Abstract
1: CLINICAL RELEVANCE OF THE WORLD HEALTH ORGANIZATION CLASSIFICATION OF LYMPHOID NEOPLASMS IN DOGS.
The current (2001) World Health Organization (WHO) classification scheme is recommended for human lymphoid neoplasms. This system is based primarily on the clinical presentation together with the immunophenotype, anatomic site, morphology, cytogenetics, and clinical aggressiveness. Sixty-two dogs seen at the University of Florida Veterinary Medical Teaching Hospital with lymphoproliferative disease were identified using cytologic or histologic evaluation. Patients were staged (I–V) and substaged clinically (a or b) according to the WHO staging system. Specimens were immunophenotyped (T or B) by a battery of cell surface markers (CD3, CD4, CD8alpha, CD18, CD21, CD45RA, CD79a, IgG) and assigned a category according to the WHO lymphoid classification. Survival time derived from the date of initial diagnosis until death or last known contact date was compared as median days for each category. Patients were treated with the Wisconsin chemotherapy protocol (49), prednisone only (7), lomustine (1), or no treatment (6). Highly aggressive conditions (< 50 days survival) included 14 Diffuse Large B-cell, subtype b (24 days), 3 B-Lymphoblastic (48 days), 3 T-Lymphoblastic (17 days), and 7 Peripheral T-cell, subtype b (17 days). Aggressive conditions (> 50 < 150 days survival) included 30 Diffuse Large B-cell, all subtypes (139 days), 9 Peripheral T-cell, all subtypes (56 days), and 3 Subcutaneous Panniculitis-Like T-cell (75 days). Indolent conditions (> 150 days survival) included 16 Diffuse Large B-cell, subtype a (314 days), 2 B-Splenic Marginal Zone (161 days), 2 Peripheral T-cell subtype a (328 days), 2 T-LGL leukemia (218 days), and 2 Hepatosplenic T-cell (174 days). Data indicate immunophenotype alone is not an important prognostic factor for survival time compared with the new WHO classification by disease category, since frequent overlap of B and T cases occurs within survival groups.
2: FIV INTER-SUBTYPE RECOMBINATION CREATES CHIMERIC VARIANTS OF EPIDEMIOLOGICAL IMPORTANCE IN ONTARIO.
Genetic variation in lentiviruses, such as the feline immunodeficiency virus (FIV), is extensive in part due to the lack of proofreading activity of the reverse transcriptase that allows frequent mutations. Also, recombination is known to occur at a very high rate, but since the 2 RNA strands of the viral genome are usually identical, homologous recombination is usually inconsequential. However, when a cell is co-infected with more than one FIV subtype, inter-subtype recombination may result, thus creating new and more complex variants faster than mutations will. In this study, we investigated inter-subtype recombination in samples from FIV-infected cats from Ontario. Sequencing of 30 % of the proviral genome, including fragments of the LTR-gag and env-LTR regions, identified A/B recombinants in more than 10% of infected cats. Recombination was assessed by inconsistent clade assignment in phylogenetic trees, visual inspection of sequences, and analysis of similarities to reference sequences of the subtypes known to be prevalent in Canada (A, B and C). Patterns of recombination were identified on the basis of homologies to reference sequences. A common recombination site was identified in gag, specifically at the junction between the regions coding for the p15 and p24 proteins. These findings indicate that inter-subtype recombination contributes to the genetic diversity of FIV strains in Ontario. The identification of common patterns of recombination in samples from unrelated cats suggests that a circulating recombinant form of FIV might be transmitted among cats in Ontario. These findings have implications for DNA diagnosis of FIV infection and for vaccination strategies.
3: THE MOLECULAR BASIS FOR GLANZMANN'S THROMBASTHENIA IN A QUARTERHORSE.
A two-year old quarter horse presented with epistaxis to the Auburn University, College of Veterinary Medicine. Initial findings indicated that the horse had normal coagulation screening test results, normal platelet numbers, and normal vWF antigen levels. Clot retraction and platelet aggregation responses to ADP, collagen, platelet activating factor, and thrombin were impaired. Flow cytometric studies indicated a reduction in the alpha IIb-beta 3 complex on platelet surfaces. Based on these findings, a diagnosis of Glanzmann's thrombasthenia was made. Glanzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder described in humans and dogs that is caused by a quantitative or qualitative deficiency in the platelet glycoprotein complex alpha IIb-beta 3. A mutation in either of the genes encoding for the alpha IIb or beta 3 subunits can result in GT. The purpose of this study was to determine the normal cDNA sequences encoding for the alpha IIb and beta 3 subunits in horses and compare the normal equine cDNA sequence to the sequence obtained from the affected horse. First-strand cDNA synthesis was performed using total RNA isolated from normal and affected equine platelets. cDNA was amplified via PCR and subjected to electrophoresis on agarose gels. Harvested bands were sequenced directly. The cDNA sequence obtained from the GT horse showed a single guanine to cytosine substitution at position 122 in exon 2 of the gene encoding for alpha IIb. This change would result in the substitution of a proline for an arginine. This represents the first case of GT to be characterized at the molecular level in a horse.
4: IDENTIFICATION OF ABERRENT EXPRESSION OF CD79A IN CANINE LYMPHOMA SAMPLES BY FLOW CYTOMETRY.
Determination of the lineage of canine lymphomas has important prognostic value, since T cell lymphomas have a poorer prognosis than B cell lymphomas. Flow cytometric methods of immunophenotyping have gained increased popularity because fresh samples can be analyzed and multiple CD molecules can be assessed simultaneously. We adapted a flow cytometry technique to immunophenotype fine needle aspirates of lymph node samples from 37 dogs with confirmed lymphoma (WHO stages III-V, substage a or b). An indirect immunofluorescent procedure was performed using a panel of monoclonal antibodies to canine cluster differentiation (CD) molecules. CD3, CD4 and CD8 surface molecules were used to define T cell lineage. CD79a (cytoplasmic portion of the B cell receptor) and CD21 were used to define B cell lineage. Twenty-two cases were determined to be B cell lineage (CD79a+ CD3-), six T cell (CD3+CD79a-) and nine cases had CD3 and CD79a expression. Because immunophenotyping results of CD79a and CD3 expression provided a diagnostic dilemma, PCR testing to identify T cell receptor or immunoglobulin gene rearrangements were performed on slide preparations. PCR results identified T cell clonality in three of the cases with CD79a and CD3 expression and multi-systemic disease. We speculate that these cases represent an aggressive form of T cell lymphoma similar to that reported for humans and in a recent canine case with chromosomal aberrations. Supportive evidence for T cell origin in two cases was reflected in the expression of CD4, but not CD21. When performed in parallel these diagnostic assays (immunophenotyping of fine needle aspirates by flow cytometry and PCR for antigen receptor rearrangement) improve our diagnostic ability to determine the lineage of canine lymphomas and to identify aberrant expression of CD molecules.
5: EVALUATION OF PANCREAS SPECIFIC LIPASE ASSAY UTILIZING THE SUBSTRATE 1,2-O-DILAURYL-RAC-GLYCERO-3-GLUTARIC ACID- (6METHYL RESUROFIN)-ESTER (DGGR) FOR THE DIAGNOSIS OF CANINE PANCREATITIS.
Lipase is an enzyme produced mainly in the pancreatic acinar cells. Increased serum lipase activity has historically been used to support the diagnosis of acute pancreatitis, a common disease in dogs that varies from mild to severe. Most of the lipase assays that are currently in use lack optimum sensitivity and specificity. Therefore, a new colorimetric method (DGGR) for determination of serum lipase in dogs is being evaluated on the Hitachi 911 analyzer. The kinetic evaluation of the DGGR assay showed low Km and high Kcat and Vmax, which indicates good substrate specificity for pancreatic lipase. Precision, linearity and interference are being evaluated in addition to determination of the canine reference range and stability of lipase activity using the DGGR assay. The linear range of the assay is between 19.6 and 862 mg/L, showing a R2 of 0.9948. The precision studies are being performed in three steps: within run, within day, and day to day for 20 days using three serum pools; low, normal (within established reference range), and high. The within-run precision study showed coefficients of variation (CV) of 4.318%, 2.445% and 1.524% for the respective pools. The within-day precision had CVs of 2.588% (low), 1.96% (reference range) and 2.26% (high). Data from the interference, stability, and reference range studies are ongoing and will be presented. Concurrent to the method validation studies, a prospective study of dogs with naturally occurring pancreatitis based on history, clinical signs, and ultrasonographic findings, was also undertaken. The sensitivity and specificity of the DGGR assay for diagnosing pancreatitis will be presented and compared to the existing assay.
6: APTAMER SELECTION FOR THE O-POLYSACCHARIDE OF E. COLI O157.
Background: Critical to public health diagnostics in a climate of potential bioterrorism are robust diagnostic tools. The objective of this project is to discover whether aptamers can be used to detect E. coli O157, a recognized bioterrorism threat and causative of hemorrhagic colitis. Aptamers are oligonucleotides that bind targets specifically by ligand affinity. Oligonucleotides are traditionally employed as hybridization probes. In contrast, aptamers bind targets on a structural basis. Aptamers will be selected that bind the O-polysaccharide, a unique linear tetrasaccharide, of E. coli O157. Methods and Results: The lipopolysaccharide (LPS) target was isolated by a modified hot phenol extraction. LPS was biotinylated and bound to streptavidin-coated paramagnetic particles. The appropriate load of biotinylated LPS for the streptavidin-coated particles was determined in an ELISA-like format. Fully substituted LPS represented the positive target, and an identically prepared LPS lacking O-poly-saccharide extracted from an O157 mutant represented the negative target. Aptamers specific for the positive target as well as the negative target were excluded, leaving a subtracted pool specific for the O-polysaccharide. Aptamers were selected from a random oligonucleotide pool using SELEX systemic evolution of ligands by exponential enrichment—an in vitro iterative protocol of isolation and PCR amplification of aptamers possessing affinity for the ligand. Binding conditions were increasingly stringent in each round of SELEX. Twelve rounds of SELEX were completed; the enriched pool exhibited two-fold affinity for E. coli O157 LPS over E. coli O55 LPS. Twenty-eight aptamers were sequenced and exhibited a conserved GTG motif and GC content of 57%. Conclusion: Aptamers from the enriched pool exhibit conserved sequence motifs and higher affinity for the O157 O-polysaccharide. The selection strategy is valid for further enrichment.
7: CULTURE AND CHARACTERIZATION OF FELINE DENDRITIC CELLS.
Dendritic cells (DC), the most potent antigen-presenting cells in the immune system, are critical in the initial activation and recruitment of resting T lymphocytes. Research suggests that DC are one of the initial cells to encounter immunodeficiency-inducing lentiviruses such as the human, simian, and feline immunodeficiency viruses (HIV, SIV, FIV). Thus, DC likely have a major role in both immune defense and viral propagation during the initial phases of all lentivirus infections. To gain insight into the role of DC in FIV infection and immunity, we developed methods to culture feline DC from CD14+ monocytes using a combination of recombinant human (rh) GM-CSF and rhIL-4. These cells demonstrated increased expression of markers previously shown to be present or enriched on DC, including MHC II, CXCR4, CD11c, and CD1a. Concurrently, expression of CD14 was decreased by comparison with feline macrophages cultured in the presence of rhGM-CSF alone. Morphological features typical of DC were observed, most importantly the presence of abundant fine cytoplasmic processes. Cultured feline DC demonstrated increased expression of IL-12 mRNA but similar uptake of FITC dextran by comparison with macrophages. Finally, feline DC induced an allogeneic mixed leukocyte reaction as indicated by marked uptake of tritiated thymidine at ratios of 1:30 or 1:60 ratios of DC to responder T cells. Feline DC will be used to stimulate immunity against FIV infection.
8: VALIDATION OF A BAYESIAN NETWORK CREATED FROM FUNCTIONAL MECHANISTIC EXPLANATIONS EMBEDDED WITHIN SNOMED.
SNOMED is applicable for use in electronic medical record systems for representing clinical information such as diagnoses and laboratory results. Bayesian prediction models in medicine are well suited for predicting outcomes (diagnoses) along pathways of causation given relevant pieces of evidence (laboratory results). While SNOMED does represent clinical information very well, it does not currently provide such pathways of causation between diagnoses and laboratory concepts. This research reports on an approach to enhance SNOMED with such causation reasoning. Specifically, functional mechanistic explanation pathways have been developed, and formulated for total bilirubin, alanine aminotransferase, and alkaline phosphatase and embedded within SNOMED; these pathways contain the expert knowledge relating the various concepts necessary to reason about the serum chemistry of these three analytes. In order to determine the validity of this causation information, the embedded knowledge is extracted from SNOMED into a Bayesian Network and tested against rodent laboratory data. Preliminary results using 10-fold cross-validation with the rodent serum chemistry laboratory data are promising, with a test accuracy of greater than 80% for prediction of involved physiologic functional processes and disorders.
9: COMPARISON AND CONTRAST OF ATRIAL AND BRAIN NATRIURETIC PEPTIDES IN FELINE HYPERTROPHIC CARDIOMYOPATHY.
Atrial and brain natriuretic peptides (ANP and BNP) are cardiac hormones that produce diuresis, natriuresis and peripheral vasodilation. Our laboratory is investigating potential cardiac markers and possible mechanisms of increased plasma levels of these peptides in cats with hypertrophic cardiomyopathy (HCM). We assessed mRNA expression of ANP and BNP in five different cardiac regions (left and right atria, interventricular septum, left and right ventricles) of ten normal hearts and seven hearts from HCM cats. Total mRNA from each cardiac region was extracted to perform Northern blot analysis with specific feline probes, followed by optical densitometry and normalization to beta-actin. Commercial kits designed to human proANP (Biotop OY, Oulu, Finland) and canine BNP-32 (Phoenix Pharmaceutical Inc, Mountain View, CA) were previously validated to tissue extracts and used in three normal hearts and three hearts from HCM cats. ANP and BNP mRNA, as well as peptide concentrations, were mainly found in the atria of normal cats (P<0.05), with low levels of BNP mRNA found in the ventricles as well. With HCM, tissue ANP concentrations were lower, but mRNA ANP levels did not change significantly (P>0.05). However, BNP mRNA expression was remarkably increased in the ventricles (P<0.05), and tissue BNP protein levels were increased 2 and 43-fold in the atria and ventricles, respectively. Moreover, cats with HCM presented a switch in overall BNP production from the atria to the ventricles. Our results suggest that different mechanisms drive the increase of ANP and BNP in feline HCM. In addition, atrial and ventricular cardiomyocytes respond differently to HCM. Due to more dramatic alterations, BNP is likely to perform as a better marker of ventricular hypertrophy in feline HCM.
10: PREVALENCE OF EHRLICHIA CANIS INFECTION IN THROMBOCYTOPENIC DOGS FROM RIO DE JANEIRO, BRAZIL.
Thrombocytopenia is a common laboratory finding in canine ehrlichiosis, which is most consistent in E. canis infection. There are few reports about the occurrence of E. canis in Rio de Janeiro and most are based on direct microscopy examination. In order to evaluate E. canis prevalence, we performed PCR assays with Ehrlichia spp. and E. canis specific primers (16S rRNA gene) in 226 animals following DNA purification. EDTA-anticoagulated blood samples sent to CAD for Complete Blood Count were used for PCR assay. Included in the study were samples from 112 thrombocytopenic (< 200,000 platelets/mm3) dogs (TD) and 114 non-thrombocytopenic (> = 200,000 platelets/mm3) dogs (NTD) which served as a control group. Thirty-six (32.1%) of the TD animals and 4 (3.5%) of NTD animals were positive for Ehrlichia spp. gene sequences. Furthermore, 30 (26.8%) and 4 (3.5%) TD and NTD animals were positive for Ehrlichia canis specific gene sequences. Although E. canis positive results were more frequently found in TD animals (p<0.0001), most members of this group (73.2%) were negative for this pathogen. It remains a possibility that even for PCR detection, too few organisms were present in blood to give a positive result. However, the PCR assay was clearly more sensitive for detecting infected animals (p<0.0001) than direct microscopy; only 4 (3.5%) TD and 2 (1.5%) NTD animals were positive by this method. Although thrombocytopenia has been previously described in canine ehrlichiosis, it is not specific for Ehrlichia canis infection and, therefore, other diseases must still be considered.
11: COMPARISON OF A URINE DIPSTICK DETERMINATION OF URINE PROTEIN:URINE CREATININE RATIO WITH CALCULATED URINE PROTEIN:URINE CREATININE RATIO FROM AUTOMATED WET CHEMISTRY INSTRUMENTS MEASUREMENTS.
Consecutively submitted urine samples from dogs (n = 100) and cats (n = 50) that were patients of the Auburn University Small Animal Teaching Hospital had complete urinalyses, including specific gravity determined by refractometry; dipstick quantification of pH, glucose, protein, blood, ketones, and bilirubin; and sediment examination. Protein was semi- quantitatively determined by sulfos-alicylic acid precipitation. Urine microprotein and urine creatinine were quantitatively determined by automated wet chemistry instrument analysis. Results from a newly available dipstick capable of determining urine proteimurine creatinine ratio were compared with results from the previously mentioned methods of protein, creatinine, and urine protein:urine creatinine ratio. Influence of pH of urine and presence of an active urine sediment were determined on urine protein:urine creatinine ratio. Preliminary results suggest good correlation between methods of protein and creatinine determination with a few notable exceptions. Predictive value for the dipstick results may prove its most useful application.
12: C-REACTIVE PROTEIN AND HAPTOGLOBIN, MARKERS OF INFECTION, INFLAMMATION, HYPERADRENOCORTICISM AND STRESS IN DOGS.
The serum concentration of C-reactive protein (CRP) and haptoglobin (Hp) are established markers of the acute phase response in dogs and there has been a growing number of reports in diagnosis of infectious or inflammatory disease. For instance CRP has been proposed as a valuable addition to a scoring index for canine inflammatory bowel disease and elevated CRP or Hp have been reported in cases of canine ehrlichiosis, canine leishmaniasis and in a group of hospital patients with confirmed infectious diseases. However, in our recent studies, elevated concentrations of Hp in sera were consistently measured in samples from dogs in which the primary condition was not of infectious or inflammatory aetiology. For example, 24 out of 33 dogs with hyperadrenocorticism had serum Hp concentration greater than the upper reference range limit of 3.0 g/L, while other groups of animals treated with steroids had a higher Hp concentration than similar groups prior to treatment. These results indicate that Hp production, as well as being stimulated by proinflammatory cytokines such as interleukin-6, is also induced by corticosteroids, which has been shown experimentally when dexamethasone injection caused serum Hp to increase in concentration. This is important, not only for the interpretation of Hp as a marker of the acute phase response, but as it suggests the possibility that Hp determination could aid in the diagnosis of hyperadrenocorticism or even be a marker of Cortisol related stress. Thus, the combined determination of the concentration of Hp and CRP may have the greatest diagnostic value to differentiate between alternative pathological states.
13: USE OF THE ADVIA 120 IN DIFFERENTIATING EXTRACELLULAR FROM INTRACELLULAR HEMOGLOBIN.
Frequency of acellular hemoglobin-based blood substitute usage is increasing in veterinary medicine. Use of these substances interferes with colorimetric tests including hemoglobin, which makes monitoring of the anemic patient more difficult. The Advia 120 hematology analyzer evaluates hemoglobin by two methods: the standard cyanmethemoglobin colorimetric method and flow cytometry. Theoretically, intracellular and extracellular hemoglobin can be differentiated using these two methods. This study determined the accuracy of the Advia 120 in differentiating intracellular from extracellular hemoglobin. Blood samples from 10 healthy dogs were run in triplicate to measure hemoglobin using both the cyanmethemoglobin and the flow cytometric methods on the Advia. Linear regression to compare these had a slope of 0.96 and intercept of 0, indicating there is no significant difference between these methods (R2 = 0.99). Quantitation of extracellular hemoglobin in the same ten dogs was done by making serial dilutions of oxyglobin in whole blood (1:1, 1:2, 1:4, 1:8, 1:16 and 1:32). Linear regression was used to analyze both the predicted and actual cellular hemoglobin concentration. The correlation of the measured value versus predicted (R2 = 0.96) suggested these methods are comparable. However at the smaller dilutions where low levels of extracellular hemoglobin are present, an increasing bias (from 0.26g/dl HGB at 1:1 to 3.5g/ dl at 1:32) suggests the Advia 120 has difficulty identifying small quantities of extracellular hemoglobin. This may be important in future studies evaluating the half life of Oxyglobin in circulation. In conclusion these methods appear compatible and the Advia 120 is capable of differentiating cellular from extracellular hemoglobin. Studies are ongoing that evaluate anemic patients which have received Oxyglobin.
14: NUCLEATED RED BLOOD CELLS IN CLINICALLY ILL DOGS AND CATS MAY INDICATE INCREASED RISK OF MORTALITY.
Circulating nucleated red blood cells are not considered a normal finding in mature mammals. Recent studies in humans describe a high association of nucleated red blood cells in clinically ill patients and increased risk of mortality. A retrospective study of one hundred nineteen hospitalized dogs and cats with circulating nucleated red blood cells (nRBCs) was undertaken to evaluate mortality rates of these patients and also to examine other parameters including signalment, hematocrit, polychromasia, leukocyte numbers, clinical diagnosis and appropriate versus inappropriate nRBC release. These findings were analyzed by logarithmic regression for trends and compared to a control population of thirty dogs and twenty-nine cats. The control animals were hospital patients that did not have nucleated red blood cells in circulation. The overall mortality rate of nRBC positive dogs and cats was 26%. The mortality rate of the nRBC negative dogs and cats was 5%. The absolute numbers of nucleated red blood cells did not correlate with mortality. The three most frequent diagnostic categories of non-surviving dogs with nRBCs in circulation were immune-mediated hemolytic anemia, untreated neoplasia and dogs receiving chemotherapy for previously diagnosed neoplastic diseases. The two most frequent disease categories of non-surviving cats were untreated neoplasia and cats receiving chemotherapy. Nucleated red blood cells were noted in some clinically healthy animals, however, in clinically ill animals, this finding was associated with increased mortality (p<.005). Inappropriate release of nRBCs into peripheral blood may prove to have prognostic value in clinically ill veterinary patients and may aid the clinician with diagnosing potential underlying disorders.
15: UTILIZATION OF ALKALINE PHOSPHATASE STAINING TO DIFFERENTIATE OSTEOSARCOMA FROM OTHER VIMENTIN POSITIVE TUMORS.
