cells excessively proliferated in the glomeruli during the course of rat Thy-1 nephritis. Also, regulated expression of bax in the glomeruli might contribute to the reconstruction of the damaged glomeruli in Thy-1 nephritis.

NATURAL DISEASE

First Place:

HISTOLOGIC AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF SPONTANEOUS PITUITARY ADENOMAS IN 13 CYNOMOLGUS MACAQUES (MACACA FASCICULARIS).
A.K. Remick1, C.E. Wood2, J.A. Cann2, E.A. Feiste1, J.M. Cline2, and N.D. Kock2. Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC1 and Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Winston-Salem, NC2.

Pituitary adenomas were identified in 13 cynomolgus macaques necropsied at the Wake Forest University School of Medicine from 1994 to 2004. The affected monkeys included both males (8) and females (5) and ranged from 18 to 32 years of age. The pituitary adenomas were either focal, causing gross enlargement of the pituitary visible on postmortem examination, or multifocal microadenomas identified on histologic examination. A total of 34 adenomas were identified in the 13 macaques. Immunohistochemical stains for follicle stimulating hormone, luteinizing hormone, prolactin, human growth hormone, thyroid stimulating hormone, and adrenocorticotropic hormone were applied to pituitary tissue from all cases. Immunostaining results revealed 22/34 (64.7%) lactotroph cell adenomas, 5/34 (14.7%) plurihormonal cell adenomas, 2/34 (5.9%) corticotroph cell adenomas, 2/34 (5.9%) null cell adenomas, 1/34 (2.9%) somatotroph cell adenoma, 1/34 (2.9%) mixed corticotroph-somatotroph cell adenoma, 1/34 (2.9%) mixed lactotroph-corticotroph cell adenoma, 0/34 (0%) gonadotroph cell adenomas, and 0/34 (0%) thyrotroph cell adenomas. This study represents the first extensive retrospective case series performed to evaluate the histologic and immunohistochemical characteristics of pituitary adenomas in cynomolgus macaques. Our findings demonstrate that macaque pituitary adenomas frequently have mixed histological appearance and hormone expression. Similar to human pituitary adenomas, prolactin-secreting neoplasms were the most prevalent type.

Second Place:

THE EFFECT OF BOVINE VIRAL DIARRHEA VIRUS ON EXPRESSION OF DEFENSINS IN BOVINE TRACHEAL EPITHELIAL CELLS.
M.H. Al-Haddawi, G.B. Mitchell, R.D. Wood, B. Jefferson, and J.L. Caswell. Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.

Co-infections of viruses and bacteria are important in the pathogenesis of respiratory disease in cattle. Bovine viral diarrhea virus (BVDV) infection is a risk factor for development of shipping fever pneumonia in feedlot cattle, but the mechanisms are not well defined. We hypothesize that BVDV predisposes to bacterial pneumonia by downregulation of innate immune responses in airway epithelial cells. The aim of this investigation was to compare the effect of noncytopathic BVDV infection on the LPS-stimulated expression of tracheal antimicrobial peptide (TAP) in bovine tracheal epithelial cells. Bovine tracheal epithelial cells were isolated from healthy calves and cultured on collagen-coated wells. The primary epithelial cell line was divided into four groups in triplicate. Groups 1 and 2 were infected with BVDV after 90% confluence, and groups 3 and 4 did not receive virus. After 48 hours, groups 1 and 3 were treated with LPS. Sixteen hours later, total RNA was extracted from cells in all four groups, and TAP mRNA expression was determined by real-time PCR, relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of tracheal epithelial cells with LPS alone resulted in a 50-fold increase in TAP mRNA relative to GAPDH, whereas TAP mRNA was increased only 2-fold in BVD-infected cells exposed to LPS. These preliminary data suggest that BVDV infection inhibits the LPS-induced upregulation of TAP mRNA in bovine tracheal epithelium, and this may be a mechanism by which this virus abrogates innate immune responses and predisposes to bacterial pneumonia in cattle.