Aspiration of lytic bone lesions is an excellent initial diagnostic test in the diagnosis of primary bone neoplasia. However, cytologically it can be difficult to differentiate osteosarcoma from other bone neoplasms including fibrosarcoma, chondrosarcoma, synovial cell sarcoma and plasma cell myeloma. Alkaline phosphatase is a hydrolytic enzyme present in multiple tissues including liver, kidney, intestine, placenta and bone. Hypothetically, neoplasms actively producing bone should be positive for alkaline phosphatase staining. The purpose of this study is to determine the sensitivity and specificity of alkaline phosphatase staining to differentiate osteosarcoma from other vimentin positive neoplasms in dogs. Unstained, cytologic specimens were incubated for 5–10 minutes with BCIP/NBT phosphatase substrate. A positive reaction stains the membrane of the cells dark purple to black. Negative samples were counterstained with a Romanowsky stain, to determine if the sample was of representative cellularity. Twenty-eight vimentin positive neoplasms have been evaluated and confirmed histopathologically. Alkaline phosphatase positive tumors include 11 osteosarcomas, 1 multilobulated tumor (MLT) and 1 amelanotic melanoma. All other vimentin positive neoplasms including chondrosarcoma, fibrosarcoma and synovial cell sarcoma are alkaline phosphatase negative. The sensitivity is 100% and the specificity is 85%. In conclusion, alkaline phosphatase appears to be a highly sensitive and fairly specific marker in the diagnosis of osteosarcoma. This is an ongoing study, and many more neoplasms as well as reactive bone need to be evaluated.
16: FELINE MULTIPLE MYELOMA: A REVIEW OF 12 CASES (1996–2003).
Multiple myeloma is an infrequent diagnosis in cats with few reports in the literature. We evaluated 12 cases diagnosed over 8 years. Diagnosis required two of the following: marrow plasmacytosis, monoclonal gammopathy, light chain proteinuria, and radiographic evidence of osteolysis. Ages ranged from 1–18 yrs (median of 13.5 yrs). All cats were FeLV/ FIV negative. FIP titers, when done, were less than 1:40. Clinical signs included weakness (7/12), anorexia (6/12), vomiting (4/12), orthopedic lameness (2/12) and epistaxis (1/12). Physical exam findings included heart murmurs (5/12), pale mucous membranes (4/12), palpable cranial abdominal organomegaly (3/12), skin masses (2/12), and retinal hemorrhages (1/12). Laboratory abnormalities included hyperproteinemia (11/ 12), hyperglobulinemia (10/12), non-regenerative anemia (10/12), hypocholesterolemia (8/12), thrombocytopenia (6/12), light chain proteinuria (4/7), leukopenia (3/12), hypoalbuminemia (3/12), renal azotemia (3/12), hypercalcemia (1/12), and regenerative anemia (1/12). Hyperviscous serum was established in one case with cardiovascular abnormalities but no retinopathies. Ten cases had serum protein electrophoresis performed with 8 monoclonal and 2 biclonal gammopathies found. Osteolytic lesions were noted in three of 11 radiographed cats while generalized osteopenia was found in one. Bone marrow plasmacytosis was detected in nine of ten cases examined. Extraosseous sites included spleen (4/12), liver (3/ 12), lymph nodes (2/12) and skin (2/12). Three cases were unusual in that one had a cutaneous malignant plasma cell tumor that progressed to multiple myeloma with 3,000/microliter circulating plasmablasts; another had multiple cutaneous plasmacytomas concurrent with marked marrow plasmacytosis at presentation; the third had a 9-year history of a monoclonal gammopathy of undetermined significance that progressed to symptomatic multiple myeloma. The twelve cases presented here display the wide spectrum of findings possible in feline multiple myeloma.
17: PLASMA PROPHENOLOXIDASE IN AMERICAN LOBSTERS (HOMARUS AMERICANUS): VARIATION IN A NATURAL POPULATION AND CHANGES ASSOCIATED WITH TWO INFECTIOUS DISEASES.
Phenoloxidase (PO) and its precursor prophenoloxidase (proPO), located in haemocytes, are integral components of the defence system of invertebrates. PO activity is measured in whole haemolymph and haemocyte lysates to evaluate responses to immunostimulants and disease. Current techniques were modified to measure PO activity in haemocyte-free haemolymph (plasma) from lobsters, Homarus americanus, to determine if plasma PO activity could be used as a biochemical marker of haemocyte degranulation during an inflammatory response. PO activity was only detected in plasma after addition of the trypsin activator and so attributed to proPO. Plasma proPO activity in 121 lobsters ranged from < 5 to 49.5 mOD/min. After exclusion of lobsters with total haemocyte counts < 10 × 10 9 cells/L or haemolymph total solids < 50 g/L, activities ranged from < 5 to 17.4 mOD/min (n = 74). Elevated plasma proPO activities (30.6 to 81.2 mOD/min) were recorded in 3/3 lobsters in the terminal stages of naturally acquired infections with the scuticociliate Anophryoides haemophita (Bumper car disease) and 3/6 lobsters (29.5 to 78.1 mOD/min) with experimental infections. Other indicators of disease (haemocytopenia and parasitaemia) preceded the increased enzyme activity. Plasma proPO activity was not significantly increased in lobsters with naturally acquired or experimental infections with the bacterium Aero-coccus viridans (gaffkemia). Although not a sensitive indicator of inflammation in these two disease models, determination of plasma proPO activity may be useful in other diseases or as a tool for examining the defence response. Further study will be required to determine the cause(s) of the variation observed in the natural population and in the two disease models.
18: INCREASED ATRIAL NATRIURETIC PEPTIDE IN KIDNEYS OF CATS WITH HYPERTROPHIC CARDIOMYOPATHY.
Atrial natriuretic peptide (ANP) is a hormone produced mainly in cardiomyocytes that once released into the circulation causes diuresis, natriuresis, and peripheral vasodilation. Although ANP is present in human kidneys, renal peptide and mRNA concentrations are respectively 10e4 and 10e7-fold lower than in the atria. Our laboratory is investigating possible sources for the increased levels of plasma ANP we have found in cats with hypertrophic cardiomyopathy (HCM). In the present study, we assessed the mRNA expression and peptide levels of ANP in kidneys of six normal cats and two cats with HCM. Protein extraction was conducted using acetic acid precipitation. A commercial kit was validated for tissue extracts (linearity R2 = 0.9905 and recovery 104% ±21) and used to measure ANP tissue levels (Biotop OY, Oulu, Finland). A specific feline ANP probe of 900 base pairs was used for Northern blot analysis. ANP concentrations in normal cat kidneys were 0.56 ± 0.12 pmol/g, whereas in kidneys of cats with HCM they were significantly higher (1.15 ± 0.16 pmol/g; p = 0.0007). Renal ANP mRNA expression was below detectable limits for Northern blot analysis with 5 ul of total mRNA. Renal ANP levels were 5,000-fold lower than atrial ANP in controls (atria 5423.29 ± 1425.83 pmol/g) and 3,000-fold lower in kidneys than in atria of cats with HCM (atria 3332.67 ± 1834.47 pmol/g). Renal ANP concentrations in normal and HCM cats were lower than in normal human beings (2.53 ± 0.19 pmol/ g). Our results suggest that renal ANP is likely to function locally in cats since mRNA and tissue levels are too low to contribute to plasma concentrations. Cats with HCM may have an increased activity in renal ANP.
19: EVALUATION OF BEAGLE POG CITRATEP PLASMA USING THE ACL APVANCE COAGULATION ANALYZER.
The objective of this work was to verify the use of the random access ACL Advance automated coagulation analyzer (Beckman Coulter, Inc.) on Beagle dog citrated plasma for FT, APTT, and fibrinogen. Precision, accuracy, and preliminary reference intervals were determined for each test; linearity of dilution and sample stability were also checked for fibrinogen. Accuracy was determined for each test using the appropriate manufacturers’ controls (Assess normal, low, high, calibration plasma, and IL Test low fibrinogen) with IL Test reagents on the ACL Advance automated coagulation analyzer. Intra-assay precision was calculated from 10 consecutive measurements of a dog citrated plasma sample; %CVs of 1.1, 2.5, and 2.9%, respectively, were obtained for PT, APTT, and fibrinogen. Linearity of dilution for fibrinogen on a dog sample showed a correlation coefficient of 0.9982 and a slope of 1.0523 for a concentration range of 144.1–421.1 mg/dL. Preliminary reference intervals were calculated from 9 dogs (PT) and 13 dogs (APTT, fibrinogen) as follows: PT (5.20–5.80 s.), APTT (9.30–12.60 s.), fibrinogen (214.0–545.8 mg/dL). Fibrinogen stability was verified with 5 dog citrated plasma samples; a mean sample difference of 2.9%, 3.9%, and 5.6%, respectively, was obtained following sample storage for 6, 24, and 48 hours at 2–8° C. In conclusion, the accuracy, precision, and preliminary reference intervals obtained on the ACL Advance show acceptable reactivity for testing dog citrated plasma samples.
20: THE EFFECTS OF TRIPOTASSIUM EDTA AND LITHIUM HEPARIN ON CELL PRESERVATION AND SAMPLE PROCESSING OF LOGGERHEAD SEA TURTLE BLOOD.
Studies were conducted to determine the most effective anticoagulant (K3 EDTA versus lithium heparin) for hematologic evaluation of sea turtle blood and to investigate the effects of each anticoagulant over time. The blood from ten loggerhead sea turtles (Caretta caretta) was examined for changes over time (4 hr to 4 days). In addition, an evaluation of leukocyte quantitation methods for this species of reptile was performed. Leukocyte counts on the Abbott Cell Dyn 3500 were determined using a setting established at the University of Florida for alligator blood. Leukocytes were counted on CDC Hemavet 1500 analyzer using a manufacturer setting for tortoises. Precision tests on two automated systems, Cell Dyn 3500 and Hemavet 1500, and a manual hemacytometer technique indicated marked differences between each method (coefficient of variation was 3.0%, 8.7%, and 22.9%, respectively). Automated systems demonstrated an expected higher degree of precision. Statistical analysis* was performed for anticoagulant and time effects using descriptive statistics, ANOVA for repeated measures followed by a Tukey pairwise comparison (SigmaStat). Lithium heparin was shown to be the anticoagulant of choice for cytologic evaluation of blood cells and for packed cell volume analysis related to reduced hemolysis and cellular swelling when compared with EDTA. The two analyzers, Cell Dyn 3500 and Hemavet 1500, had different leukocyte counts, but showed consistent changes over time with each anticoagulant with significant changes after 24 hours. High variability in the manual method affected all corresponding results obtained when using that particular technique. As expected relative to biochemical analytes over time, analysis should be performed as soon as possible, as evident by decreases in glucose after 4 hours.
21: COMPARISON OF INSTRUMENT AND MANUAL PLATELET COUNTS AND PLATELET ESTIMATES FROM BLOOD SMEARS IN DOGS AND CATS.
Instrument and manual platelet counts and platelet estimates from blood smears in 25 dogs and 20 cats were compared. Additionally, platelet estimates done on microscopes with different field numbers (FN = 20 and FN = 22) were compared. In 16 of the 25 dogs and in all cats, the instrument platelet count was less than the manual count. In two of the nine canine cases, in which the instrument count exceeded the manual count, the manual count was less than the reference range, but the instrument count was within the established reference range. When corrected for field size, the average number of platelets counted agreed in 23 of 25 dogs and 19 of 20 cats. Multiplication by published factors for total platelet estimate exceeded the manual count in 22 of 25 dogs and 19 of 20 cats. When estimates were lower than the manual count, both were either lower than reference range (three cases) or were within reference range (one case). The correlation between platelet estimates from smears and the manual count was good. More general comments (markedly decreased, slightly decreased, or adequate) taken from technologist assessment of smears provided accurate clinical information.
22: DETERMINATION OF ACUTE STAGE PROTEINS (CRP, SAA AND FIBRINOGEN) IN AFRICAN GREEN MONKEYS.
Purpose of study: To evaluate the specificity and sensitivity of acute stage protein assays as unspecific markers of inflammatory disease in African green monkeys. A population of 50 adult African green monkeys [Cercopithecus aethiops sabaeus] (28 males and 22 females) of St Kitts origin, with different types of infectious/inflammatory conditions (infectious skin disorders, pneumonia, peritonitis, pyelonephritis, UTI, GI disorders) were included in this study and compared to 40 normal healthy animals of corresponding breed, age and sex. All animals were born in captivity in a breeding program at the SKBRF. In this study, serum CRP, SAA and plasma Fibrinogen were measured. For CRP a passive latex agglutination technique (Dade-Behring Rapi-Tex) was used and compared to quantitative measurement by Radial Immunodiffusion (RID) technique. The SAA concentration in serum was measured by RID and ELISA was used as a reference method. Plasma Fibrinogen was measured by heat precipitation (QBC Fibrinogen precipitator, IDEXX). In general the Fibrinogen assay was not sensitive enough in discriminating normal animals (mean ± SD, 310+48 mg/dl) from those with inflammatory conditions (345 ± 68 mg/dl). Only in animals with very severe inflammatory conditions (as pneumonia, peritonitis) the elevation in Fibrinogen concentration was considered diagnostic. Both serum CRP and SAA showed rapid increase in concentration in most inflammatory conditions (10–30 times normal serum level). A pronounced elevation in CRP and SAA concentration (often 30–60 times normal level) was seen in inflammatory conditions of severe nature as pneumonia, pertitonitis and acute nephritis. The latex agglutination technique for CRP as used in this study was useful in the discrimination of normal animals versus animals with inflammatory disease. Serum CRP and SAA protein are therefore considered valuable markers of inflammatory/infectious diseases in the African green monkey.
23: A DVM SUMMER SCHOLARS PROGRAM-TRAINING FUTURE SCIENTISTS.
A summer research training program supported by Merck Company Foundation and Merial Ltd. (11 years) and a NIH T35 grant (4 years) has sponsored 8–17 DVM students per year at Iowa State University. In this program, mentor project abstracts and DVM student applications are matched by a ranking process. The goals are to introduce students to research by using critical thinking, preparing hypothesis-based proposals, designing statistically sound experiments, and interpreting data. The program has resulted in national and international presentations and posters, publications, and student enrollment in PhD graduate programs. With 72 participating scholars, during a period of 8 years, 25 students pursued education beyond the DVM degree and 16 completed MS or PhD degrees. Four current PhD graduate students in the Department of Veterinary Pathology were formerly in this program. Even though not all participants will choose a research career, this program provides a positive experience in recruiting potential future scientists. The websites for the two programs are http://www.vetmed.iastate.edu/research/merck/, and, http://www.vetmed.iastate.edu/research/biomedical/.
24: VIRTUAL SLIDEBOXES FOR TEACHING NORMAL HISTOLOGY AND CANCER HISTOPATHOLOGY.
Virtual slide technology now provides the capability to rapidly digitize a whole glass slide at high resolution and save the file in a highly compressed file format. A web-based pan-and-zoom viewer, by accessing these giant virtual slide files, can nearly perfectly emulate viewing an entire glass slide under a traditional microscope. To date we have digitized 145 animal and 119 human histology slides, and 63 animal and 286 human cancer slides. The home page is at http://www.path.uiowa.edu/virtualslidebox. Virtual slides can be annotated with lines and arrow overlays, descriptions, and gross images. Sets of virtual slides have been organized into computer-based educational programs for teaching histology and histopathology, and for comparing cancer across species. Evaluation in histology and pathology courses indicate that incorporation of virtual slides into laboratory exercises results in a marked increase in efficiency and accessibility for student study in these disciplines, without a decrease in performance on photomicrograph and traditional glass slide practical examinations. Additionally, students are better able to grasp the morphologic features of cancer with the traditional microscope when instructors demonstrate using virtual slides rather than photomicrographs. The virtual slides of veterinary neoplasms will be incorporated into residency training and will be made available to other training programs outside of Iowa. Our experience to date demonstrates that virtual slide technology can revolutionize the way students learn microscopic morphology. We expect that increased availability of virtual microscopy will reduce the reliance of histology and pathology departments on expensive glass slide sets and traditional student microscope laboratories, and will also increase the accessibility of a wider range of common and rare histopathology slides to pathology residents in training.
25: MULTIPLE USES OF A SENIOR YEAR ASSIGNMENT BY ASSESSMENT OF THE PATHOLOGY CURRICULUM.
Senior veterinary students are required to take a two-week rotation in necropsy laboratory. The necropsy rotation is brief and provides an inconsistent caseload, so additional exposure to necropsy cases has been achieved through the Case Correlation Assignment (CCA). The CCA requires students to review a medical chart from a necropsied patient and assimilate all of the clinical data (history, hematology, chemistry, radiology, biopsy, treatment, response) and correlate the antemortem diagnoses to postmortem findings. The reports are submitted as computer files, rather than printed paper, to accommodate multiple media. The reports are graded in part on the completeness, interpretation, and correlation of ante- and postmortem data. The educational impact of the report (i.e. illustrations, diagrams, use and explanation of accurate terminology) is also considered in the grade. Examples of reports will be displayed in this presentation. This assignment educes higher-order thinking from students and emphasizes the range and limits of data to be gained from necropsies. Beyond the educational value for senior students, the CCA will be an evaluation tool for the department. A Miller Fellowship has funded our group to incorporate the CCA in an assessment plan of the four-year pathology curriculum. Additionally, in spring 2004, junior veterinary students will provide peer-assessment of the senior students’ reports. In doing so, we will assess the outcomes and effectiveness of our teaching efforts in the general, systemic, special and clinical pathology courses.
26: AFIP ON LINE VETERINARY SYSTEMIC PATHOLOGY.
The Department of Veterinary Pathology, Armed Forces Institute of Pathology, has implemented a web-based resource in veterinary systemic pathology with partial funding from a Department of Education, Fund for the Improvement of Post-secondary Education grant. This database utilizes the same eleven-organ system training archive as the AFIP residency training. Each organ system is divided into bacterial, fungal, metabolic and miscellaneous, neoplastic, parasitic, toxic, and viral entities. Each case also has a unique alphanumeric code. The database can be sorted by organ system, animal species, and disease category for more than six hundred seventy-five entities in numerous species. The cases can also be viewed as unknowns. Linked sequential images (with legends) are provided to simulate examination of the tissue at the microscope. Lower magnification images contain rectangular hyperlinked areas or hotspots that delineate the viewable area contained in the next higher magnification image. Each case manuscript is reviewed and updated every three years, including recent references, by residents under the supervision of ACVP board-certified staff members. Future projects include linking virtual slides to each case for the remaining nine organ systems (cardiovascular and reproductive organ systems are completed); addition of gross pathology images, cytology and clinical pathology data, electron photomicrographs, immunohistochemistry photomicrographs, and links from references to on line publications; and development of on line testing modules. An on line discussion board is available to allow users to share their thoughts, ideas, or submit comments and questions about the web site to the Department of Veterinary Pathology project team.
27: EXOTIC AND EMERGING DISEASES OF ANIMALS: AN INTERNET COURSE.
An Internet course was developed by veterinarians at Iowa State University, University of Georgia, University of California Davis, and the USDA to instruct veterinary students in the recognition and response to exotic and emerging animal diseases (EEAD). Funding was provided by a Higher Education Challenge Grant from the USDA CSREES. The course is designed to provide a resource, be flexible for adaptation, and effectively meet requirements for veterinary colleges that need to train students and veterinarians in EEADs. Modules address the role of international, national, and state agencies; modes of introduction of EEADs; appropriate responses to EEADs; descriptions of recent incursions of EEADs; global animal agriculture issues; and scenarios of EEAD outbreaks. The course also includes a searchable database with information on 76 reportable EEADs in the U.S. The course was offered to veterinary students at ISU, and the University of Minnesota through the Veterinary Information Network (VIN). Additionally, it will be offered as a continuing education course for veterinarians. Preliminary evaluations found the students enjoyed the format, flexibility, and on-line resources. Students also made recommendations for improvements to the course. Overall, the course was found to be efficient and strongly recommended, since there is a growing need for veterinarians to recognize and respond to emerging disease threats.
28: A NATIONAL CANCER INSTITUTE CENTER FOR CANCER RESEARCH (NCI CCR) COMPARATIVE MOLECULAR PATHOLOGY RESEARCH TRAINING INITIATIVE.
The NCI CCR intramural research program recognizes a need for investigators capable of translating an understanding of molecular mechanisms of disease in animal models to clinical applications in cancer research. To address this need, NCI CCR seeks to establish a program for training in animal pathophysiology, human cancer pathology, molecular biology and medical research in partnerships with domestic universities having pathology-training programs for graduate veterinarians. The NCI CCR will support students jointly recruited for graduate training by a partnership university and the NCI CCR for up to 5 years. The initial 2 years pathology training and graduate course work is undertaken at a partnership university followed by 3 years of molecular pathology and research training in the intramural laboratories of the NCI CCR. A complimentary pathway exists to support research training for those with previous residency experience. Completion of the program would anticipate conferral of the Ph.D. degree by the partnership university and the program seeks to allow students to achieve eligibility for specialty board certification as a component of training. Completion of such a training program will prepare comparative pathologists for careers in discovery and translational cancer research. The website is at http://ccr.nci.nih.gov/resources/training/default.asp
29: A SIMIAN/HUMAN IMMUNODEFICIENCY VIRUS INFECTED MACAQUE MODEL OF AIDS RELATED EPSTEIN-BARR VIRUS ASSOCIATED LYMPHOMAS.
Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis and is associated with the development of several human malignancies, including AIDS-associated lymphomas. Rhesus macaques are naturally infected with lymphocrytptovirus (LCV), a gamma herpes virus closely related to EBV. It has been shown that there is a high degree of homology between the EBV and rhesus LCV genome. In order to investigate the viral and host factors involved in the development of AIDS associated EBV positive lymphomas we utilized a SHIV-infected rhesus macaque model. LCV seronegative rhesus macaques were inoculated intravenously with SHIV-89.6P and subsequently infected with LCV-transformed autologous B-cells via the oral or intravenous route. After oral inoculation, the animals exhibited early, prolonged viraemia, failed to develop an LCV-specific humoral response and there was no evidence of lymphoproliferation. Intravenously challenged animals also failed to seroconvert but in contrast to orally inoculated animals they did exhibit evidence of lymphoproliferation. One animal which was euthanised for a nosocomial infection 2 months post intravenous LCV inoculation had infiltration of LCV-infected lymphocytes in multiple organs. A second animal developed a rapidly growing submandibular mass five months post LCV inoculation. This mass was confirmed on histological examination to be a LCV- positive lymphoma. These studies suggest that the SHIV-infected rhesus macaque may prove a valuable model for studying EBV infection in AIDS patients.
30: GUINEA PIG MODEL OF EQUINE CONGENITAL HYPOTHYROIDISM.
Congenital hypothyroidism of foals is an important cause of reproductive loss and neonatal mortality in western Canada and other areas of North America. To accelerate, and lower the costs associated with, the study of this disorder; we investigated the suitability of the guinea pig as an animal model. Pregnant guinea pigs were administered either 0.1% propylthiouracil (PTU) in drinking water at 3 or 40 days gestation, 1131 (100uCi or 200uCi) at 40 days gestation, or a diet with marginal iodine before and throughout gestation. In contrast to control animals, pups from dams treated with PTU were delivered after a longer gestation (2 days), however, because of the small sample size, this difference was not statistically significant (p = 0.08). These pups were often stillborn (24/27); had grossly enlarged thyroid glands (goitre) characterized by enlarged, variably sized follicles composed of tall columnar epithelium and little or no colloid; and had radiographic evidence of skeletal dysgenesis. Many of these lesions are present in congenitally hypothyroid foals. Pups from dams in the other treatment groups had no grossly or radiographically detectable lesions. Histologically, thyroid glands of pups from dams treated with radioactive iodine had smaller follicles with taller epithelium and small amounts of colloid. The thyroid gland of pups from dams fed a marginal iodine diet had a slightly taller follicular epithelium. The guinea pig appears to be a suitable model for the future study of congenital hypothyroidism in horses.
31: CHARACTERIZATION OF HYPOTHALAMIC LESIONS IN THE PAHENU2 MOUSE MODEL: POSSIBLE ROLE IN THE NEUROPATHOGENESIS OF PHENYLKETONURIA.
Phenylketonuria (PKU) is one of the most common single gene disorders in man that occurs with an overall frequency of 1 in 10-20,000 births. The metabolic defect arises from deficient activity of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination, and these functions are associated with various dopaminergic tracts. Hyperphenylalanemia results in competitive inhibition of amino acid transport across the blood brain barrier and within dopaminergic neurons, altering normal tyrosine and dopamine metabolism. The purpose of this study was to identify neuropathologic lesions in the Pahenu2 mouse model, to eventually use in determining the efficacy of AAV-mediated gene therapy, while gathering information regarding the neuropathogenesis of the disease. In this study, 6 groups of Pahenu2 adult mice were used, varying in sex and genotype, with n = 6 for each group. Using morphometric microscopic analysis, it was determined that cell density was significantly increased within the female homozygous recessive genotype in the vicinity of arcuate and premamillary nuclei of the hypothalamus, which contain dopaminergic cell bodies. Immunohistochemical results show that the influx of cells in this region does not express tyrosine hydroxylase, therefore the increased cellularity may possibly be due to induction of gliosis due to oxidative DA catabolism. These lesions may provide a useful marker to detect amelioration of pathological processes by gene therapy and provide insight into the neuropathogenesis of the disease.
32: CUSTOMIZED CELL TYPE SPECIFIC AUTOIMMUNITY IN HEMAGGLUTININ-TRANSGENIC MICE.
The regulatory mechanisms that control autoimmune phenomena are of considerable interest for a variety of disorders but available models are limited. In our work, we developed three lines of transgenic mice with a partial influenza virus hemagglutinin (HA) coding sequence under expressional control of one of three cell type-specific gene promoters. Restricted HA expression was obtained by transcriptional control under the surfactant protein C (SPC) promoter (lung), the villin (VIL) promoter (intestine) or the insulin (INS) promoter (pancreas). No morphological or functional abnormalities were seen in these mice. However, when the mice were crossed with mice transgenic for an HA-specific T cell receptor (TCR-HA), the double transgenic mice developed severe autoimmune inflammation in the respective tissues. The SPC-HA x TCR-HA-, the VIL-HA x TCR-HA- and the INS-HA x TCR-HA- double transgenic mice will serve as models for cell-type specific immunological disorders including diabetes mellitus and inflammatory bowel disease. Furthermore, the experimental approach should be useful for generating models for tissue-specific autoimmune inflammatory disorders in a variety of other cell types.
33: THE EFFECTS OF BACTERIAL AND VIRAL PNEUMONIA ON SP-D mRNA EXPRESSION.
Surfactant Protein D (SP-D) is a collagenous calcium-dependent lectin constitutively expresses by type II pneumocytes and Clara cells. Its role in lung innate immunity involves binding to the surface glycoconjugates expressed by a wide variety of microorganisms such as Gram-negative bacteria, Influenza A virus, and various fungi, leading to pathogen inactivation or enhanced neutrophil and macrophage activity. In vivo deficiency of SP-D is associated with increased risk for infection. The purpose of this study was to determine and compare SP-D mRNA expression during the progression of bacterial and viral pneumonia in lambs. The first group of animals, healthy weaned lambs, was inoculated intrabronchially with either pyrogen-free saline (controls) or M.haemolytica by using a fiberoptic bronchoscope. The animals were subsequently euthanized on day 1, 15, or 45 post-inoculation. The second group, healthy neonatal lambs, received either pyrogen-free saline (controls) or Parainfluenza Type 3 (PI-3) virus intratracheally and intranasally, and the animals were euthanized on day 3, 6, or 17 post-inoculation. The lung tissues were collected and homogenized lung lesions as well as healthy lungs were analyzed by real-time relative quantitative reverse transcriptase PCR (TaqMan). SP-D mRNA levels were not increased or significantly altered by M.haemolytica infection, and they showed a trend of decreased expression when compared to control animals. In contrast, the SP-D mRNA levels were significantly increased in PI-3 virus inoculated lambs at all time points when compared to controls. These results suggest that SP-D expression is not increased by M. haemolytica, which was expected for SP-D, because it is constitutively regulated. The mechanistic basis for increased SP-D expression during PI-3 infection is under investigation.
34: ULTRASTRUCTURAL MORPHOLOGY AND IMMUNOGOLD LOCALIZATION OF P-SELECTIN AND FACTOR VIII RELATED ANTIGEN IN DEER AND CATTLE PLATELETS.
Platelet alpha granules contain a variety of substances that play a role in hemostasis, inflammation, or tissue repair. P-selectin and factor VIII related antigen (FVIIIRA), which mediate leukocyte and platelet adhesion, respectively, are two that could be important in the pathogenesis of diseases that cause endothelial damage. The objectives of this study were to compare platelet morphology and P-selectin and FVIIIRA distribution in resting and phorbol myristate acetate activated white-tailed deer and cattle platelets. Resting deer platelets were discoid and contained small numbers of large, round to oval alpha granules. Alpha granules had an incomplete electron-dense peripheral layer and a flocculent, less electron-dense, core. Platelets immunostained for P-selectin had a few gold particles localized at the periphery of the alpha granules. Larger, albeit small, numbers of gold particles localized over the flocculent areas of the alpha granules in platelets immunostained for FVIIIRA. Activated platelets were greatly distorted, with thin pseudopodia, vacuolated cytoplasm, and collapsed angular alpha granules composed of electron dense material. Platelets immunostained for P-selectin and FVIIIRA had gold particles localized over alpha granules, throughout the cytoplasm, and on the plasma membrane. Cattle platelets were morphologically similar, however, the electron-dense layer at the periphery of the alpha granules had prominent banding. Resting platelets immunostained for P-selectin and FVIIIRA had gold particles localized primarily over the flocculent portions of the alpha granules in relatively larger amounts than deer. Although activated platelets developed pseudopodia, they were not as distorted or vacuolated as activated deer platelets. Ultrastructurally, deer and cattle platelets showed differences in response to an agonist and P-selectin and FVIIIRA alpha granule content. These findings could help explain interspecific variability in susceptibility to certain diseases.
35: EVALUATION OF A COMMERCIAL METHYLCELLULOSE MEDIUM AND RECOMBINANT BOVINE GRANULOCYTIC COLONY-STIMULATING FACTOR FOR CULTURING BOVINE BONE MARROW CELLS.
The purpose of this study was to evaluate a commercial methyl-cellulose culture medium for use in a colony-forming unit (CFU) assay for bovine bone marrow cells. Bone marrow mononuclear cells were isolated and cultured in a methylcellulose-based commercial medium, Methocult GF H4534. This medium contains a variety of recombinant human cytokines. Cultures were done with or without 100 ng/ml of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF). Bone marrow mononuclear cells were added to the two media on culture day 0. The size and mean number of colonies per plate from culture days 3 to 9 were compared. We concluded that bovine bone marrow colony growth was supported by this culture medium. The addition of rbG-CSF yielded larger and more numerous colonies. The mean number of colonies in cultures supplemented with rbG-CSF significantly increased at several times during the culture period. The highest colony counts in cultures with or without rbG-CSF were on day 5. This is the first report of a commercial human bone marrow culture medium and rbG-CSF for culturing bovine bone marrow cells. This combination is convenient to use in a CFU-assay to study bovine hematopoietic cytotoxicity such as that associated with acute bovine viral diarrhea virus infection.
36: STUDIES FOR THE NEURAL TRANSMISSION OF NEOSPORA CANINUM FROM PERIPHERAL NERVE TO THE BRAIN IN MICE.
Neospora (N.) caninum is an apicomplexan parasite that causes encephalomyelitis, polyradiculoneuritis, polymyositis and abortion in multiple animal species. But it is unknown whether this parasite disseminates to the brain via the route of peripheral nervous system. Inbred 5-6-week-old male Nude mice (BALB/cA Jcl-nu, N = 12) and wild type male BALB/c mice (N = 50) were used for all experiments. BALB/c mice were divided into 3 groups (nonimmunized group and 2 immunized groups). Nonimmunized nude and BALB/c mice inoculated with tachyzoites of BT-3 strain N. caninum both eye (IE) and intra nerve (right sciatic nerve) at 6 week of age and were killed every 1 week postinoculation (PI) by Nembutal anesthesia. Immunization BALB/c groups were divided into 2 different methods such as subcutaneous (SC) and intracerebral (IC) immunization using killed BT-3 antigen. After immunization, all of BALB/c mice inoculated with BT-3 and euthanized same manner as in nonimmunized groups. Histopathologic and immuohistochemistry were performed all the tissue samples from dead or sacrificed mice. In nonimmunized groups, the hemtogenous spreading of the N. caninum was predominant than nervous spreading. However in immunized groups the distribution and severity of the lesions milder than non-immunized Balb/c group. The distribution of lesions and parasites in optic pathway and spinal cord strongly suggest that nervous spreading of N. caninum is occurred in mice. Hence, immunization for mice may be influence on the prevention of hematogenous spreading of this parasite and enhance the neuro-invasion of N. caninum.
37: IMMUNOHISTOCHEMICAL EVALUATION OF SODIUM/IODIDE SYMPORTER EXPRESSION IN TRANSGENIC MOUSE MODELS OF BREAST CANCER.
The sodium/iodide symporter (NIS) is a membrane glycoprotein that actively transports iodide into several tissues, including lactating mammary gland epithelium. NIS has also been detected in over 80% of human mammary carcinomas, where it could serve a useful therapeutic role in radioiodide ablation. Identification of NIS-expressing mouse models of breast cancer will be informative in the elucidation of signal transduction pathways involved in mammary gland NIS regulation. In this study, we used immunohistochemistry to detect NIS expression in 13 mouse models of breast cancer: C3(1)-TAg, WAP-p53R->H (DMBA and pituitary iso-graft treated), WAP-des-IGF-1, WAP-p53R->H x WAP-des-IGF-1 bi-transgenic, p53 null, MMTV-Cox-2, MMTV-HPV16E6/beta-casein, MMTV-Her-2/neu, WAP-TAg x WAP-maspin bitransgenic, MMTV-c-myc x MMTV-v-Ha-ras, UbiC-hCGbeta, alpha-LHbeta/CTP, and DMBA and pituitary isograft treated mice. Using rabbit anti-rat NIS antibody, paraffin-embedded sections representative of each model were processed and evaluated for membrane and cytoplasmic staining using a scale of 1+ to 4+. Negative control slides were processed without primary antibody. Hematoxylin and eosin staining was performed on additional sections in order to determine the microscopic type of each neoplasm. Two models, MMTV-Her-2/neu and UbiC-hCGbeta, had strongly positive membrane staining. One model, MMTV-Cox-2, had moderate membrane and cytoplasmic staining. Four models, C3(1)-TAg, WAP-p53R->H, p53null, and alpha-LHbeta/CTP, had moderate to weak cytoplasmic staining. The remaining five models were negative. While no single signal transduction pathway has been identified as responsible for NIS expression in breast cancer based upon our study, pathways leading to cellular proliferation with retention of differentiation appear to be important.
38: OSTEOBLASTIC AND OSTEOLYTIC BONE LESIONS BY A HIGHLY METASTATIC AND INVASIVE CANINE PROSTATE CARCINOMA IN NUDE MICE.
Bone metastases are common in advanced prostate cancer, and there is often significant new woven bone formation induced by the metastases (osteoblastic metastases). Dogs are a commonly used animal model for investigations of hyperplastic and neoplastic diseases of the prostate in vivo. We have successfully developed a serially transplantable canine prostate carcinoma (ACE-1) in nude mice. The primary tumor was an intra-alveolar prostate carcinoma with foci of urothelial differentiation. The ACE-1 cells grew in a highly invasive fashion in the normal mouse subcutis, and stimulated both new periosteal bone formation and lysis of cortical bone when tumor cells invaded into bone. Spontaneous lung and lymph node metastases occurred in 30% of the ACE-1 implanted mice. Immunohistochemical studies revealed the production of parathyroid hormone-related-protein (PTHrP) and cathepsin K by both the primary prostate carcinoma and ACE-1 cells. Quantitative RT-PCR analysis of the 3 prime UTR and the coding region of canine PTHrP revealed production of the 141 amino acid isoform by the xenograft. However, mice with the ACE-1 xenograft were normocalcemic and their plasma PTHrP concentrations were not different from controls. Radiographs of mice injected intra-tibially with ACE-1 cells demonstrated a mixed osteoblastic/osteolytic reaction in cortical bone. The ACE-1 xenograft is a novel model for investigating the pathogenesis of human prostate cancer bone metastases because it is invasive into soft tissues and bone, induces a marked increase in new woven bone formation in addition to bone lysis, and metastasizes spontaneously.
39: MURINE CUTANEOUS INNATE IMMUNITY: CD14, TOLLLIKE RECEPTOR AND BETA-DEFENSIN EXPRESSION IN MURINE SKIN.
Innate immunity is responsible for the earliest defense to infection and orchestrates acquired immune responses. Despite this fundamental role, little is known about innate host defense mechanisms in the skin. This experimental study assessed murine skin for expression of key components of innate immunity including pathogen recognition receptors (CD14, toll-like receptors (TLR)) and antimicrobial peptides (mouse beta-defensins (MBD)). Total cellular RNA was extracted from normal skin and primary keratinocyte cultures from fetal and adult female NIH/Swiss mice and from mouse keratinocyte cell-line (PAM 212) cultures. mRNA expression was determined by reverse transcriptase-polymerase chain reaction utilizing murine gene-specific primer-pair sequences. Fetal and adult mouse skin constitutively expressed mRNA for CD14, (Fetal 10/10, Adult 6/10), TLR2 (Fetal 10/10, Adult 10/10), and TLR4 (Fetal 10/10, Adult 10/10). Keratinocyte expression of these receptor mRNAs was supported by their detection in nearly all primary keratinocyte cultures and PAM 212 cultures. MBDs were expressed differentially by murine skin and cultured keratinocytes. MBD1 mRNA was detected in all skin samples, primary keratinocyte cultures and PAM 212 cultures. MBD2 mRNA was detected in fetal (10/10) and adult skin (7/10) samples and adult primary keratinocyte cultures (3/3), but was not often detected in fetal primary keratinocyte cultures (3/ 10 culture day 3, 0/10 culture day 7) or PAM 212 cultures (0/5). MBD3 mRNA was detected in all primary keratinocyte cultures and PAM 212 cultures, but not in skin. MBD4 was detected in all primary keratinocyte cultures only. This study provides evidence of TLR and MBD expression in murine skin and supports a primary role for skin keratinocytes in innate host resistance to infection.
40: 14–3–3 PROTEIN IN CSF: AN EARLY PREDICTOR OF SIV CNS DISEASE.
In ischemia as well as chronic CNS diseases including Creutzfeldt-Jakob disease, the presence of 14–3–3 proteins in CSF is believed to reflect neuronal damage. In neurons, 14–3–3 proteins may regulate signal transduction, apoptosis, and neurotransmitter production by binding to target proteins. To establish the relationship between presence of 14–3–3 protein in CSF and the development of SIV CNS disease, CSF levels of 14–3–3 were measured by quantitative immunoblot throughout infection in 6 macaques inoculated with SIV. From day 28 post-inoculation until sacrifice at 3 months post-inoculation, persistently elevated 14–3–3 levels were present in 4 of 6 animals, including all 4 macaques that developed encephalitis. In contrast, 14–3–3 protein was not detected in the CSF of 2 animals that did not develop encephalitis. Animals with 14–3–3 in CSF had elevated body temperatures (mean increase = 2.0 degrees C) and a marked decline in motor activity (mean decline of 60% from baseline) measured by telemetry in contrast to the animals without CSF 14–3–3. Levels of CSF 14–3–3 were associated with extent of CNS viral load and microglial/macrophage activation measured by quantitative immunohistochemical staining for SIV and CD68 respectively. To examine whether activated microglia or infiltrating macrophages could be a source of 14–3–3 protein, western blots were performed on supernatants from uninfected and SIV-infected primary cell cultures. 14–3–3 was not present in these supernatants, suggesting that damaged neurons are also the cellular source of 14–3–3 in the CSF of SIV–infected macaques.
41: PHENOGENOMIC DATA INTEGRATION AND VISUALIZATION (PDIV): AN EMERGING DISCIPLINE TO DRIVE INTEGRATION OF PATHOLOGY DATA WITH MEDICAL IMAGING, GENE EXPRESSION, AND LABORATORY DATA FOR MOUSE MODELS OF HUMAN DISEASE.
OBJECTIVE(S): Targeted and random mutagenesis approaches, either gene-driven or phenotype-driven, promise to identify new clinically relevant mouse models of human disease. Each approach is designed to generate a useful phenotype, and each phenotype represents the complex and combined characteristics of the model system. Morphology-based pathology phenotype is a major contributor to these heterogeneous and dynamic datasets and generation of pathology-based phenogenomic data on any individual mouse model is generating massive and complex datasets. In order to improve access to, and utility of these models of human disease, innovative new approaches are required.
MATERIALS and METHODS: To advance utility of these model systems, we need to move beyond cataloguing the components of a given genomic alteration with a limited representation of the associated pathology phenotype to meaningful integration and visualization of complete phenotype datasets, in real-time and accessible format. The tasks involved include display, visualization, and query of 2D and 3D gross-, histo-, and molecular pathology results (e.g., high resolution whole slide scans) co-registered across multiple heterogeneous medical imaging (MRI, mCT, ultrasound, radiology) and clinical laboratory data sources, along with gene nomenclature and expression datasets. An approach is required that combines the disciplines of pathology, biology, genomics, imaging, scientific visualization, human-computer interfaces, information technology, and data integration. Examples of representative data, data warehousing, visualization, integration, and data analyses approaches will be presented.
RESULTS and CONCLUSIONS: As pathology-based phenotype mapping of mutant mice continues to generate staggering amounts of data, our ability to process, visualize, analyze, integrate, and share that information has become one of our biggest challenges. To be successful, there must be a pathology-based field of scientific endeavour that will provide these tools.
42: CLINICAL AND PATHOLOGICAL PARAMETERS OF PARAINFLUENZA VIRUS-3 INFECTION IN NEONATAL LAMBS WITH CONCURRENT ADENOVIRAL-MEDIATED BETA-DEFENSIN GENE THERAPY.
Beta-defensins are a class of antimicrobial peptides with wide range of microbicidal activity, including enveloped viruses. The purpose of this experiment was to evaluate the clinical and pathological features of parainfluenza virus-3 (PIV-3) infection during co-inoculation of a non-replicative human adenovirus (Ad) with or without the human beta-defensin-6 (HBD-6) gene insert. Four groups of neonatal lambs (n = 4/group, 3–5 days of age) were inoculated intratracheally (40cc/lamb) with: 1) sterile media, 2) PIV-3, 3) PIV-3/Ad or 4) PIV-3/Ad-HBD6. On day-1 post-inoculation (PI), rectal temperatures and respiratory rates were mildly increased in all lambs, especially groups 3–4. By day-2 PI, group 3 and 4 temperatures were reduced similar to group 2 and remained steady until increasing from day-4 PI until necropsy. At day-2 PI, respiration rates were similar in groups 2–4 with a gradual increased respiration rate starting day-3 PI and an elevated respiratory rate in group 4 on days 5 and 6 PI. Groups 2–4 had reduced average daily gain and milk consumption compared group 1 though the experiment. Subjective dyspnea scores gradually increased in group 2–4 from 1-day PI to necropsy. Gross lesions in groups 2–4 consisted of multifocal, hilar to cranial ventral plum-red consolidation and atelectasis. Affected bronchiolar epithelium was hyperplastic with rare necrotic foci. Bronchiolar lumens contained degenerate neutrophils, macrophages and sloughed epithelial cells. Adjacent alveolar septa were thickened by macrophages and neutrophils. Groups 3–4 had moderately increased numbers of bronchiolar luminal neutrophils compared to group 2. Adenoviral-mediated gene therapy during PIV-3 infection slightly enhanced lesion severity possibly due to increased antigenic exposure.
43: IMMUNOHISTOCHEMICAL AND MORPHOMETRIC PHENOTYPING OF TRANSGENIC AND SPONTANEOUS MAMMARY ADENOCARCINOMAS IN THE MOUSE: A STEP TOWARD GENOTYPIC CHARACTERIZATION OF CANCER.