Third Place:

A SURVEY OF TESTICULAR LESIONS IN HORSES.
S.M. Birch, U. Blas-Machado, and G.R. Holyoak. Oklahoma Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK.

Because of its invasiveness, testicular tissue collection for microscopic evaluation is not routinely performed in the stallion as a common diagnostic procedure. Very little is known or has been published about the incidence of testicular lesions in the general equine population. The aim of this survey was to assess the incidence of microscopic lesions in stallions slaughtered for food consumption at a facility in Fort Worth, Texas. From March to June 2003, testicular tissue from either the left or right testis was collected from 65 adult stallions. Tissue samples were fixed in Modified Davidson's solution for 24-72 hours, processed routinely, stained with HE and evaluated microscopically. A board-certified pathologist (UBM) evaluated the distribution and graded the severity of testicular lesions present, if any, from minimal to severe. Tissue alterations were grouped as occurring in the seminiferous tubules, testicular interstitium, rete testis, epididymis, or capsule. In the seminiferous tubules, 89% of the sampled stallions had evidence of tubular degeneration and 31% had evidence of tubular atrophy. Tubular dilation, intraepithelial cysts, and intratubular giant syncytial cells characterized the degenerated seminiferous tubules. Intratubular granulomatous inflammation was present in 38%. A malignant seminoma was identified in only one stallion. Of the 65 stallions, 92% had significant tissue alterations in the testicular interstitium; these consisted of interstitial edema, Leydig cell hypocellularity, perivascular lymphocytic inflammation, and interstitial fibrosis. The majority of the lesions graded as minimal to mild; some were moderate and occasionally severe. No significant tissue alterations existed in the rete testis, epididymis, or capsule. The survey indicates that the incidence of testicular lesions in the general equine population is high but that their severity is minimal to mild.

TOXICOLOGIC PATHOLOGY

First Place:

BRONCHOSCOPIC ADMINISTRATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR INDUCES A DOSE-DEPENDENT MONOCYTE RECRUITMENT IN NEONATAL LAMBS.
D.K. Meyerholz1, B. Grubor1, T. Lazic1, J.M. Snyder2, P.B. McCray Jr.3, and M.R. Ackermann1. 1Department of Veterinary Pathology, Iowa State University, Ames, IA; 2Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA; and 3Department of Pediatrics, University of Iowa, Iowa City, IA.

Preterm birth is an increasing statistic and premature infants are more prone to respiratory distress syndrome due to lack of adequate surfactant synthesis. Vascular endothelial growth factor (VEGF) is a growth factor that stimulates vascular development. In recent studies of preterm mice, intratracheal instillation of recombinant human VEGF (rhVEGF) in mice was shown to improve clinical scores and survivability with lack of described side-effects. To further evaluate this mechanism, a preliminary study was designed in neonatal lambs to assess various rhVEGF dosages for extrapolation to a perinatal lamb model. Lambs were given 20cc of rhVEGF (dosages - 0.05 mg/ml, 0.005 mg/ml, or 0.0005 mg/ml) or sterile bovine serum albumin (BSA, 0.5 mg/ml) by sterile bronchoscope to the right pulmonary bronchus. Lung tissues were collected at 16, 24, and 32 hours post inoculation. Control lungs (with BSA) had no lesions at any time point. However, the rhVEGF treated groups had a dose-dependent deep red, well-defined consolidation in the right lung that was consistent in location with bronchoscopic deposition of rhVEGF. Microscopically, alveolar septa and alveolar spaces of lesions contained monocytes (>95%) with a small number of neutrophils. The lambs given the lowest rhVEGF dose had relatively sparse monocytic infiltration. This preliminary study demonstrates a dose-dependent and rapid recruitment of monocytes following rhVEGF administration. This acute monocytic infiltration following pulmonary rhVEGF deposition is a novel finding that warrants caution for the prophylactic or therapeutic intervention with rhVEGF in preterm infants.

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