Regression analysis was performed on immunohistochemical and morphometric data of spontaneous and transgenic mouse mammary neoplasms. Evidences of myoepithelial differentiation, defined by the presence of basal neoplastic cells expressing a-smooth muscle actin (SMA), was detected in B6SJL-TgN(Wntl)1 Hev (Wnt1), and C57BL6/J-TgN(WapTAg)1 Knw (TAg) transgenic mice, and in most types of spontaneous tumors with the exception of a subset of papillary adenocarcinomas. Myoepithelial differentiation was absent in tumors of FVB/N-TgN(WapHRAS)69Lln YSJL (Ras), FVB/N-TgN(WapNotch4)10Rnc (Notch4), and FVB/N-TgN(MMTVneu)202Mul (Neu) mice, as well as in most tumors arising in FVB/N-TgN(WapMyc)212Bri (Myc) mice. Expression of keratins 5, 14 and 17, three markers of myoepithelial differentiation when expressed by basal cells, correlated with SMA expression. Expression of keratins 8 and 18 was most prominent in suprabasal cells of a subset of Wntl tumors and a subset of spontaneous tumors characterized by the formation of structures resembling the terminal end bud of the pubertal mammary gland. Epithelial/mesenchymal transition with occasional vimentin expression by neoplastic cells was a feature of a subset of Myc tumors. Squamous metaplasia was observed in some tumor types of mice transgenic for Myc, Ras, and Wntl, but not in tumors of mice transgenic for Notch4, TAg, or Neu. Spontaneous adenoacanthomas were the only tumors that consistently expressed filaggrin, loricrin or trichohyalin in areas of squamous metaplasia. These data suggest that Neu, Ras and Notch4 mammary tumors originate from a progenitor cell committed to the luminal compartment of the mammary gland, while the cell of origin of most other transgenic and spontaneous tumor types is not yet committed to a myoepithelial or luminal phenotype. Also, these results suggest that the Neu/Ras and Notch4 pathways are not activated in most types of spontaneous mouse mammary tumors.
44: MECHANISM OF NITRIC OXIDE REGULATION OF PLATELET GRANULE EXOCYTOSIS.
Nitric oxide (NO) inhibits platelet activation and aggregation by mechanisms that are not completely defined. Platelet activation includes exocytosis of platelet granules and the release of mediators that regulate interactions between platelets, leukocytes, and endothelial cells. I hypothesized that NO inhibits platelet granule exocytosis by inhibiting N-ethylmaleimide Sensitive Factor (NSF), an ATPase that regulates exocytosis. I demonstrate that exogenous and endogenous NO inhibits exocytosis of dense, lysosomal, and a-granules from platelets ex vivo. NO inhibits platelet granule exocytosis by pathways independent of guanylate cyclase but dependent upon S-nitrosylation of NSE NO inhibits NSF regulation of platelet granule exocytosis by increasing the affinity of NSF for Soluble NSF Attachment Receptor (SNARE) molecules that mediate exocytosis. Animals lacking endothelial NO synthase have increased platelet interactions with vascular endothelial cells, and increased thrombosis. Regulation of NSF is a novel mechanism by which NO regulates thrombosis.
45: MURINE AIRWAY HYPERREACTIVITY MODELS USING INHALED ANTIGEN.
Murine models of airway hyperreactivity used to study asthma have typically relied on a sensitization dose of antigen given intraperitoneally and a subsequent inhaled challenge dose. The objective of this study was to develop and characterize a more natural model that relied strictly on inhaled antigen in order to test the effects of environmental tobacco smoke (ETS) exposure. In this study 3 strains of female mice, A/J, Balb/ c, and C57B1/6, were exposed daily to either ETS or filtered air for 6 hr per day for 9 weeks. At three time periods relative to initiation of ETS, mice were sensitized by nose-only exposure to aerosolized 1% ovalbumin for 20 min daily for 10 days. At 6–8 weeks after sensitization, the mice were challenged with 5% ovalbumin for 5 min. Negative control mice received aerosolized PBSS only. Positive control mice were sensitized by intraperitoneal inoculation. Immediately after exposure, airway responsiveness was determined. The mice were euthanatized the following day. Blood, bronchoalveolar lavage (BAL) specimens, and perfusion-fixed lungs were collected. Differential cell counts were performed on the BAL specimens. Serum and BAL fluids were evaluated for antibody and selected cytokine responses. The lungs underwent histopathological evaluation and a 20-point scoring system was developed. Significant strain differences were seen in all responding parameters. A/J mice were most responsive, Balb/c were intermediate, and C57B1/6 mice were minimally responsive. The effects of ETS in this model were mild and primarily seen in the Balb/c mice sensitized late in the ETS exposure period. The histological lesions produced in this model were subtle yet yielded significant differences in scoring. Obvious inflammatory airway disease was seen in the classically primed mice and in occasional A/J mice only.
46: GASTRIC LAMINA PROPRIAL FIBROBLASTS ARE AN IMPORTANT INTERMEDIARY IN DEVELOPMENT OF GASTRIC EPITHELIAL PROLIFERATION IN HELICOBACTER PYLORI GASTRITIS.
The goal of this study was to evaluate the role of lamina proprial fibroblasts in the pathogenesis of gastric epithelial proliferation induced by chronic Helicobacter pylori gastritis. Primary lamina proprial stromal fibroblasts from gastric tissue of H. pylori infected and uninfected SCID mice recipients of congenic splenocytes expressed transcripts for keratinocyte growth factor (KGF), keratinocyte growth factor receptor (KGFR), and interferon-gamma receptor as determined by RT-PCR. ELISA showed that KGF secretion was significantly elevated (p<0.05) in fibroblasts treated with TNF-alpha, IL-1 alpha, H. pylori LPS, and nifedipine (positive control) compared to untreated cells. Interferon-gamma did not elicit secretion of KGF by the primary fibroblast cell line. KGF immunocytochemistry supported the ELISA results. To determine interactions between epithelial and fibroblast-derived mediators, GSM06 cells (murine gastric epithelial cells) were evaluated by RT-PCR and found to express transcripts for transforming growth factor-alpha (TGF-alpha), and KGFR without expression of interferon-gamma receptor transcripts. Both cell lines were stained immunocytochemically for phospho-STAT-1, and phospho-IKK alpha/beta (NF-kB pathway). Results showed that only the primary fibroblast cell line signaled through the JAK-STAT pathway when treated with interferon-gamma, and both cell lines signaled through the NF-kB pathway with treatment with TNF-alpha. TNF-alpha is important in the development of gastric epithelial proliferation based upon the strong association between signaling through the NF-kB pathway and the KGF secretion induced by TNF-alpha in both the KGF ELISA and immunocytochemistry. Interferon-gamma secreted in response to H. pylori causes activation of macrophages which are a source of TNF-alpha, and TNF-alpha is also secreted by stimulated CD4+ T-cells. A multifactorial pathogenesis of the gastric epithelial proliferation seen in chronic H. pylori gastritis is suggested.
47: A NEW MOUSE MODEL OF INTESTINAL TUMORIGENESIS— CELL LINEAGE STUDIES IN THE INTESTINAL MUCOSA.
The Id proteins are considered to have important functions in promoting proliferation and inhibiting differentiation. They form heterodimers with E proteins and prevent DNA binding. This sequestration of E proteins by Id proteins inhibits cell differentiation. We describe tumors in the small intestine, and occasionally the colon, in twenty-six of twenty-seven Id2 -/- mice (96%). No tumors or intestinal abnormality were observed in seventeen mice that were either Id2 heterozygote or wild type. We report the characteristics of the intestinal tumors using special stains (Alcian blue and PAS) and by immunohistochemistry markers including cell cycle proteins p21, cyclin D1 and p27. β-catenin was overexpressed in the intestinal tumors and in the adjacent mucosa with normal architecture by Western blot and immunohistochemistry. The mucosa of the intestine with normal architecture showed alterations at the cellular level in differentiation of Paneth cells, indicating an underlying derangement in the orchestration of intestinal epithelial cells along the crypt villus axis. BrdU labeling demonstrated increased proliferation in the intestinal tumors, indicative of dysregulation of the cell cycle. The re-entry of cells into the cell cycle contributed to the adenoma formation. Although the percentage of proliferating cells was not altered in the crypts in the normal intestinal mucosa, the location of the proliferating compartment in the crypts was shifted. The observations in these mice provide new insights into potential early events in tumorigenesis in the intestine, and indicate the complexity of pathways important for homeostasis of intestinal differentiation.
48: ROLE OF INDUCIBLE NITRIC OXIDE SYNTHASE AND NADPH PHAGOCYTE OXIDASE IN IMMUNOPATHOLOGY INDUCED BY TICK-BORNE ANAPLASMA PHAGOCYTOPHILUM IN A MURINE MODEL.
Human anaplasmosis and animal ehrlichioses are severe, potentially fatal, emerging tick-borne diseases caused by Anaplasma phagocytophilum. Blood phagocytes mediate inflammatory injury and destroy intracellular bacteria by NADPH oxidase generating reactive oxygen intermediates (ROI) and inducible nitric oxide synthase (iNOS) leading to release of reactive nitrogen intermediates (RNI). A. phagocytophilum stimulates a poorly restrained response dominated by IFNg and iNOS. In vitro A. phagocytophilum is also known to inhibit NADPH oxidase activity by downregulating the gp91phox. Whether activation of iNOS and NADPH oxidase are the basis for tissue injury was tested. Using a murine model and intraperitoneal inoculation of A. phagocytophilum-infected HL60 cells, mild to moderate hepatic histopathology with inflammatory responses similar to infected patients was induced. A. phagocytophilum-infected iNOS -/- (n = 3), gp91phox -/- (n = 3), and wild type C57BL/6 (n = 3) mice were examined at days 16, 25, and 43. Results indicate hepatic histopathologic lesions are more frequently encountered in gp91phox-/- mice, and hepatic pathology was more severe in gp91phox -/- mice compared to control (p = 0.006) and iNOS -/- mice (p = 0.008). No significant differences were observed in bacterial burden of various tissues of all three mouse strains at different time-points after infection. Loss of NADPH oxidase, but not iNOS, leads to a decreased ability of the host to control the inflammatory response, which is likely to cause an exacerbation of IFNg- and RNI-mediated injury, similar to that seen in chronic granulomatous disease. As previously demonstrated, tissue injury was not associated with increased pathogen burden.
49: IMMUNOHISTOCHEMICAL AND CLUSTER ANALYSIS CONFIRMS A ROLE FOR CXC CHEMOKINES IN ANAPLASMA PHAG-OCYTOPHILUM INFECTION IN VIVO.
The tick-borne pathogen Anaplasma phagocytophilum has the ability to induce expression of the neutrophil chemokine, IL-8. This mechanism allows the organism, which resides in neutrophil vacuoles, to attract other susceptible, uninfected cells into close proximity to allow spread of infection. Prior studies indicate that antibody blocking of CXCR2, an IL-8 receptor, leads to decreased A. phagocytophilum propagation. We hypothesized that the morphological basis for decreased propagation could be demonstrated by immunohistochemical (IHC) staining of tissues after A. phagocytophilum infection in control and anti-CXCR2 (MIP/IL-8 receptor) antibody-treated mice. Infected neutrophils were identified by light microscopy in splenic tissues by IHC and were recorded by location using paired x/y coordinates. Image analysis allowed superimposition of the coordinates of infected cells for each tissue exactly as observed by light microscopy. Cluster analysis was accomplished using a computer algorithm that evaluated the distance between the x/y coordinates recorded for each infected cell on a single slide. A significant cluster of infected cells was defined as more than 3 cells not separated by more than 200 microns. Cluster analysis significance was determined by comparing experimental results with tissue- and cell number-matched computer-generated randomized cell distributions. Clustering of infected cells was found to occur in control antibody treated mice (functional MIP/IL-8) (p< 0.001), but not in mice treated with anti-CXCR2, as compared to randomly distributed infected cells. With less stringent criteria (clusters separated by less than 500 microns), no significant differences from random distributions were observed. Immunohistologic observations confirm a critical role for A. phagocytophilum-induced CXC chemokine expression in enhanced propagation of infected cells, and potentially in disease pathogenesis.
50: HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 OPEN READING FRAME II ENCODED P30II IS REQUIRED FOR IN VIVO REPLICATION: EVIDENCE OF IN VIVO REVERSION.
Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma. It exhibits high genetic stability in vivo, with less than 10% nucleic acid divergence across geographic populations. HTLV-1 contains four open reading frames (ORFs) in its pX region. ORF II encodes two proteins, p30II and p13II, both of which are incompletely characterized. To determine the in vivo significance of p30II, we inoculated rabbits with cell lines expressing a wild-type molecular clone of HTLV-1 (ACH.1) or a clone containing a mutation in ORF II, which eliminated p30II expression (ACH.30.1). ACH.1-inoculated rabbits maintained higher HTLV-1-specific antibody titers than rabbits inoculated with ACH.30.1, and all ACH.1-inoculated rabbits were seropositive for HTLV-1, whereas only two of six ACH.30.1-inoculated rabbits were seropositive. Provirus could be consistently PCR amplified from peripheral blood mononuclear cell (PBMC) DNA in all ACH.1-inoculated rabbits but in only three of six ACH.30.1-inoculated rabbits. Quantitative competitive PCR indicated similar PBMC proviral loads between the proviral positive rabbits. Surprisingly, sequencing of ORF II from PBMC of all provirus positive ACH.30.1-inoculated rabbits revealed a reversion to wild-type sequence by week 6 post-inoculation with evidence of coexistance of mutant and wild-type sequence within the rabbits at week 2 post-inoculation. Taken together, our data indicated that in vivo pressures selected a reversion to wild-type ORF II sequence and that this reversion is necessary to maintain infection following inoculation with an HTLV-1 p30II mutant clone. Our data provide evidence in an animal model that this highly cell-associated virus must maintain its key accessory genes to survive in vivo.
51: HISTOPATHOLOGICAL FINDINGS IN Sc5d -/- (LATHOSTEROL 5-DESATURASE DEFICIENCY) MICE: AN ANIMAL MODEL FOR LATHOSTEROLOSIS.
Over the past few years, a number of identified inborn errors of cholesterol biosynthesis have emerged as a cause of human malformations. The prototype for this group of disorders is Smith-Lemli-Opitz syndrome, which was discovered to be a deficiency of 7-dehydrocholesterol reductase; one of more than 19 enzymatic and biochemical processes involved in the cholesterol biosynthesis pathway in mammals. To date nine inborn errors of cholesterol metabolism have been described in humans or in mutant mice. The most recently reported is lathosterolosis (deficiency in lathosterol 5-de-saturase) in two human patients. To further our understanding of the pathophysiological process underlying these disorders and to gain insight into the corresponding human disorder, we disrupted the lathosterol 5-desaturase gene (Sc5d), which catalyzes the conversion of lathosterol to 7-dehydrocholesterol in the next to last step of cholesterol synthesis, in mice. Five 18.5 day, Sc5d -/- embryos were processed as whole mounts and 44 longitudinal, serial H&E stained sections were evaluated microscopically for developmental defects. Lesions seen include, Polydactyly, palatoschisis, lingual shortening, aplasia of the soft palate/ nasopharynx, decreased tooth formation, pulmonary hypoplasia, small intestinal segmental hypoplasia and aplasia, cloaca formation, and hydrocephalus. Many of the malformations found in Sc5d -/- mice are consistent with impaired hedgehog signaling, and appear to be the result of decreased cholesterol rather than increased lathosterol.
52: INVESTIGATION OF LEUKOTRIENE INHIBITION ON PREMATURE RAT LUNGS EXPOSED TO HYPEROXIA.
Pre-term infants exposed to prolonged hyperoxia develop chronic lung disease and it is postulated to be the major contributor to the development of bronchopulmonary dysplasia (BPD). The lung injury mechanism is unknown, but elevated leukotrienes, which are inflammatory mediators and neutrophilic chemotaxins, are thought to promote BPD by augmenting the inflammatory response. Current therapeutic aims are directed against protection from pulmonary damage through the use of selective leukotriene inhibitors to counteract the effects of pulmonary edema, inflammation and increased airway resistance. This report investigates zileuton, a selective 5-lipoxygenase inhibitor, which prevents LTB4 neutrophil chemotaxis and LTC4, LTD4, and LTE4 smooth muscle constriction, eosinophil migration, and edema formation; and zafirlukast, a CysLT1 receptor antagonist, which inhibits the actions of LTC4, LTD4, and LTE4. Briefly, 39 five-week-old rat pups, divided into six groups, were exposed to either 21% or 95% oxygen and concurrently received a daily dose of either zileuton, zafirlukast or saline for seven days. On day eight, tissues were collected, fixed in 10% formalin and processed according to routine hematoxylin and eosin procedures and examined by light microscopy. Tissues were assigned a histological score for edema, bronchus-associated lymphoid tissue, congestion, hemorrhage, type II pneumocyte hyperplasia, or pleural, alveolar, interstitial, mesothelial and bronchiolar changes. Results indicate zileuton significantly protected against hyperoxia induced hemorrhage (P = 0.037) and type II pneumocyte hyperplasia (P = 0.027). Based on our findings, zileuton may have a protective effect against hemorrhage and type II pneumocyte hyperplasia and thus a clinical application in children exposed to hyperoxia.
53: OXIDATIVE STRESS INDICATORS IN A MURINE CARDIAC ISCHEMIA/REPERFUSION MODEL.
Oxidative stress plays a major role in the pathogenesis of I/R injury and modulation of the stress response and oxidative damage may be a mechanism by which selected agents exert cardioprotective effects. To understand the cardioprotective mechanism of certain drugs in I/R and determine if modulation of oxidative stress may contribute to protective effects, temporal characterization of the oxidative stress response and oxidative injury in a murine model of cardiac I/R injury was required. We examined changes in expression of selected oxidative stress response genes by laser capture microdissection and quantitative real-time PCR (TaqMan) and in situ hybridization (ISH) and evaluated extent of oxidative damage by immunohistochemistry (IHC) in myocardium from mice within the first 24 hrs following I/R. At 2, 4 and 24 hrs post-I/R, mRNA levels for p21 and heme oxygenase-1 (HO) were increased compared with control. Increased expression of iNOS and metallothionein (MT) was not observed until 24 hrs post-I/R. ISH revealed expression of HO, MT, and p21 in interstitial and endothelial cells, but not in myofibers, in non-infarcted myocardium 24-hour post-I/R. Expression of HO and MT was not detected in control hearts by ISH. Immunoreactivity for nitrotyrosine, a marker of peroxynitrite oxidative damage, was localized primarily to myocyte plasma membranes at 4 hrs and by 24 hrs. post I/R, was diffusely present throughout the myocardium. This study demonstrated acute upregulation of p21 and HO followed by iNOS and MT in myocardial interstitial and endothelial cells in response to I/R, with extent of oxidative damage in myofibers increasing progressively with time.
54: A TRANSGENIC MOUSE MODEL OF SECONDARY HYPERTROPHIC OSTEOARTHROPATHY CHARACTERIZED BY C-MYC OVEREXPRESSION IN B LYMPHOID CELLS.
Secondary Hypertrophic Osteoarthropathy (HOA) is a bone disease characterized in humans by symmetrical periosteitis, primarily of long bones. 90% of HOA in adult humans occur in patients that have a malignancy located in the thoracic cavity. The underlying mechanism of HOA is unknown. While HOA has been described in cattle, horses, and dogs, to our knowledge no murine models have been previously described. Here we report a murine model of HOA characterized by the selective expression of a c-myc transgene in B lymphoid tissues. Overexpression of c-myc intially results in a benign polyclonal expansion of B lymphocyte progenitors in the bone marrow and spleen (Eu-c-myc mice, Adams et. al., Nature 318, 1985). However, around 8–20 weeks of age, aggressive B cell lymphosarcomas develop which metastasize to many tissues including lymphnodes, liver, thymus, and brain. Characteristic bone lesions are observed in a subset of Eu-c-myc transgenic mice around 12 weeks of age, and are generally associated with B cell lymphosarcoma and thymic involvement. Microarray analysis of sorted pre-B cells from Eu-c-myc and littermate control mice, and Eu-c-myc tumors, indicate differences gene expression which could contribute to the development of HOA, but require further investigation. Immunohistochemistry and immunoblot analysis of lymphoid tissues and purified B cells show elevated levels of vascular endothelial growth factor (VEGF), which has been reported to be associated with HOA in humans. These preliminary results suggest the Eu-c-myc mouse may be a novel and informative model to investigate the pathogenesis of HOA in humans.
55: CYNOMOLGUS MONKEYS AS A MODEL FOR INHALATION ANTHRAX.
Anthrax is considered a serious biowarfare and bioterrorism threat because of its high lethality by the inhalation route. Rhesus macaques (Macaca mulatta) are the most commonly used non-human primate model of inhalation anthrax exposure. The availability of rhesus macaques necessitated development of an alternate model for vaccine testing and immunologic studies. This report describes the median lethal dose and pathology of inhalation anthrax in cynomolgus macaques (Macaca fascicularis). Cynomolgus monkeys were exposed to aerosolized Bacillus anthracis spores (Ames strain) to determine the median lethal dose. Blood cultures taken from deceased or moribund monkeys were used to confirm B.anthracis infection. All monkeys that died or were euthanized (n = 14) were necropsied and tissue samples were formalin-fixed for microscopic examination. The median lethal dose and 95% confidence intervals were 61,800 (34,000 110,000) colony-forming units. The most common gross lesions were mild splenomegaly, lymph node enlargement, and hemorrhages in various organs, particularly involving the meninges and the lungs. Mediastinitis, manifested as hemorrhage and/or edema, affected 29% of the monkeys. Microscopically, lymphocytolysis occurred in the lymph nodes and spleens of all animals, and was particularly severe in the spleen and in lymph node germinal centers. Hemorrhages were common in lungs, bronchial lymph nodes, meninges, gastrointestinal tract, and mediastinum. These results demonstrate that the aerosol median lethal dose of the Ames strain of B.anthracis was similar to previously reported values in the rhesus monkey. The gross and microscopic pathology of inhalation anthrax in the cynomolgus monkey is remarkably similar to that reported in rhesus monkeys and humans.
56: EFFECTS OF EXPERIMENTAL INFECTION OF CALVES WITH NONCYTOPATHIC TYPE 2 BOVINE VIRAL DIARRHEA VIRUSES ON HEMATOPOIETIC TISSUE.
To investigate the hematological abnormalities observed with noncytopathic type 2 bovine viral diarrhea virus (ncpBVDV-2), 6-8 month old calves were inoculated with either high (HV24515) or low (LV11Q) virulence isolates. Blood and bone marrow samples were collected to assess the effects of the viruses on hematopoietic tissue. Calves were viremic from day 4–10 post-inoculation. Neutrophil, lymphocyte and platelet counts decreased concurrently in all inoculated calves, but were significantly lower and remained decreased longer in calves given HV24515. For each isolate, a decrease in mature bone marrow myeloid cells coincided with the development of neutropenia, but depletion persisted significantly longer in calves given HV24515. Proliferating myeloid cells increased in proportion to the decrease in mature bone marrow myeloid cells in calves given LV11Q. In contrast, increased proliferation took 4 to 6 days longer in calves given HV24515. BVDV antigen in bone marrow cells was observed in calves inoculated with HV24515 when blood cell counts were lowest. Megakaryocytes and myeloid cells exhibited positive BVDV staining. Viral antigen was not observed in the bone marrow of calves given LV11Q. These experiments demonstrated that both high and low virulence ncpBVDV-2 isolates caused decreased leukocytes and platelets but the HV24515 isolate caused a delay in the production of proliferating myeloid cells. The delay may contribute to the ability of certain ncpBVDV-2 isolates to induce severe disease.
57: COMPARISON OF IMAGING MODALITIES AS PATHOLOGY TOOLS FOR EVALUATING SOFT TISSUE NEOPLASMS: APPLICATION TO A RAT MODEL OF HUMAN UTERINE FIBROID DISEASE.
Noninvasive in vivo imaging enables longitudinal assessments of experimental disease in the same animal. Animal studies typically utilize a single imaging modality, and paradigms for modality selection are not well established. Using a combination of MRI, CT and US, we evaluated each modality for general benefits and disadvantages in detecting and tracking solid soft tissue tumors. For this purpose spontaneous myometrial leiomyomas in eleven 16 to 20 month-old Eker rats served as a model. Uterine fibroids are the most common reproductive tract neoplasm in women, with a reported incidence near 77%, and are the leading indication for hysterectomy. Approximately 65% of female Eker rats with germline mutation in the tuberous sclerosis 2 (tumor suppressor) gene develop uterine fibroid-like lesions. MRI pulse sequences with and without abdominal cavity fat signal suppression resulted in complimentary series of images. Some lesions visible by MRI were difficult to detect by CT due to lack of contrast between urinary bladder and colon. Typical uterine body and pericervical lesion topography enabled reproducible assessment of fibroids in an axial view plane by US. In-time measurements and relative high throughput of US were seen as advantages, however advantages were diminished by file management difficulties experienced. Threshold US cervical axial plane area to body weight ratios correlated with mass presence at necropsy. Complete agreement of quantitative image analysis was not always obtained among modalities, however. While throughput and imaging processing are important considerations, lesion biology will also continue to influence modality selection.
58: MODULATION OF CYP2E1 AND CYP3A EXPRESSION BY THE POTENT INDUCERS, PHENOBARBITAL AND DEXA-METHASONE IN SZ-INDUCED DIABETES OF APA HAMSTERS.
Although the influences of diabetes on the metabolism of xenobiotics and/or the expression of cytochrome P450 (P450) have been characterized by many investigations, the modulation of P450 expression by some xenobiotic inducers in the diabetic animals have not been discussed. Therefore, we examined the effects of phenobarbital (Phe) and dexamethasone (Dex) on the expression of CYP2E1 and CYP3A in the liver of control (CB) and streptozotocin (SZ)-induced diabetic APA hamsters. Only Phe treatment caused about a 140% increase of P450 content compared to the untreated animals. No significant change in the activity of 6-hydroxylase of chlorzoxazone was seen in SZ hamsters treated with Phe compared to that in non-treated SZ hamsters, whereas Dex treatment induced this activity in both CB and SZ hamsters, and anti-CYP2E1 antibody markedly inhibited this activity. The activity of 6b-hydroxylase of testosterone was induced only by Dex treatment in both CB and SZ hamsters and anti-CYP3A antibody inhibited almost completely this activity. The content of CYP2E1 protein was induced only by Dex treatment, although the expression of CYP2E1 mRNA was increased in Phe-treated SZ hamsters and Dex-treated hamsters. Both Phe and Dex treatment changed the distributions of CYP2E1 and CYP3A in the acinus of the livers in SZ hamsters. These data suggest that in APA hamster CYP2E1 and CYP3A expression and these activities are differently induced or suppressed by diabetes from those of rats and mice, and the diabetes can alter the inducibilities of CYP2E1 and CYP3A by potent inducers.
59: OUTFITTING THE IMMUNOHISTOCHEMISTRY LABORATORY FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY CASES: WHERE PERCEPTION MEETS REALITY.
No uniform protocols for biohazard containment, infectious material disposal, and personnel health and safety practices are used in veterinary diagnostic histology/immunohistochemistry laboratories. While it is important that procedures and safety precautions for staff in the laboratory be guided by accurate scientific information (rather than sensationalized media reports), laboratory supervisors must attend to the real and perceived safety concerns of the staff. The South Dakota State University Animal Disease Research & Diagnostic Laboratory has made the decision to maximize caution relative to TSE-suspect cases. A small room and adjacent laboratory counter space within our histology laboratory is the designated work area for TSE-suspect material. Dedicated equipment includes a microtome, slide dryer, tissue processor, embedding station, flotation bath, refrigerator/freezer, timer, scale, immunohistochemistry-autostainer, paraffin dispenser, slide racks, pedal-operated sink, tissue digester, propar/xylene/xylene-substitute recycler, combination fume hood/ biological safety cabinet, heating tank to boil appropriate equipment in 1M sodium hydroxide, and carboys to treat waste water with 1M sodium hydroxide. Personnel entering the dedicated area wear disposable lab coats, hair caps, shoe covers, face shields, disposable respirators, glove liners, and disposable nitrile gloves. The carcasses and all non-brain viscera, used paraffin from the embedding station, paraffin-embedded tissue blocks ready to be discarded, and disposable materials such as plastic biohazard bags, non-porous lab coats, and gloves are incinerated. Distilled residue from the xylene and propar, liquid waste from the autostainer, used formalin, and alcohol undergo alkaline hydrolysis before incineration. All wastewater is treated with sodium hydroxide prior to entering the sewer. Finished slides are soaked in 1M sodium hydroxide before being removed from the designated area by the pathologist.
60: PATTERNS OF PRP-CWD DISTRIBUTION AND SPONGIFORM ENCEPHALOPATHY IN THE OBEX AND LYMPHOID TISSUES OF ROCKY MOUNTAIN ELK WITH CHRONIC WASTING DISEASE.
Sections of obex, palatine tonsil, and medial retropharyngeal lymph node were examined with immunohistochemical staining (IHC) using monoclonal antibody F99/97.6.1 for detection of protease resistant prion protein (PrP-CWD) in 10,078 captive Rocky Mountain elk, 158 of which had chronic wasting disease (CWD). Brain sections also were stained with hematoxylin and eosin and examined for spongiform encephalopathy (SE). Based upon the location and abundance of IHC and the location and severity of SE, elk were placed into 6 groups. Animals with no detectable PrP-CWD in the obex and lymphoid tissues were placed within group 1. Animals with no IHC in the obex and positive staining in the lymphoid tissues were placed within group 2. Animals with staining only around several neurons in the lateral aspect of the middle third of the vagus nucleus (VN) up to filling half of this nucleus were placed within group 3. Animals with staining filling the VN, but not found outside of this nucleus were placed within group 4. Animals with staining filling the VN and minimal to moderate PrP-CWD detection in surrounding nuclei and white matter tracts were placed in group 5. Animals with heavy staining throughout the section of obex and within surrounding nuclei and white matter tracts were placed within group 6. Mild to moderate SE was found in groups 5 and 6. These grades (2–6) may represent spread of the abnormal prion protein within the brainstem at the level of the VN. Clinical signs of CWD were observed only in elk placed in grade 6.
61: LESIONS ASSOCIATED WITH AN OUTBREAK OF MAREK'S DISEASE IN GREEN JUNGLEFOWL (GALLUS VARIUS).
Marek's disease virus (MDV) is a gamma-herpesvirus often associated with neoplastic transformation of lymphoid cells in avian hosts (primarily chickens). Over an 18-month period, 12 captive-bred green junglefowl died with evidence of atypical or neoplastic lymphoid infiltrates in multiple organs. Clinical signs observed prior to death included lymphocytosis (> 100,000 cells/microliter in some cases), weakness, ataxia, lameness, and palor of the comb. Birds averaged approximately 8 months of age at the time of death. Gross lesions were most commonly observed in the pectoral muscles (7/ 12), kidney (5/12), spleen (4/12), liver (3/12), adrenal gland (3/12) and included patchy pale tan infiltrates, organomegaly and pallor, and distinct tan masses. Enlargement of the sciatic nerves was observed in 3 birds. Histopathology revealed neoplastic lymphoid infiltrates in peripheral nerves (10/12), skeletal muscle (10/12), spleen (9/12), gonad (9/12), kidney (9/12), lung (9/12), liver (9/12), heart (6/12), skin (6/12), intestinal tract (6/12), adrenal gland (6/12), pancreas (6/12), proventriculus (4/12), and brain (3/12). MDV infection was confirmed via PCR amplification of the oncogenic MEQ gene from lesional tissue collected from 4 birds. Transmission electron microscopy confirmed the presence of viral particles in neoplastic lymphoid cells from 1 bird. Three of the affected birds had been vaccinated against MDV serotypes 2 and 3 (Ft. Dodge, MD-VAC, 0.2ml SQ) at <24 hours of age, but still succumbed to disease at 5–8 months post vaccination. Since MDV was first diagnosed in this flock, 100% of the on-site hatched juvenile junglefowl have died of this disease. It is unknown whether green junglefowl are uniquely susceptible to MDV or if the MDV strain responsible for this outbreak is especially virulent. Attempts are underway to isolate virus from plasma collected from affected birds.
62: ROLE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 p30II IN REGULATION OF CELLULAR GENE TRANSCRIPTION.
Human T-Lymphotropic virus type 1 (HTLV-1), the etiologic agent of Adult T-cell Leukemia/Lymphoma, is a member of the deltaretrovirus group, along with Bovine Leukemia Virus and Simian T- Lymphotropic virus. HTLV-1 encodes various accessory genes in four pX open reading frames. PX ORF II encodes two proteins, p13II and p30II, which are not defined clearly in terms of virus life cycle or pathogenesis. Selected mutations in pX ORF II that abolish p30II, and p13II expression diminished the ability of HTLV-1 to maintain high viral loads in infected rabbits. We have shown that the nuclear localizing protein, p30II differentially modulates CREB responsive element and Tax responsive element mediated transcription through its interaction with CREB binding protein (CBP)/p300. p30II target genes and other not yet identified direct p30II-responsive DNA elements may include promoters of genes critical for T-cell function, such as the IL-2 promoter, which contains Oct-1-responsive elements. We hypothesized that p30II has functional significance as a regulator of various cellular and viral genes. In this study, we have further characterized the role of p30II in regulation of cellular gene expression, using a stable p30II expression system employing lentiviral vectors and Affymetrix U133A arrays, representing ∼39000 genes. We have identified that p30II regulates expression or activity of several cellular genes, involved in many biological and molecular functions, such as apoptosis, cell proliferation and cell cycle. This is the first study showing the effect of an HTLV-1 accessory protein, on cellular gene expression. Collectively, our data suggest that p30II, modulates cellular gene expression to allow the virus to maintain proviral loads in vivo.
63: CRYPTOCOCCOSIS DUE TO CRYPTOCOCCUS NEOFORMANS GATTI IN STRANDED HARBOR AND DALL'S PORPOISES IN THE PACIFIC NORTHWEST.
As with the recently recognized outbreak of cryptococcosis in humans and terrestrial animals on Vancouver Island, British Columbia, over the last 4.5 years, an increased incidence of Cryptococcus neoformans gatti infection has been observed in wild Dall's (Phocoenoides dalli) and harbor porpoises (Phocoena phocoena) stranded along the Pacific northwest. Although C neoformans gatti and a closely related fungus, C. neoformans neoformans have sporadically been reported in captive dolphins in the United States, this reported outbreak is believed to be among the first due to C neoformans gatti in wild cetaceans. The condition has been identified in 2 subadult and 7 adult animals consisting of 4 harbor and 5 Dall's porpoises. Animals typically wash ashore dead and are in fair to moderate body condition with no apparent external lesions. Internal examination reveals severe pneumonia and attendant lymphadenopathy. Microscopically, there is a nodular to diffuse granulomatous infiltrate predominantly within the lung and lymph nodes with florid multisystemic accumulations of yeast. Polymerase chain reaction for morbillivirus has consistently been negative. Intensive environmental investigations have disclosed that the Douglas fir, alder and cedar trees, particularly along the Coastal Douglas Fir Biogeoclimatic zone of Vancouver Island are the probable sources of contamination. Exposure is attributed to air borne dissemination of fungal elements. From an ecological perspective, these tree species are highly mobile, but similarities between the isolates from these porpoises and other animals, people and the environment suggest that exposure was in this Vancouver Island endemic focus. This fungus can survive in salt water and infection has likely occurred along coastal foreshores. The contribution of this pathogen to changes in population dynamics remains unknown.
64: PULMONARY ULTRASTRUCTURE IN SPONTANEOUS FELINE IDIOPATHIC PULMONARY FIBROSIS: EVIDENCE FOR A PRIMARY TYPE II PNEUMOCYTE DEFECT.
Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disorder of humans characterized by progressive remodeling of the alveolar parenchyma. Recently, we reported on spontaneous IPF in a group of domestic cats. Current information on the pathogenesis of IPF in humans suggests that the disease may be due to a defect in alveolar repair. Recently, a kindred of humans with a defect in the pro-surfactant protein C gene developed idiopathic interstitial lung disease associated with abnormal type II pneumocytes. Based upon this information, and the previously reported histopathologic findings, we hypothesized that IPF in cats is associated with a defect in type II pneumocytes. Tissues from 6 cats with spontaneous IPF were examined using electron microscopy. The tissues were routinely processed, embedded in Araldite 501, and examined using a Phillips 301 electron microscope. In the areas of active remodeling, the airspaces were lined by numerous hypertrophic, hyperplastic type II pneumocytes. In the plastic-embedded lung, the type II pneumocytes contained many large basophilic inclusions within the cytoplasm. The ultrastructure of the pneumocytes identified the bodies as lamellar body-like inclusions. These inclusions were densely osmiophilic and condensed, with little ultrastructural similarity to normal type II pneumocyte lamellar bodies. Many of the altered pneumocytes were sloughed into the alveolar lumen. Dysplastic lamellar bodies were seen within alveolar macrophages and rarely within the fibrotic pulmonary interstitium. These findings share similarities with the ultrastructure of type II pneumocytes of human IPF. We conclude that spontaneous feline IPF is a defect in type II pneumocyte function that leads to the progressive fibrosis characteristic of IPF.
65: VERTEBRAL FRACTURES IN MINK.
Department of Veterinary Diagnostic Medicine, College of Veterinary Medicine, University of Minnesota.
Posterior paresis/paralysis in mink is recognized as an important problem by mink ranchers, affecting up to 1% of the animals; however, this entity has not been studied in detail. The objectives of this study were to determine the natural history of the disease in growing mink and to determine its cause and pathogenesis. Necropsies, including radiographs of the entire vertebral column and histological examination of sections of thoracic spine, were performed on 150 mink. The animals ranged in age from newborn to 3 years of age, and included clinically normal as well as affected animals. Mink became clinically affected during the fast growth period (July and August) in an age range of 7–10 weeks. Animals of all coat colors were affected, with no obvious familial predisposition. Nearly all clinically affected animals had an isolated, explosive thoracic vertebral fracture that was centered on an intervertebral disk space and was characterized by marked callus formation and was sometimes accompanied by suppurative inflammation. Approximately 45 percent (5 out of 11) of the vertebral sites that were cultured for bacteria revealed a pure growth of Streptococcus sp. In many cases, the vertebral lesion was accompanied by myelomalacia and suppurative myelitis. Affected and unaffected animals had variable osteopenia of the dorsal thoracic vertebral cortices that became apparent histologically at approximately 6 weeks of age, but was not apparent radiographically in any of the animals. A review of husbandry practices revealed no history of trauma and did not implicate dietary factors. Necropsy examinations revealed no evidence of trauma or hemivertebra. In summary, no clearly identifiable etiology was determined for this puzzling disease entity, which clearly merits further study.
66: NEOPLASTIC DISEASES OF MILITARY WORKING DOG PERSIAN GULF VETERANS COMPARED TO NON-DEPLOYED CONTROLS.
The prevalence and histogenesis of neoplasia at necropsy of US Military Working Dogs (MWD) were studied to determine if deployment to the Persian Gulf (PG) in support of Operations Desert Shield and Desert Storm affected the number or types of neoplasms present. Since deployed dogs were exposed to the same natural and war-related environmental hazards as human veterans and have similar physiology, they can be considered a sentinel species, and their neoplastic disease outcome may predict a similar outcome in human veterans. Canine veterans and controls were compared in a matched cohort study designed to equalize the confounding effects of breed, sex, and age at death. Histopathological diagnoses rendered on 117 primary neoplasms collected at necropsy of 104 deployed MWD were compared to those of 465 neoplasms of 406 non-deployed controls. Analysis revealed veterans and controls had the same mean number of neoplasms (1.1), and the prevalence difference between groups for 76 histologically distinct neoplasms was less than 5%. There were no statistically significant differences identified when the relative risk of neoplastic disease was calculated using broad histogenic classifications (epithelial, mesenchymal, hemolymphatic, melanocytic, endocrine/neuroendocrine, neuroepithelial, germ cell) irrespective of location, or by organ system irrespective of histogenesis. Relative to controls, veterans risk for neoplastic disease (1.04), malignant neoplasms (1.08), metastasis (1.08) and dying due to neoplasia (1.39) were not significantly different. If deployed MWD are a valid sentinel model for human combatants, these findings indicate human PG veterans may not develop significantly more neoplasms later in life than will non-veterans.
67: EXPRESSION OF DIFFERENTIATION-SPECIFIC MARKERS IN KERATINOCYTES FROM NORFOLK TERRIER DOGS WITH A HERITABLE KERATIN 10 DEFECT.
A recessive cornification defect resulting from a keratin 10 mutation has been identified in Norfolk terrier dogs. Affected dogs have generalized hyperkeratosis and superficial epidermolysis that resembles a mild form of epidermolytic hyperkeratosis. Real-time PCR was used to compare the expression of genes associated with terminal epidermal differentiation between affected dogs and normal controls to define the abnormal cornification process. The most accurate method of evaluating gene expression is through in vivo extraction of high quality RNA; however, because the canine epidermis is generally 1–2 nucleated cell layers thick it is difficult to obtain sufficient quantities of RNA to analyze multiple genes from standard punch biopsies without amplification. Conversely, cell culture provides ample high quality RNA, but may not simulate completely normal cornification. In this study, we analyzed keratinocytes from both skin biopsies and organotypic cultures to determine if similar changes in gene expression occurred in each system. Initially, expression levels were obtained for keratin 10 to determine if the mutation resulted in decreased transcription levels. A 119-fold decrease in keratin expression was detected in cultured keratinocytes while a 5.0- to 5.5-fold decrease was detected in tissue keratinocytes. Although both results indicated post-transcriptional mRNA degradation, the larger decrease in culture suggested additional in vitro constraints on keratin 10 expression. Further analysis was performed for keratin 1, keratin 2e and transglutaminase 1. Keratin 1 was increased over normal in biopsies from affected dogs but decreased over normal in cultured cells. Keratin 2e was variably expressed while transglutaminase expression did not change between normal and affected keratinocytes in tissue or cell culture.
68: PARANEOPLASTIC PEMPHIGUS IN A DOG WITH SPLENIC HEMATOPOIETIC NEOPLASIA.
Paraneoplastic pemphigus (PNP) is a rare but severe autoimmune blistering disease that is associated, in humans, with lymphoproliferative malignancies. One case of canine PNP, which was associated with a thymic lymphoma, has been reported. Herein, we report a second dog with PNP that developed in association with a splenic hematopoietic neoplasia. The patient was a seven-year-old male castrated Golden Retriever presenting with severe generalized mucocutaneous erosions and ulcerations. Microscopic examination of skin biopsy specimens revealed suprabasilar clefting and apoptotic keratinocytes suggestive of pemphigus vulgaris and erythema multiforme, respectively. The splenic tumor was characterized by a nodular proliferation of lymphoid and neoplastic fibrohistiocytic cells intermixed with hematopoietic elements. Direct immunofluorescence (IF) revealed intercellular deposition of IgG in both epidermal and follicular epithelia. Indirect IF confirmed the presence of circulating IgG autoantibodies that bound the membrane of epithelial cells of canine gingival and bladder substrates. Immunoprecipitation established that IgG autoantibodies targeted desmoplakin I and II, envoplakin, periplakin and BPAG1. Additionally, immunoblotting studies revealed that IgG autoantibodies also recognized the extracellular segments of recombinant human desmoglein-3 and canine desmoglein-1. The clinical, histopathological, and immunopathological characteristics of the disease affecting this dog are all diagnostic for PNP. Additionally, this is the first report of canine PNP occurring in association with malignant splenic hematopoietic neoplasia and circulating anti-desmoglein autoantibodies.
69: ENDOCRINE DIFFERENTIATION IN METASTATIC PANCREATIC EXOCRINE ADENOCARCINOMA.
Biopsies were taken from the pancreas, liver, and mesenteric lymph nodes of a nine-year-old Shetland sheepdog with multiple abdominal masses and a history of hypoglycemia. The histopathological diagnosis was pancreatic exocrine adenocarcinoma with metastases to the liver and lymph node. Due to the hypoglycemia with a concurrent normal insulin level observed in this dog, the biopsies were further evaluated by insulin immunohistochemistry and transmission electron micrography. Scattered insulin-positive cells were identified by insulin immunostaining and electron micrography identified individual cells with ultrastructural features consistent with pancreatic endocrine cells. These findings are consistent with previous studies suggesting a common stem cell origin of pancreatic endocrine and exocrine cells, and may help explain why hypoglycemia is sometimes observed in dogs with pancreatic exocrine adenocarcinomas.
70: RHODOCOCCUS EQUI- INFECTED MACROPHAGES ARE RECOGNIZED AND KILLED BY CD8+ T LYMPHOCYTES IN A NON -ELA-A (MHC CLASS I) -RESTRICTED FASHION.
R. equi is an intracellular bacterium closely related to Mycobacterium tuberculosis (MTb). MTb-specific CD8+ cytotoxic T lymphocytes (CTL) recognize peptide antigens presented by MHC class I molecules or non-peptide antigens presented via the CD1 system. The goal of this research project is to define the role of equine CTL in the control of R. equi. We tested the hypothesis that R. equi-specific CD8+ are present within the blood of immune horses. Following expansion of effector cells with either live R. equi or soluble R. equi antigen, equine peripheral blood mononuclear cells efficiently lyse autologous and ELA-A mismatched R. equi-infected monocyte-derived macrophages. Lysis is significantly decreased by depletion of CD8+ T cells, whereas depletion of CD4+ cells results in increased or unaltered cytolysis. To evaluate potential target antigens, target cells were infected with either virulent (plasmid bearing) or avirulent (plasmid cured) R. equi. Lysis was not altered by the absence of the plasmid, providing evidence that the virulence plasmid is not necessary for recognition and killing of R. equi infected cells. These data indicate that adult horses have CD8+ CTL, which may play a role in immunity to R. equi. The apparent lack of restriction via classical MHC molecules suggests a novel or non-classical method of antigen processing and presentation such as lipid antigen recognition by the CD1 system.
71: CHEMOKINES IN THE SKIN OF CHRONIC PROLIFERATIVE DERMATITIS (CPDM/CPDM) MICE.
Chronic proliferative dermatitis (cpdm) mice develop chronic eosinophilic inflammation in tissues, most prominently in the skin. To understand the mechanism underlying the accumulation of eosinophils in the skin, the expression of chemokines in skin samples from mutant mice and control littermates was determined. RNA was isolated from the skin and analyzed by ribonuclease protection assay. Expression of mRNA was significantly increased for CCL1 (TCA-3), CCL2 (MCP-1), CCL11 (eotaxin), and CXCL10 (IP-10), but not for CCL3 (MIPl-alpha), CCL4 (MIP1-beta), CCL5 (RANTES), XCL1 (lymphotactin), and CXCL1 (MIP-2) in the skin of cpdm/ cpdm mice. Skin lysates were prepared and the concentrations of CCL2 and CCL11 were determined by ELISA. The concentration of CCL2 and CCL11 was increased 4–5 fold in the skin of cpdm/ cpdm mice. In vitro culture of primary dermal fibroblasts from cpdm/cpdm and control mice with TNF-alpha, IL-4, and IL-13 did not reveal differences in the ability to secrete CCL2 and CCL11. These studies suggest that the cpdm/cpdm mouse is a useful model to determine the role of chemokines in chronic eosinophilic inflammation.
72: CALCIFYING ONDONTOGENIC CYST AND HETEROTOPIC POLYODONTIA IN A MALE CYNOMOLGUS MONKEY (MACACA FASCICULARIS).
A 5-year-old, naive, male cynomolgus monkey (Macaca fascicularis) was submitted to necropsy with an enlargement of the left mandible as determined by physical examination and radiography. The monkey was seronegative for Herpes B, SIV, STLV-1, and SRV. Hematology values were within normal limits. A mildly decreased albumin to globulin ratio, indicative of inflammation, was seen in the clinical chemistry panel. Macroscopically, a well circumscribed 3.5 × 2.5 × 2.0 cm mass was present in the left mandible. A fistulous tract, 0.3 cm in diameter, was present on the left mandible rostral to Molar 2. Microscopically, an non-erupted, supernumerary tooth (heterotopic polyodontia) was detected, and was within normal histological limits. A periodontal epithelial cyst with communication to the surface was also present. This epithelial cyst was characterized by marked epithelial proliferation with hyperkeratosis containing ghost cells, and complex epithelial cords extending into the surrounding tissue. In several foci the epithelial morphology was suggestive of odontogenic origin. Foci of dysplastic dentin or osteodentin material were found adjacent to the periodontal cyst. A severe mixed inflammatory infiltrate, and marked mandibular osseous proliferation were also present. A diagnosis of calcifying odontogenic cyst was made based upon the presence of epithelial proliferation and osteodental differentiation adjacent to the cyst. Primary dental neoplasms are uncommon in the cynomolgus monkey. A previous report identified an ameloblastic odontoma in this species, while a single case of heterotopic polyodontia was also found. To our knowledge, a calcifying odontogenic cyst has not been previously described in the cynomolgus monkey.
73: GRANULOMATOUS PNEUMONIA, PLEURITIS, MYOCARDITIS, AND LYMPHADENITIS CONSISTENT WITH ZYGOMYCOSIS IN A CYNOMOLGUS MONKEY (MACACA FASCICULARIS).
A 4-year-old, female cynomolgus monkey (Macaca fasciculahs) died unexpectedly during handling. The monkey was serologically negative for Herpes B virus, SIV, STLV-1, and SRV, but was positive for measles. A hemogram and clinical chemistry panel were within normal limits. At necropsy, an 8cm diameter, mediastinal mass was present. The mass was multilobulated and surrounded the trachea, esophagus, and aorta, and invaded the right lung and left atrium. Microscopically, there were severe multifocal to coalescing chronic granulomatous pneumonia, pleuritis, myocarditis, and lymphadenitis with numerous intralesional fungal hyphae. The fungal hyphae had thin non-parallel walls (5–15um in diameter), bulbous dilatations and non-dichotomous branching, often at right angles. These histological features were consistent with the Zygomycetes group. An acid- fast stain (Ziehl-Nielsen) did not demonstrate acid-fast bacilli. The death of this monkey was due to a combination of respiratory and cardiac insufficiency secondary to the chronic fungal infection. The source of the fungal infection is unknown, but may have been caused by a penetrating wound. In monkeys, fungal infections have been frequently described, including cutaneous Zygomycosis in rhesus monkeys. To our knowledge, this is the first report of invasive thoracic Zygomycosis in a cynomolgus monkey.
74: SPONTANEOUS PROSTATIC LESIONS IN RHESUS MACAQUES.
Prostatic diseases, including benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma affect a large proportion of aged men. There has been extensive research into the pathogenesis of prostatic proliferative diseases, but efforts need to be sustained in order to find therapeutic approaches for treatment and prevention. There are few spontaneous veterinary prostatic lesions. Canine BPH and adenocarcinoma occur spontaneously, but the behavior of the lesions and the anatomic differences make the dog a problematic model. Rodent models generally lack spontaneous hyperplastic prostatic changes and anatomically are markedly different from man. Previous surveys of prostatic lesions in rhesus macaques (Macaca mulatto) utilized small numbers of aged animals and produced conflicting results. This study evaluated the frequency and character of prostatic lesions in the rhesus macaque. The survey included sections of the cranial and caudal prostatic lobes in animals ranging from 2–27 years old. The majority of prostates evaluated had a minimal to mild peritubular and occasional interstitial lymphocytic infiltrate occurring predominantly in the caudal lobe. Fewer prostates contained minimal intralumenal neutrophilic infiltrates. Approximately 10 percent of the prostates contained hyperplastic epithelial lesions, ranging from 100–300 microns in diameter, randomly arranged in the caudal prostate. Focal stromal proliferation or urethral impingement was not seen. Recently, hormonally induced prostatic lesions in rhesus have been used to evaluate toxic effects of potential therapeutic agents. Other studies reveal a potential link between low-grade chronic prostatitis and BPH in man. Our results demonstrate a low-grade background prostatitis in the majority of mature rhesus and infrequent nodular epithelial hyperplasia in fewer macaques of advanced age. We suggest these results should be considered background or contributory changes when evaluating the rhesus prostate as a model for human disease.
75: A TRANSITIONAL MENINGIOMA IN A FEMALE RHESUS MACAQUE (MACACA MULATTA).
Tumors of the central nervous system (CNS) are an infrequent finding in macaque monkeys. Malignant tumors of the CNS that have been reported in macaques include ependymomas, astrocytomas, lymphomas and oligodendrogliomas; however, little information is available concerning benign tumors of the CNS in macaque species. Meningiomas, which are tumors that originate from the meningeal cap (meningothelial) cell, are reported in significant numbers in humans, cats, dogs, F344 rats, and less frequently in other species. This is a report of a transitional (mixed) meningioma in a female rhesus macaque. Approximately one week before death, the monkey was inactive, appeared blind and was ataxic. Despite supportive care, the animal died. Grossly, there was a discrete, nonen-capsulated, friable, tan/pink mass in the region of the right piriform lobe that extended from the region of the optic chiasm to the anterior portion of the cerebellum and brainstem. The mass was easily dissected free of the underlying brain parenchyma, and had induced severe atrophy of the piriform lobe. Histologically the mass was comprised predominantly of sheets of spindle-shaped cells that whorled around blood vessels and included multifocal mineralized concretions (psammoma bodies) throughout. Based upon the gross appearance and histologic pattern, a diagnosis of a transitional (mixed) meningioma was made. While meningiomas are a common neoplastic lesion in humans, they are rarely reported in nonhuman primates.
76: GASTROINTESTINAL STROMAL TUMOR IN A CHIMPANZEE (PAN TROGLODITES).
At necropsy of a 22-year-old male chimpanzee, a round, firm 2 cm in diameter tumor was found at the level of mid stomach and diagnosed as a gastrointestinal stromal tumor (GIST). Histologically, the mass was composed of spindle to polygonal epithelioid cells arranged in short to intermediate-length, interlacing streams, bundles, and nodular whorls often separated by hyalinized eosinophilic matrix. Tumor cells had indistinct borders with moderate amounts of pale, fibrillar, eosinophilic cytoplasm, and round to elongate nuclei with finely stippled chromatin and indistinct nucleoli. The mitotic rate was less than 1/50 HPF. Immunohistochemically, tumor cells were diffusely positive for CD117 (KIT) and CD34, focally positive for smooth muscle actin, and negative for muscle specific actin, desmin, S-100 protein, synaptophysin, and glial fibrillary acidic protein. Gain-of-function KIT mutations are considered to be a driving force in the development of human GISTs. A great majority of these mutations occur in exon 11 (juxtamembrane domain); however, alterations of exon 9 (extracellular domain) and exon 13 and 17 (tyrosine kinase domains) have also been reported in a few cases. Exons 9, 11, 13, and 17 of chimpanzee KIT gene were PCR amplified using intronic primers based on human KIT gene sequences. PCR products were sequenced directly and analyzed using DNA STAR software. Although chimpanzee and human KIT gene sequences revealed high homology, no mutations were identified in mutational hot spots. More cases should be studied to identify molecular genetic mechanisms causing KIT activation in chimpanzee GISTs. However, this case report is the first complex, histomorphologic, immunohistochemical and molecular genetic study of chimpanzee GIST.
77: CEREBRAL INFARCTION IN A MALAYAN SUN BEAR (HE-LARCTOS MALAYANUS).
Wyeth Research, Chazy, NY; Cornell University, Ithaca, NY; and White Oaks Conservation Center, Yulee, FL.
A 17-year-old Malayan Sun Bear (Helarctos malayanus) developed an acute onset of left hemiparesis and proprioceptive deficits. A diagnostic workup that included a CT scan was unrewarding. Following steroid therapy, the left hemiparesis improved, but the proprioceptive deficits did not. Sixteen months later, quadriparesis, proprioceptive deficits in all four limbs, circling to the left, and stumbling were noted. The bear was then euthanized. Macroscopically, there was bilateral discoloration in the cerebrocortical white matter at the level of the centrum semiovale, extending caudally. Atrophy and white discoloration of the crus cerebri, longitudinal fibers of the pons, pyramid, and deep dorsal aspect of the left lateral funiculus of the first cervical spinal cord segment were also present. Microscopically, two distinct cerebral infarcts were identified. The right cerebral infarct was older, more extensive, and induced the left hemiparesis and proprioceptive deficits. Leukoencephalomalacia, reactive astrocytosis, hemorrhage, hemosiderin-laden macrophages, and cholesterol clefts were present. The left cerebral infarct was more recent, and was cavitated. Gitter cells were found. The left cerebral infarct likely affected the right limbs leading to quadriparesis, and proprioceptive deficits. Numerous arteries at the terminal distribution of the middle cerebral arteries contained atheromatous plaques. Extensive Wallerian degeneration and gliosis caused the atrophy of the descending motor pathways. The Sun Bear appears to be similar to primates with the cerebral lesions causing a gait deficit. The loss of the thalamocortical projection and the projection fibers from the motor cortex at the level of the cerebral infarct caused the contralateral loss of proprioception and hemiparesis, respectively. To our knowledge, cerebral infarction in the Sun Bear has not been previously described.
78: EPIDEMIOLOGICAL AND PATHOLOGICAL SURVEY OF CAPTIVE CHEETAHS (ACINONYX JUBATUS) IN JAPAN.
An epidemiological and pathological study of 340 cheetahs that died in captivity in Japan between 1935 and 2002 was carried out under the Cheetah Species Survival Plan of Japan. Of the 340 cheetahs, 140 were imported from Africa and the rest were bred in Japan. Of the total, 167 were male, 168 were female, and the sex of 5 was unknown due to cannibalization shortly after birth. At the time of death, 241 were adults aged 2 years or more. Average age at death for the adult group was 6.8 years for all adults, 7.5 years for imported adults, and 5.8 years for adults bred in Japan. Moreover, the average age at death in two facilities housing 20 or more animals was 9.4 years for imported adults, and 4.9 years for locally bred adults, while the average age at death in three facilities housing 5 or less animals was 12.2 years for imported adults, and 8.8 years for locally bred adults. Pathological studies carried out on 60 of the adult cheetahs showed that a high percentage (70%, 42/60) had renal failure. Systemic amyloidosis was observed in 53/58 cheetahs (91%), affecting particularly the kidneys, with papillary necrosis frequently observed. The degree of amyloidosis was numerically scored, and the average scores compared for imported and locally bred adult animals. Amyloid deposits tended to be more severe and present in more organs in the locally bred group, which scored 4.1 compared with 2.3 for the imported group. The amyloid deposits were immunohistochemically identified as AA-type protein. Our results indicate that amyloid-induced renal and/or multiple organ failure is a significant cause of morbidity and mortality in captive cheetahs in Japan, particularly those bred in multi-animal facilities. The results support the hypothesis that amyloidosis is a transmissible disease, and that its progress may be exacerbated by 1) exposure from infancy, and 2) in the case of cheetahs, the stress of housing in multi-animal facilities.
79: SYSTEMIC AMYLOIDOSIS IN CAPTIVE BRED CUVIER'S AND SLENDER HORNED GAZELLES.
Over a 22 month period, histopathologic examination was performed on tissues collected at necropsy from 14 captive bred gazelles: 8 Cuviers gazelles (Gazella cuvieri) and 6 slender horned gazelles (Gazella leptoceros). Systemic amyloidosis of varying severity was present in 12/14 animals from 1–6 years of age. Wasting was the most common clinical sign. Variable organomegaly was observed in liver (7/12), kidney (5/12), and spleen (1/12). Renal medullary amyloidosis was present in all affected animals except for a 1 -year-old slender horned gazelle, in which significant amyloidosis was present only in the small intestine. Concurrent glomerular amyloidosis occurred in all Cuviers’, but in only 2/4 slender horned gazelles. Hepatic sinusoidal amyloidosis was present in 10/12 animals and splenic amyloidosis was found in 8/9 animals. Amyloid deposits were also common in the abomasum (6/7), forestomach (3/4), small intestine (8/8), and large intestine (7/8). Other organs affected were adrenal (4/7), esophagus (2/2), heart (2/9), lymph node (1/2), and pancreas (1/3). Hepatic, splenic, renal, abomasal and small intestinal mucosal amyloidosis were often severe, resulting in effacement of architecture. Mild lesions in affected organs were associated with blood vessels and basement membrane zones, and were best detected with Congo red stain. Chronic inflammation, most often pododermatitis, was associated with systemic amyloidosis in 4/7 Cuvier's gazelles, and Arcanobacterium pyogenes infection was detected in 3 of these gazelles. Minimal to no evidence of chronic infection was found in affected slender horned gazelles. Systemic amyloidosis was considered the major cause of death in 1/7 Cuviers’ and in 4/5 slender horned gazelles. Systemic amyloidosis with significant involvement of the gastrointestinal tract is common in captive bred Cuvier's and slender horned gazelles.
80: BOVINE PAPILLOMA VIRUS DETECTION USING IMMUNOHISTOCHEMISTRY AND ELECTRON MICROSCOPY AND THE PREVALENCE OF BOVINE TEAT PAPILLOMA IN KOREA.
Bovine papilloma is a benign epithelial neoplasm caused by infection with bovine papilloma virus (BPV). Atypical flat papillomas of the bovine teat are often difficult to distinguish from lesions caused by Foot and Mouth disease (FMD); therefore, reliable diagnostic methods to detect papillomavirus antigen and to differentiate lesions from FMD are important. Toward this aim, we surveyed the prevalence of bovine teat papilloma by gross and histopathological examination, immunohistochemistry and electron microscopy. Teats were collected from slaughtered cattle during January 2001 to November 2002. Out of 880 total animals with teat lesions, 432(49.1%) cases were from Holstein cattle and 448(50.9%) were from native Korean cattle. Of the Holstein cases, 263(60.9%) were papilloma, 19(4.4%) were attributed to teat trauma, and 2(0.5%) were mixed infections of BPV and pseudocowpox. Of the native Korean cattle, 33(7.4%) were papilloma and 11(2.5%) cases were traumatic. Gross and histopathological examination of teat papillomas in Holstein cattle revealed 225/263(85.6%) cases of atypical flat papilloma, 12(4.6%) cases of typical fibropapilloma, and 7(2.7%) cases of atypical filiform papilloma. By immunohistochemistry (IH) for bovine papilloma virus antigen, 35/153(22.9%) cases exhibited positive staining confined to nuclei of the granular layer of keratinocytes. Of 79 cases examined by electron microscopy, 31(39.2%) were positive. In summary, we conclude: 1) the prevalence rate of papilloma in Korean Holstein cattle is higher than reported in US Holsteins, 2) native Korean cattle appear more resistant to papilloma virus infection compared to Holstein cattle, 3) atypical flat papillomas were the most prevalent morphologic type, 4) viral replication occurs within granular keratinocytes, and 5) immunohistochemistry and electron microscopy can be used to detect bovine papilloma virus diagnostically. The sensitivity of electron microscopic detection was higher than IH, most likely because most cases were first stage papilloma lesions at which time virus was not actively replicating and therefore not detectable by IH.
81: FELINE LEPROSY SYNDROME IS ASSOCIATED WITH SEVERAL MYCOBACTERIUM SPECIES: A HISTOLOGIC AND GE-NOTYPIC RETROSPECTIVE STUDY ON FORMALIN FIXED AND PARAFFIN EMBEDDED TISSUES.
In order to investigate Mycobacterium species associated with histologic lesions characteristic of feline leprosy, we performed a retrospective study of feline cutaneous mycobacteriosis using formalin-fixed paraffin embedded tissues from the archives of the Western College of Veterinary Medicine (SK, Canada) from 1971 to 2001. Other samples were obtained from Australia, New Zealand, the UK, and the USA. Cutaneous lesions from more than 30 cases were characterized histologically as feline leprosy (lepromatous or tuberculoid) using haemotoxylin and eosin and Fite's acid fast stains. DNA sequence analysis of a 450bp section of the mycobacterial 16S rRNA gene was performed to identify the Mycobacterium in the tissue sections. In addition to M. lepraemurium and M. intracellular, M. szulgai, and M. visibilis-like species were found to be associated with cutaneous lesions from cases previously identified as feline leprosy. An M. tilburgii-like species similar to that which has been previously identified in Australia and the USA causing canine leproid granuloma syndrome was also demonstrated. Based on this study, feline leprosy should be considered as a syndrome and not as a specific infection caused by M lepraemurium. Since a variety of Mycobacterium species were found to produce lesions remarkably similar to those produced by M. bovis, M. bovis should be considered as a differential in regions where this organism is prevalent. Therefore, molecular assays for species identification are essential for rapid and accurate diagnosis.
82: IMMUNOHISTOCHEMICAL AND ENZYME HISTOCHEMICAL STAINS FOR THE DIAGNOSIS OF CANINE CUTANEOUS MAST CELL TUMORS AND HISTIOCYTOMAS.
We applied a panel of monoclonal antibodies (tryptase, chymase, serotonin for mast cells; CD18, HLA-DR for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one enzyme histochemical stain (for chymase activity using naphthol AS-D chloroacetate as the substrate) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III mast cell tumors were positive for both tryptase antibody and chymase enzyme. Both the tryptase monoclonal antibody and the chymase enzyme histochemical stain were equally effective in diagnosing mast cell tumors. Serotonin antibody was negative in most mast cell tumors and not useful in the diagnosis of mast cell tumors. Chymase immunohistochemical staining was positive for both tumor and non-tumor cells and was not useful in the diagnosis of mast cell tumors. CD18 and HLA-DR were equally effective in diagnosing histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.
83: REOVIRAL TENOSYNOVITIS IN BROILER CHICKENS: REAL TIME RT-PCR DETECTION AND SEQUENCING OF VIRAL GENOMIC RNA EXTRACTED FROM FORMALIN FIXED PARAFFIN EMBEDDED TISSUES.
Avian reovirus (genus orthoreovirus family Reoviridae) infects chickens and produces multiple disease syndromes, including an economically significant tenosynovitis (viral arthritis). Infection occurs either by vertical or horizontal exposure of antibody-free neonatal chickens to arthrotropic reoviral strains. Virus initially localizes and replicates in the intestine and cloacal bursa within 2 to 12 hours of exposure. Multisystemic dissemination occurs within 24 to 48 hours, and initial infection of the hock and surrounding tendon sheaths occurs during this dissemination. This initial infection results in an acute inflammatory response, but these changes are often not grossly evident and produce no immediate clinical signs. Chronic persistence of reovirus in tendons produces intra- and peritendonal fibrosis that is grossly evident, and results in tendon weakening and subsequent rupture under loading. Reovirus is present within chronic lesions at a reduced titer; therefore, diagnosis by isolation, immunohistochemistry, or nucleic acid probes is difficult using chronically affected samples. We identified or experimentally produced, chickens with acute or chronic reoviral tenosynovitis. Tarsal tendons were collected, processed and sectioned routinely for light microscopy. Additional sections were cut from the formalin-fixed paraffin-embedded tendons, and using a real time RT-PCR with primers MK87 and MK88 to amplify a 532 base pair product of the reoviral S1 gene, we detected reovirus in these samples. Resulting cDNA was sequenced and found to be homologous to the S1 sequence of reovirus strain S1133. These result show reoviral RNA persists in tissues within routinely processed paraffin tissue blocks at sufficient levels for RT-PCR detection and sequencing of reovirus.
84: NATURALLY-OCURRING TOXIC SHOCK DUE TO STREPTOCOCCUS PYOGENES IN A RHESUS MONKEY (MACACA MVLATTA).
Resurgence of serious infections caused by group A streptococci has recently been recognized worldwide. Streptococcus pyogenes, a group A Streptococcus, is one of the most common human pathogens among children and is responsible for both a wide variety of suppurative infections such, as pharyngitis, and nonsuppurative infections with sequela of rheumatoid fever and rheumatic heart disease. Streptococcus pyogenes produces several superantigen-like erythrogenic toxins that are believed to be associated with pyrogenicity and erythromatous skin reactions in addition to various immunological and cytotoxic effects. Furthermore, these toxins may also cause myocardial necrosis. Recent reports of streptococcal infection in obstetric human patients appear to be clinically different from classic puerperal sepsis. Here, we report a spontaneous case of streptococcal infection in a pregnant female rhesus monkey (Macaca mulatto). In addition to lesions compatible with bacteremia and toxic shock, this animal had severe cardiac lesions resembling those described in humans with rheumatic heart disease. Streptococcus pyogenes was isolated from heart blood, liver, placenta and fetal tissues. PCR analysis was then used to determine the presence of the specific pyogenic exotoxin genes in this isolate.
85: CANINE DISTEMPER VIRUS INFECTION OF CANINE FOOTPAD KERATINOCYTES.
Canine distemper virus (CDV) infection can cause massive thickening of footpad epithelium due to hyperkeratosis. Early descriptions also included hyperplasia of basal keratinocytes. Footpad epithelia from dogs infected with A75/17 strain of distemper, which contained viral antigen and mRNA, had more mitotic figures, and higher staining indices for proliferation markers Ki67 and PCNA compared to virus-negative epithelia. As such keratinocyte proliferation could have been induced by dermal cells or epidermal cells other than keratinocytes, we performed in vitro studies with pure keratinocyte cultures. Canine footpad keratinocytes were infected with A75/17 distemper strain and cell proliferation was analyzed by automatic cell count, immunohistochemical staining for proliferation markers Ki67 and PCNA, incorporation of thymidine analogue BrDU, and measurement of total DNA. Controls consisted of mock- and non-infected cultures. Canine footpad keratinocytes were successfully infected with virulent A75/17 strain as demonstrated by immunohistochemistry and in situ hybridization. CDV-infected cells contained infectious virus, which was also released into the supernatant. The percentage of infected cells increased throughout the life of the culture. Infected cultures had higher cell numbers from day 3 to day 7 post-infection (pi). The percentage of cells positive for Ki67, PCNA, and BrDU incorporation was increased on day 3, 5, and 7 pi as was the amount of total DNA. Double-labeling for Ki67 and CDV nucleoprotein by immunofluorescence staining revealed Ki67 positivity in cells adjacent to CDV-positive cells, suggesting that proliferation may at least partly be due to a bystander effect. In contrast, most CDV-positive cells were negative for Ki67. In summary, canine footpad keratinocyte cultures can be successfully infected by virulent CDV strain A75/17, produce infectious virus, and proliferate as a result of infection.
86: FUNCTIONAL ANALYSIS OF HUMAN T LYMPHOTROPIC VIRUS TYPE 1 P13 ACCESSORY PROTEIN IN HUMAN LYMPHOCYTES.
Human T Lymphotropic Virus-1 (HTLV-1), a member of the deltaretrovirus group that includes bovine leukemia virus (BLV), is the etiological agent of adult T-cell lymphoma/leukemia (ATL). It encodes unique accessory proteins in four open reading frames (ORF) of its pX region of the genome. The pX ORF II encodes two proteins, p13 and p30. Though the selective ablation of the ORFII products from infectious HTLV-1 molecular clones results in reduced viral load and immune responses in rabbit model, functional roles of either protein in HTLV-1 infection have not been defined. p30 localizes to the nucleus, and functions as transcriptional factor. In contrast, p13 localizes to mitochondria via a conserved mitochondrial localization signal. To investigate the functional roles of p13 in lymphocytes, we constructed recombinant lentiviral vectors that stably express p13 in Jurkat T cell lines. The vectors were designed to allow detection of p13 expression by combining a bicistronic eGFP coding gene, facilitated by a picornaviral IRES element. Lymphocyte proliferation measured by MTS assay revealed no significant difference in growth of p13 expressing cell line under basal conditions or in response to broad T-cell stimulation. Other preliminary results indicate p13 cell lines have increased susceptibility to apoptosis and lower tumorgenicity in mouse model. These data suggest that p13 functions to modulate the proliferation of HTLV-1 infected lymphocytes.
87: EVALUATION OF SHIGA TOXIN 2E BINDING TO PORCINE LEUKOCYTES.
Shiga toxin 2e (Stx2e), a variant of Stx2, is produced by host-adapted strains of Escherichia coli and is responsible for the clinicopathologic manifestations of edema disease of swine, namely vascular necrosis, edema, and neurological signs. Edema disease has been proposed as a model for hemolytic uremic syndrome (HUS), an uncommon complication of Escherichia coli 0157:H7 infection in human beings. Recently, Stx2e has been shown to bind porcine erythrocytes, which is believed to be crucial to the pathogenesis of edema disease. In contrast, Stx2, the primary toxin implicated in the pathogenesis of HUS, has been shown to bind human neutrophils more consistently than human erythrocytes. The purpose of this study was to evaluate Stx2e binding to porcine leukocytes in vitro and to determine the potential role of these cells as toxin carriers in edema disease. Fluorescence-activated cell sorting (FACS) was used to evaluate Stx2e binding of cell populations following immunofluorescent staining. Whole blood was collected in EDTA from 2–3 month old pigs. Preparations of either porcine erythrocytes or leukocytes were incubated with Stx2e, which was identified using indirect immunofluorescence. Our results indicate that although binding of Stx2e to erythrocytes was represented by a shift in mean fluorescent intensity, no significant binding of Stx2e to lymphocytes, granulocytes, or monocytes was observed. These results suggest that porcine leukocytes do not bind Stx2e in vitro and therefore are not likely to be a significant means of Stx2e delivery to target tissues in edema disease.
88: PCR ANALYSIS FOR MYCOPLASMA HAEMOFELIS AND “CANDIDATUS MYCOPLASMA HAEMOMINUTUM” IN CATS FROM SASKATCHEWAN AND ALBERTA.
Cats with haemobartonellosis typically develop clinical and laboratory changes consistent with regenerative anemia. Two strains of the organism previously known as Haemobartonella felis, namely, Mycoplasma haemofelis (Ohio/Florida strain) and “Candidatus Mycoplasma haemominutum” (California strain) have been reported. The purpose of this study was to determine if both strains exist in naturally infected cats from Saskatchewan and Alberta, and whether disease manifestation corresponds to strain. Cats with regenerative anemia and suspected haemobartonellosis, cats with non-regenerative anemia (NRA) attributable to other illnesses, and cats with normal complete blood cell counts (CBCs) were examined for M. haemofelis and “Candidatus M. haemominutum” using PCR analysis. Primers were used that differentiated the two strains. Thirteen of 18 cats in the regenerative anemia/suspect haemobartonellosis group were infected, 12 with M. haemofelis and 1 with “Candidatus M. haemominutum”. Eight of the 22 cats in the NRA/other illnesses group were infected, 4 with M. haemofelis and 4 with “Candidatus M. haemominutum”. Of the 20 cats with normal CBCs, 2 were infected with “Candidatus M. haemominutum.” The strain of the organism was related to the severity of clinical disease, with most sick cats being infected with M. haemofelis. “Candidatus M. haemominutum” infection was usually considered incidental; however, this strain was occasionally associated with anemia.
89: WEST NILE VIRUS INFECTION IN EASTERN FOX SQUIRRELS (SC/URUS NIGER).
Since the initial outbreak of West Nile virus (WNV) in the northeastern United States in 1999, the virus has rapidly spread westward and southward across the U.S., causing high mortality in crows, as well as sporadic mortality in horses, humans, and a wide variety of birds. In 2002 the epidemic widened as hundreds of equine and human cases, and sporadic cases in other mammalian species were reported. We report an infection with WNV in three Eastern fox squirrels (Sciurus niger). Neurological signs included head tilt, uncoordinated movement, paralysis, and tremors. Gross lesions were absent. Microscopic lesions consisted of lympho-plasmacytic inflammation involving the brain, heart, kidney, and liver. Formalin-fixed tissues from the three squirrels were tested for WNV-antigen by IHC and for WNV-specific RNA by RT-PCR. The kidneys of all three squirrels stained positive with IHC for WNV. Two of the 3 squirrels were positive for WNV by RT-PCR. In our experience with surveillance of hundreds of free-ranging crows with WNV infection during the last two years, microscopic lesions tend to be mild or absent, while IHC staining confirms the presence of large amounts of viral antigen in various tissues. Interestingly, Eastern fox squirrels had large amounts of WNV antigen associated with moderate to severe microscopic lesions in different tissues. It is possible that squirrels are somewhat more resistant to infection than are crows, which means that infection may persist longer in squirrels than in crows and stimulate a greater inflammatory response prior to illness and death.
90: PHIALEMONIUM SP. INFECTION IN TWO DOMESTIC CATS: A NOVEL FUNGAL PATHOGEN.
Two domestic cats from different households in Baltimore County, Maryland developed recurrent pyogranulomatous dermatitis and cellulitis in the interscapular region. Lesions in both cats contained numerous intra and extracellular yeast-like forms accompanied by occasional short, septate hyphae. Repeat surgical excision and antifungal therapy were unsuccessful in eliminating the infections over a 5-year time period and both cats were euthanized. On post-mortem examination, fungal elements were limited to the subcutis in one cat but had disseminated to cervical and mediastinal lymph nodes in the second cat. In the cat with disseminated infection, fungal elements were more numerous. On culture, fungal isolates from both cats were strongly similar in colonial and microscopic features. Microscopically, the isolates produced narrow, ellipsoidal, obovate to slightly curved conidia mainly from adelophialides (inconspicuous to cone-shaped intercalary phialides) consistent with the genus Phialemonium, but differed in growth habit from known species. DNA sequences were obtained from the small subunit (SSU) and internal transcribed spacer (ITS) regions of the nuclear ribosomal rRNA gene to confirm conspecificity of the cat isolates and their placement in Phialemonium. Results showed the cat isolates to be identical and grouped them together in a clade with P. obovatum, the type species of the genus, thus confirming their placement within Phialemonium. A new species is being proposed.
91: IKK SIGNALOSOMES, USED BY THE CELL, ABUSED BY THEILERIA.
Theileria parasites are protozoan organisms that are transmitted by ticks and infect predominantly ruminants. Within their mammalian host, Theileria spp. affect cells of the immune system such as T-cells, B-cells or monocytes and the schizont stage of the parasite has the unique capacity to transform its host cell resulting in a mye-lo- or lymphoproliferative disease. In Theileria-transformed leukocytes, parasite-dependent activation of NF-kappaB is critical for survival of the infected cell. Whereas the biological relevance may be well understood, the precise mechanism by which NF-kappaB is induced remains to be elucidated. NF-kappaB can be activated through a range of pathways which all converge onto a multi-subunit kinase complex, called IKK (IkappaB kinase). IKK typically consists of two catalytic subunits, IKK1 and IKK2 (or IKKalpha and IKKbeta), and a modulating unit NEMO (or IKKgamma). Theileria parasites interfere with the NF-kappaB pathway by direct activation of the IKK complex. This is supported by the observation that large foci containing activated IKK can be found in close apposition to the surface of the Theileria schizont. In addition, when epitopetagged forms of bovine IKK1, IKK2 or NEMO were expressed in Theileria-transformed cells, they became incorporated into the parasite-associated foci. The fact that a wide range of dominant negative mutant forms of upstream components in the NF-kB pathway do not block NF-kappaB transcriptional activity, indicates that they do not participate in parasite-induced constitutive NF-kappaB activation. The pathway can be blocked, however, by direct interference with the IKK complex, either at the level of NEMO or by blocking the catalytic unit IKK2. The assembly of IKK foci is specific for the transforming stage of the parasite life cycle and is downregulated as the parasite undergoes merogony.
92: FATTY LIVERS IN MOUSE HEPATITIS VIRUS (MHV) INFECTED SCID/NCr MICE.
We identified a polytrophic infection by mouse hepatitis virus in unmanipulated SCID mice, 1.5 to 3 months of age. These mice may have contracted the virus through fomites generated by mice inadvertently exposed to a transplantable cell line contaminated with MHV. A history of dead cagemates prompted the necropsy of surviving animals from which tissues were collected for complete assessment. Submitted animals exhibited rough hair coat and listlessness; some were moribund. Grossly, livers were diffusely enlarged, pitted on the surface and pale yellow. Histopathologic findings revealed focal to massive coagulation necrosis of hepatocytes with severe fatty change and multinucleated giant cells in the parenchyma and blood vessels. Syncytia were also present in intestinal villous tips and lymphoid tissues. Livers contained excessive fat accumulation as determined by frozen section Oil red O stain. MHV was confirmed by serology, immunohistochemistry and electron microscopy (EM). EM identified large numbers of viral particles budding from hepatocyte vesicles and membranes.
93: 2-DIMENSIONAL ELECTROPHORESIS AS A METHOD OF ANALYZING PROTEINS IN BOVINE BRONCHOALVEOLAR LAVAGE FLUID.
Proteomics is a promising method of broadly characterizing alterations in protein expression in diseased tissues or fluids. This study establishes a technique of analyzing bovine bronchoalveolar lavage fluid by 2-dimensional electrophoresis, as a strategy to identify lung defense proteins that are altered in calves at risk of developing bacterial pneumonia. Bronchoalveolar lavage fluid was repeatedly harvested from clinically normal, sedated calves. The proteins were concentrated by ultrafiltration, with a molecular weight cut-off of 10 kDa. 8 mg of protein was loaded onto immobilized pH gradient strips and separated by isoelectric focusing using the following conditions: 12 hours rehydration, 1 hour at 500V, 1 hour at 1000V, and 3 hours at 8000V. The strips were subsequently applied to 12% SDS-PAGE gels and separated in the second dimension for 6 hours at 225V. Gels were stained with either silver stain or Coomassie blue. This technique resulted in reproducible gels containing 150–200 protein spots, including 20–30 proteins with multiple iso-forms.
94: PREVALENCE OF OVINE PROGRESSIVE PNEUMONIA VIRUS INFECTION IN CULLED EWES IN ALBERTA.
Ovine progressive pneumonia virus is a relatively common chronic infection in sheep of North America. It causes illthrift, pneumonia and mastitis, resulting in economic losses to the sheep industry. The objectives of this study were to: 1) measure the prevalence of ovine progressive pneumonia virus (OPPV) infection in culled ewes in Alberta, Canada by histological examination of the lungs and udder and by serological evaluation of antibodies to OPPV using an agar gel immunodiffusion (AGID) test, 2) examine geographic differences in the prevalence within the province, 3) evaluate the agreement between histopathology and serology, and 4) correlate presence of histological lesions in the lungs and udder in the same animal. Based upon a histopathological case definition that required the presence of lesions specific to OPPV infection in either the lung or udder, the prevalence of OPPV infection was 26.1%, compared to 13.0% using serology (antibody detection). There were no significant differences in prevalence of the disease among the seven zones. There was fair agreement (kappa = 41.8%) between the histopathological case definition and serology. Sensitivity and specificity of the AGID test were 38.9% and 96.1%, respectively, using histopathology as the ‘gold standard’. There was poor agreement (Kappa = 11.5%) between the presence of lung and udder histological lesions within the same animal, suggesting that OPPV may affect either lung or udder alone or that time of onset of lesions in the two organs may differ. This study indicates that OPPV infection is relatively common in culled ewes in Alberta, with no significant geographic variation. The poor sensitivity of the AGID test, compared to histology, should be taken into consideration when interpreting serology results.
95: HISTOCHEMICAL, IMMUNOHISTOCHEMICAL AND ULTRA-STRUCTURAL CHARACTERIZATION OF CONGESTIVE HEART FAILURE IN THE CANINE LUNG.
Pulmonary consequences of chronic passive congestion include alveolar septal thickening by extracellular matrix and accumulations of alveolar macrophages containing hemoglobin breakdown products. To better define septal lesions, we characterized the changes of chronic pulmonary congestion by histochemistry, immunohistochemistry and ultrastructure in seven dogs with clinically evident left heart failure (CHF) compared with two clinically normal animals. Septa from CHF dogs had increased trichrome stained collagen and increased Verhoeff's Van Gieson stained elastin that isolated alveolar capillaries. The extracellular matrix was immunoreactive for collagen I and III; however, the staining was not in excess of the control dogs. CHF dogs had variable numbers of macrophages and lymphocytes present throughout the septa and within the alveolar spaces. Cases with acute alveolar damage also had moderate numbers of neutrophils within the alveolar spaces. There was intense smooth muscle actin immunoreactivity throughout the septa. Ultra-structurally, septa were expanded by a mixture of amorphous matrix, disorderly arrays of elastin and processes of interstitial cells. While the epithelial basal lamina remained intact, capillaries were often surrounded by a thick band of electron dense finely fibrillar matrix. Few fibrils of periodic collagen were evident. The increase in elastin, trichrome stained collagen and expression of smooth muscle actin suggests that pulmonary chronic passive congestion in dogs stimulates matrix production by alveolar septal myofibroblasts.
96: CLARA CELLS AND THEIR SECRETORY PRODUCTS ARE REDUCED IN THE LUNG OF HORSES WITH SMALL AIRWAY DISEASE.
Clara cells are nonciliated epithelial cells located predominantly at the transition between the distal conducting airways and gas-exchange areas of the lung. Their principal secretory product is Clara cell protein 10 (CC10), which belongs to the secretoglobin family. Secretoglobins are expressed by pulmonary, uterine and prostatic epithelial cells, and have potent anti-inflammatory, immunomodulatory, and fibroblast-suppressive activity. Clara cells also possess abundant cytochrome p-450 activity, and are progenitors for epithelial cells after bronchiolar injury. The objectives of this study were to assess the number of Clara cells and their production of CC10 in the lung of horses with normal and compromised pulmonary function parameters due to Small Airway Inflammatory Disease (SAID or heaves). Transthoracic biopsies were obtained from 10 normal and 10 horses with severe active SAID. Equine CC10 expressing cells were identified with a rabbit polyclonal antibody against goat CC10. The bronchiolar perimeter, the area comprised by bronchiolar epithelium, and the density and intensity of CC10 expressing cells, were measured with image analysis software. A numerical value was derived from the number of CC10 positive cells per 100 mm2 of bronchiolar epithelium. A minimum of five bronchiolar regions per slide was assessed. Horses with SAID had epithelial hyperplasia, goblet cell metaplasia, peribronchiolar fibrosis and mononuclear cell inflammation in the lung. Clara cells, and the intensity of CC10 immunoreactivity, were significantly decreased in horses with SAID. These findings suggest that Clara cells and their secretory products are important components of the innate defense of the lung against deleterious inflammatory stimuli such as those resulting from the exaggerated antigenic response in horses with SAID.
97: LOW-GRADE GLIAL TUMOR WITH FEATURES OF ASTRO-BLASTOMA IN A DOG.
A 12-year-old neutered male Belgian Malinois/Great Dane cross dog presented with a 5-month history of weakness and lack of endurance, followed by acute onset of rear limb ataxia. Mild bilateral hydrocephalus of the lateral ventricles was noted at gross necropsy. A 9x16 mm, multilobular, firm, white to tan, expansile mass was found in the cerebellum. Histologically, there was a well-demarcated glial neoplasm comprised of medium-sized astrocytic elements that have homogeneous cytoplasm sometimes with ill-defined eosinophilic inclusions, irregular peripherally located nuclei with a single nucleolus, and short cytoplasmic processes. Prominent perivascular pseudorosettes with cellular processes in contact with blood vessels were present. Some blood vessels exhibited hyalinized walls. Mitotic figures were not observed. Immunohistochemically, neoplastic cells were diffusely positive for GFAP and vimentin. These features are consistent with an astroblastoma. To our knowledge, this is the first report of astroblastoma in domestic animals.
98: AGANGLIONOSIS IN A 3-DAY-OLD HOLSTEIN CALF
A 3-day-old Holstein heifer calf was euthanized after a history of depression, marked abdominal distention and no defecation since birth. At necropsy, the spiral colon was severely distended by gas and meconium (megacolon). The distal colon and rectum had a severely narrowed lumen and, histologically, lacked both submucosal (Meissner's) and myenteric (Auerbach's) ganglia of the enteric nervous system (ENS); the mucosal, submucosal and muscular layers showed normal differentiation. Sections from the portion of distended spiral colon used as controls contained prominent neurons of the myenteric and submucosal plexuses (at least l/10xfield). Congenital aganglionosis of the distal colon and rectum was diagnosed. Aganglionosis of the large intestine is well documented in humans (Hirschsprung's disease), overo paint foals, and a few mouse and rat strains, but has not been previously reported in cattle. Aganglionosis is an inherited condition associated with mutations in the genes encoding for endothelin receptor B (EDNRB), EDNRB ligand endothelin 3 (ET-3), or endothelin converting enzyme-1 (ECE-1), the protease that activates ET-3. Mutations in EDNRB and ET-3 have been reported in humans, horses and mice. Mutations in ECE-1 cause multiple neurocristopathies, including intestinal aganglionosis, in humans and mice. The EDNRB/ET-3 system is responsible for the maturation and migration of neural crest cells, including those that form the ENS and melanocytes. Distinctive depigmentation is often part of the characteristic phenotype in horses (lethal white foal syndrome), mice (Piebald lethal and spotting lethal) and rats (spotting lethal) with EDNRB/ET-3 mutations. In this calf, no abnormal pigmentation was observed. Although genetic studies were not performed, the clinical presentation, gross and histologic findings in this calf are consistent with congenital colorectal aganglionosis.
99: FAMILIAL CONVULSIONS AND ATAXIA IN ANGUS CALVES IN SASKATCHEWAN.
From March to June 2002, six Angus cross-bred calves, ranging from 4 days to 3 months of age, from a Saskatchewan herd of 22 second-calf heifers of different origins, bred to the same bull (50% Angus, 50% Limousin), were presented with similar clinical signs. At birth, the calves had marked stiffness of the limbs and could not stand; a few hours later they were affected by recurrent rigid spastic seizures that lasted for up to 10 hours. The six calves were euthanized and submitted for necropsy. No significant gross lesions were present except for the presence of corneal opacity in two calves that was interpreted as exposure keratitis. Microscopically, all six calves had cerebellar lesions confined to the vermis and most prominent in the lingula and the uvula, consisting of marked spheroidal and fusiform swelling of the proximal segment of the axons of the Purkinje cells. The history and histologic lesions are consistent with a condition seen in purebred and crossbred Aberdeen Angus calves previously reported as Bovine Familial Convulsions and Ataxia (BFCA). The disease is inherited and epidemiological data suggest that it is associated with an autosomal dominant gene with incomplete penetrance. The condition has been reported in the United Kingdom and the United States, but this appears to be the first report of the condition in Canada.
100: THE IMMUNOHISTOCHEMICAL AND MORPHOLOGIC CHARACTERISTICS OF HISTIOCYTIC AND MALIGNANT FIBROUS HISTIOCYTOMA-LIKE NEOPLASMS IN THE CENTRAL NERVOUS SYSTEM OF 18 DOGS.
Primary histiocytic and malignant fibrous histiocytoma-like tumors of the brain and meninges are uncommon neoplasms in the dog. Eighteen cases of primary central nervous system histiocytic and malignant fibrous histiocytoma-like (MFH) neoplasms were identified through the department of Toxicology and Pathobiology biopsy and necropsy service. The neoplasms had two distinct morphologic patterns. One pattern consisted of sheets of large pleomorphic cells with oval to reniform nuclei and abundant cytoplasm mixed with small numbers of inflammatory cells that typically invaded the adjacent parenchyma. The second pattern consisted of streams of plump fusiform to spindle cells mixed with moderate to large numbers of inflammatory cells, which usually remained within the meninges invading the parenchyma along Virchow-Robin spaces. The inflammatory infiltrate consisted of lymphocytes, plasma cells, and fewer neutrophils. 11/18 cases were classified as MFH-like and 7/18 as histiocytic sarcomas. In an attempt to correlate tumor morphology with histochemical staining, all neoplasms were stained with vimentin, glial fibrillary acidic protein (GFAP), CD18, CD79a, CD3 and smooth muscle actin (SMA). Neoplastic cells from 8/18 tumors were positive for SMA and 10/18 were positive for CD18, but none were positive for both antigens. All neoplastic cells were positive for vimentin and negative for GFAP, CD3, and CD79a. Of the MFH-like neoplasms, 5 were positive for SMA and 6 positive for CD18. 3/7 histiocytic sarcomas were positive for SMA and 4 were positive for CD18. Our results indicate that tumor morphology and pattern of growth alone cannot differentiate these neoplasms.
101: PATHOLOGY OF WEST NILE VIRUS INFECTION IN A SHEEP.
A 2-year-old, male, sheep developed pneumonia, high fever and stiff gait in the hind limbs. The ram had progressive weakness, stiffness of the limb and loss of weight. He died within 5 days of the onset of observed neurological signs. The carcass was submitted to the University of Kentucky, Veterinary diagnostic laboratory for necropsy. Necropsy examination was unremarkable except for congestion of all internal organs. Histopathological examination of the nervous system revealed lesions confined to the cerebrum, cerebellum, medulla and cervical spinal cord. The medulla and cervical spinal cord had multifocal, perivascular cuffing by lymphocytes with glial nodules. Immunohistochemical staining of spinal cord section demonstrated the perivascular lymphocytes were positive for CD3, indicating T cells. The histopathological findings in the brain and spinal cord were similar to the lesions of West Nile virus infection in horses. Brain tissues were positive for West Nile virus by means of RT-PCR and cDNA:RNA insitu hybridization. The remaining sheep in the herd were serologically negative for the West Nile virus antibodies. A field investigation conducted at the farm where the ram was kept revealed several house sparrows (Passer domesticus) and mosquitoes in the barn. These biological units may have been the source and vector for the infection.
102: HEMANGIOSARCOMA IN THE NICTITANING MEMBRANE OF A 15-YEAR-OLD DOMESTIC SHORT HAIR CAT.
A single 7 mm in diameter solid mass protruding from the third eyelid of a 15-year-old spayed female domestic shorthaired cat was excised after being present for several months. Histologically, the outer surface of the nictitating membrane was greatly expanded by a subepithelial non-encapsulated poorly demarcated and highly infiltrative neoplasm. A pleomorphic collection of cells that ranged from plump polygonal to spindle-shaped was supported by a fine fibrovascular stroma. The polygonal cells were arranged in sheets; the more spindle-shaped cells formed irregular streams and, occasionally, became flattened and formed blood-filled spaces. Cellular borders were fairly distinct, cytoplasm was scarce to moderately abundant, pale and foamy. Nuclei were ovoid to round with smooth palely basophilic chromatin and 1–2 prominent round magenta nucleoli; karyomegaly was frequent. Mitotic figures were infrequent, 0–2 per high power field (40x), but often aberrant. A mild lymphocytic infiltrate was admixed with the neoplastic cells. Immunohistochemical staining for factor VIII revealed moderate to intense cytoplasmic staining in many neoplastic cells. The neoplasm was diagnosed as a conjunctival hemangiosarcoma of the nictitating membrane. A previous report of a similar neoplasm in a cat, also 15 years old, found that the surgery to remove the tumor was uneventful and no recurrence was present at 7 months post-surgery. In this case, the cat presented with a recurrent mass 3.5 months after the initial surgery. Although no histologic examination was performed on the second mass, recurrence of the original hemangiosarcoma following an incomplete excision during a conservative surgery is likely. No evidence of metastasis has been found to date (3.5 months post excision).
103: CYSTIC ENDOMETRIAL HYPERPLASIA AND UTERINE ADENOMYOSIS IN YOUNG BXD RI MICE.
The purpose of this study was to identify lesions associated with aging in BXD recombinant inbred (RI) strains of mice, their progenitor strains (C57BL/6 and DBA/2), and reciprocal F2 groups between the progenitors. Gross and histologic examination of 461 female nulliparous mice at 150 days of age revealed cystic endometrial hyperplasia (CEH) in 32%, uterine adenomyosis (UA) in 10%, and 5% with both CEH and UA. There were similarities between the appearance of CEH and UA. Both may contain proliferative epithelial and stromal cells. UA was found deep within or exterior to the circular layer of myometrium and sometimes exterior to the longitudinal layer and subjacent to the serosa. Small subserosal nodules could be identified grossly as has been described in rats. UA was not contiguous with the lumen as seen with CEH, which could be found to extend from the lumen to the serosa. Both lesions have been associated with reproductive hormones and have genetic components in their pathogenesis. CEH was found in B6 (10/12) and not D2 (0/9) progenitor strains. UA was found in D2 (3/9) and not B6 (0/12) progenitor strains. The incidence of individual strains and further analysis results will be presented. The lesions are useful as biomarkers of aging and correlations can be made between tests to assess aging, the lesions identified, and quantitative trait loci that may be identified. Supported by NIA AG14.
104: UTERINE SMOOTH MUSCLE TUMORS IN POTBELLIED PIGS (SUS SCROFA) RESEMBLE HUMAN FIBROIDS: A POTENTIAL ANIMAL MODEL.
Uterine leiomyomas, commonly termed fibroids, clinically affect approximately 25 percent of women of reproductive age in the U.S., with a subclinical incidence as high as 77 percent. The pathogenesis of fibroid formation remains poorly understood, due in large part to the lack of a suitable animal model. This retrospective study characterizes the pathology of similar, spontaneously occurring uterine tumors in potbellied pigs, and their potential use as an experimental model for studying human fibroids. Medical records available through a local Potbellied Pig Spay/ Neuter Program, pig sanctuaries, and the Duchess Fund database were reviewed for evidence of reproductive disease or surgery. 95 female potbellied pigs were evaluated, and single or multiple mesenchymal neoplasms were identified in 17 animals; tissues were available for 12 of these. Clinical, gross, and histologic features were reviewed. Hematoxylin and eosin and Trichrome stained sections were evaluated for morphological features of human fibroids and scored for published differential criteria of human and animal leiomyomas and leiomyosarcomas. Smooth muscle actin stains were also performed. All affected pigs were estimated to be 5 years of age or greater. Pigs presented with clinical signs of abdominal distention, vaginal bleeding, or were subclinical and identified during spay surgery. Tumors ranged from microscopic to 100 lbs and involved the uterine body and horns primarily. The cellular pattern/morphology and degree of fibroplasia varied considerably compared to that reported for human fibroids. Uterine leiomyoma was diagnosed in 10/12 cases, leiomyosarcoma in 1/12 cases, and undifferentiated sarcoma in 1 case. The results support further investigation of uterine leiomyomas in potbellied pigs as a potentially valuable animal model for studying human fibroids.
105: UNILATERAL RENAL DYSPLASIA WITH UROGENITAL ANOMALIES IN A DOG.
During a routine ovariohysterectomy in a 1 year-old Golden retriever mix, a large abdominal mass was recognized, the surgery was stopped and the dog referred to Iowa State University's Veterinary Teaching Hospital. During exploratory surgery, a mid abdominal dilated tubular structure (6 × 6 × 14 cm) containing a translucent, yellow-tinged fluid was identified as the left uterine horn. At the cranial end of the left uterine horn was a small (1.5 × 1.0 × 2.5 cm) mass tentatively identified as ovary. The left kidney and ureter were absent while the right urogenital tract was grossly normal. Histologically, the tissue preliminarily identified as left ovary consisted of segmental fibrosis (∼80% of the section) with admixed foci of shrunken obsolescent glomeruli, dilated Bowman's capsules thickened by fibroplasia, tortuous arteries, tubular structures, lympho-plasmacytic infiltrates and hemorrhage. The remaining 20% of more normal renal tissue was separated from the fibrotic areas by a distinct zone of demarcation. Here, subcapsular glomeruli were small, lacked clear capillaries and contained cells with peripheral nuclei resembling fetal glomeruli. The dilated uterine horn was lined by simple to columnar epithelium and has a thin lamina propria with a marked reduction in uterine glands. In one section a tubular structure (0.5 mm) lined by transitional epithelium was fused with the uterus to form a common lumen. Histologically this was consistent with an ectopic ureter. In this case, unilateral renal dysplasia was associated with ipsilateral: 1) absence of the ovary, 2) ectopic ureter opening in the uterine cavity and 3) hydrometra. To our knowledge this is the first canine case report describing unilateral renal dysplasia with these concurrent urogenital lesions.
106: ULTRASTRUCTURAL FINDINGS IN GLOMERULOCYSTIC KIDNEY DISEASE IN A BELGIAN MALINOIS DOG.
Cysts in the renal cortex of a juvenile Belgian Malinois dog with acute renal failure were studied by means of light, scanning and transmission electron microscopy to determine the morphological features of the epithelial cells of these cysts. The cysts were renal corpuscles with expanded urinary space. Glomerular tufts were small with poorly developed capillary loops and relative increase in mesangial matrix. Continuity with the proximal tubule was evident in some cystic glomeruli. Two cell types lined Bowman's capsule. One was squamous with a central cilium and microvilli. The other had the immature morphological features of parietal podocytes. The later cell type was round and protruded into the urinary space; it had thick cytoplasmic projections (that resembled podocyte foot processes), microvilli, and filtration slits. The basement membrane of Bowman's capsule was irregularly thickened. Histological and ultra-structural findings in this dog are consistent with glomerulocystic kidney disease. This is the second report of canine glomerulocystic kidney disease. Features are similar to those of the human counterpart, but it is unclear whether genetic defects cause the disease in the dog. The presence of parietal podocytes in all cysts suggests that abnormal differentiation may play an important role in the pathogenesis of this type of polycystic kidney disease.
107: ACUTE RENAL TUBULAR NECROSIS IN DOG AND CAT FETUSES DUE TO ETHYLENE GLYCOL TOXICOSIS.
Ethylene glycol ingestion is a common cause of toxicity in domestic animals, usually from accidental or intentional exposure to antifreeze. Ethylene glycol is metabolized in the liver to the toxic metabolites glycolaldehyde, glycolic acid, glycoxylate, and oxalate, which are filtered by the glomeruli. Oxalate combines with calcium and precipitates as calcium oxalate crystals within tubular lumens. A Rottweiler, a mixed-breed dog, and a domestic shorthair cat were found dead or were euthanized for acute severe clinical disease including vomiting, ataxia, and high serum creatinine levels. All animals were in late-term pregnancy and were diagnosed with ethylene glycol toxicity at necropsy based upon finding characteristic bire-fringent monohydrate crystals within areas of acute renal tubular necrosis. Fetal kidneys from each of the three animals also contained acute renal tubular necrosis with similar crystals. Developmental defects resulting from repeated ethylene glycol exposure have been reported in rats, mice, and humans and are markedly worsened with metabolic acidosis. In the cases presented here, no developmental defects were observed, and the animals were most likely exposed to a single toxic dose, quickly resulting in maternal and fetal death. To our knowledge, fetal ethylene glycol toxicity has not been reported in domestic animals. Although the toxic effects were acute in this case, the possibility of developmental defects in puppies and kittens that may survive in utero exposure to ethylene glycol should be investigated.
108: THE CLINICAL AND PATHOLOGICAL CHARACTERISTICS INFLUENCING THE PROGNOSIS OF 50 CANINE PATIENTS WITH LYMPHOID MALIGNANCIES.
A prospective study was conducted to determine whether several variables including morphologic type, immunophenotype, clinical stage, or substage had an effect on remission or survival times. Fifty dogs that presented to the University of Florida Veterinary Medical Teaching Hospital with lymphoproliferative disease were identified using cytologic or histologic evaluation. Patients were staged (III, IV, V) and substaged clinically (a or b) according to the WHO staging system. A cytologic sample from each patient was immunophenotyped (T or B) by a battery of cell surface markers (CD3, CD4, CD8alpha, CD18, CD21, CD45RA, CD79a, IgG) and assigned a morphologic classification according to the updated Kiel classification. Patients were treated with either the Wisconsin chemotherapy protocol, prednisone only, or did not receive treatment. First remission length and overall survival were calculated. Data was analyzed using a Cox's regression model. Each factor was considered separately and no adjustment for multiple testing was done. The statistical analysis showed that only WHO substage had a significant effect on survival time with substage a (not sick) having a longer survival time than substage b (sick). None of the factors evaluated had a significant effect on remission length. There was no statistical difference between two morphology groups (group 1: centroblastic high grade B-cell and group 2: high grade T-cell). Similar to previous studies, WHO substage was found to be significant in predicting survival time. The lack of a statistical difference between T and B immunophenotype survival may relate to the inclusion of T-CLL (chronic lymphocytic leukemia), an indolent disease, which is often excluded in other studies. Furthermore, the B-cell group contained both early and advanced cases of disease, which can influence survival time.
109: A LEUKEMIA CLUSTER IN THREE CAPTIVE AFRICAN HEDGEHOGS.
Three adult female African hedgehogs (Atelerix albiventris) donated to the Knoxville Zoo (Knoxville, Tennessee) in May 1998 developed leukemia and died or were euthanized between August 1999 and April 2000. The animals were donated together and shared living quarters at the zoo from the time of donation to the time of death. All three hedgehogs had clinical signs of hematochezia, anorexia, and splenomegaly prior to being diagnosed with leukemia. Specific diagnoses were chronic eosinophilic myelogenous leukemia, acute myeloid leukemia (granulocytic and megakaryocytic), and plasma cell leukemia.
110: THE CLINICOPATHOLOGY AND IMMUNOPHENOTYPE OF EQUINE LYMPHOMA.
Lymphoma is the most common hemolymphatic neoplasm in horses. Clinical features are distinct from lymphoma in other species. In this study clinical and laboratory data as well as tissue specimens from 11 horses diagnosed with malignant lymphoma were examined. Polyclonal antibody against CD3, and monoclonal antibody against CD79a identified T-cell and B-cell lymphocytes, respectively, in formalin-fixed biopsies or post-mortem specimens. Sections were stained additionally with the BLA36 monoclonal antibody, and expression of nuclear sex hormone receptors was assessed with monoclonal antibodies to estrogen and progesterone receptors. The sample population included 7 female and 4 male horses of various breeds and ranging in age from 2 months to 16 years. Laboratory abnormalities consisted of severe anemia with red blood cell agglutination and a positive Coombs test (2), mild to severe thrombocytopenia (3), hyperglobulinemia with monoclonal gammopathy (2), hyperfibrinogenemia (2), elevated hepatic or muscle enzyme activities (3), and electrolyte disturbances (2). All tumors were diffusely infiltrative and composed of a heterogeneous population of lymphocytes. Independent scoring of immunohistochemically stained sections by 3 pathologists resulted in agreement for 6 cases of T-cell and one case of B-cell lymphoma, but there was discrepancy regarding the remaining 4 cases. Lymphomas comprised of more than 90% T-cells were associated with monoclonal gammopathies in two horses. Neither estrogen nor progesterone receptors were expressed by any tumor. BLA36 expression was infrequent and restricted to scattered large irregularly shaped cells. These findings indicated that lymphoid neoplasms in the horse were frequently composed of mixed cell populations and that assessment by immunochemical methods alone yielded equivocal results. Molecular studies may be valuable to further characterize the transformed lymphoid population in equine lymphoma.
111: SHEEP BETA-DEFENSIN-2 ONTOGENY: DISTRIBUTION AND EXPRESSION.
Antimicrobial peptides are a component of innate host defense. Beta-defensins are a subclass of antimicrobial peptides defined by cationic charge, conserved cysteine residues that form intramolecular disulfide bonds and microbicidal activity against bacterial, fungal and viral pathogens. The aim of this study was to define the ontogeny of sheep beta-defensin-2 (SBD-2) mRNA and peptide in selected tissues of fetal, neonatal and adult sheep by real-time PCR and immunohistochemistry, respectively. Fetal and neonatal lambs had a significantly greater SBD-2 mRNA tissue distribution compared to adult sheep. For all ages, the intestines had consistent SBD-2 mRNA expression while extra intestinal expression was sporadic and weak. In adult sheep, SBD-2 mRNA levels were highest in the jejunum and decreased caudally to the rectum. A pooled sample from all age groups showed a similar tendency. Variability of SBD-2 expression among individual animals was pronounced even in tissues with consistent SBD-2 mRNA expression. SBD-2 immunoreactive cells were predominantly in the crypts and base of villi in the small intestine and in a modest number of glands in the large intestine. Paneth cells and immature goblet cells near villus crypts often showed staining. Interestingly, ileal follicle-associated epithelium, which is associated in many species with antigen sampling, lacked detectable SBD-2 immuno-reactivity. In conclusion, SBD-2 mRNA and peptide expression are greatest in the intestinal tract and tissue distribution progressively decreases with maturity. The wider SBD-2 tissue expression in the young lamb may be related to the suggested role of beta-defensins in cell proliferation and differentiation. The lack of SBD-2 staining on ileal follicle associated epithelium may allow for enhanced microbial interaction for antigen sampling of the gut lumen.
112: PATHOLOGICAL FINDINGS IN NATURALLY INFECTED DOGS WITH ANGIOSTRONGYLUS VASORUM.
Angiostrongylus vasorum is a metastrongylid nematode that develops to adult form in pulmonary arteries of wild and domestic canids. The parasite is endemic in Newfoundland's Avalon Peninsula. In order to study pathological changes associated with infection, 18 adult dogs from St. John's, Newfoundland and Labrador were examined. Adult nematodes were found within pulmonary arteries of 3 dogs. One dog, which was minimally affected, had one visible nematode within a medium sized pulmonary artery and rare thromboses of medium sized pulmonary arteries associated with mild perivascular granulomatous inflammation. The second had chronic, severe, multifocal granulomatous interstitial pneumonia with vascular thrombosis that resulted in cor pulmonale, right heart hypertrophy, hepatic congestion, and ascites. Ten adult parasites were recovered from the pulmonary arteries. The third dog had been diagnosed and treated for angiostrongylosis. The dog developed acute, severe neurological signs 10 days after initiation of treatment and was euthanized. A severe granulomatous interstitial pneumonia with extensive vascular thrombosis, smooth muscle hypertrophy and hyperplasia, with numerous intralesional adult, larval and egg forms was diagnosed. Approximately 560 adult worms were recovered from pulmonary arteries and faecal Baermann test was positive. Multiple areas of cerebral necrosis with granulomatous encephalitis containing nematodes and eggs were seen. Eggs and larva were identified in sections of brain, kidney, liver, spleen, adrenal gland, and tracheobronchial lymph nodes. In conclusion, Angiostrongylus vasorum infections can produce severe pathological changes in infected domestic canids. This parasite should be considered when examining dogs that have visited or live in this area of Newfoundland.
113: NON-IMMUNOGLOBULIN ESCHERICHIA COLI F4 FIMBRIAL AND LIPOPOLYSACHARIDE-BINDING PROTEINS IN PORCINE MILK.
Diarrhea caused by Escherichia coli F4 is one of the most important diseases in neonatal pigs. The pathogenesis of E. coli diarrhea begins with its fimbrial attachment to enterocytes. Colostrum and milk are the first diets of mammalian neonates and are rich in proteins with known protective functions against microbial organisms. Colostrum contains a significant amount of immunoglobulins (Igs), but these decrease rapidly during the lactation period. Enteric diseases generally increase after weaning. This, together with the reduced levels of Igs in milk over the period of lactation, suggests that factors other than Igs are important in conferring protection in the intestine of suckling pigs. We hypothesized that non-immunoglobulin substances in porcine milk protect suckling pigs from enteric pathogens. This study investigated E.coli F4 fimbrial- and lipopolysaccharide-binding proteins in porcine milk by affinity chromatography. Positive columns (fimbria and LPS with sepharose beads) as well as control columns (only sepharose beads) bound proteins from porcine milk and these proteins were identified by liquid chromatography mass spectrometry (LC-MS). Results showed that a number of proteins in porcine milk such as lactadherin, butrophilin, xanthin, alpha s2 casein and beta casein bound to the E. coli F4 fimbria and lactoferrin, alpha casein, beta casein and alpha lactalbumin bound to the E. coli F4 LPS. Our results suggest interaction of innate immune defenses in porcine milk with fimbria and LPS of E.coli F4 in order to block the mucosal binding and/or aid in elimination of the bacteria.
114: A RETROSPECTIVE STUDY OF 65 CASES OF CANINE CHRONIC INFLAMMATORY BOWEL DISEASE.
Canine chronic inflammatory bowel disease (IBD) presents a challenging diagnosis for both the pathologist and the general practitioner. In this retrospective study, 67 cases of chronic IBD were examined to determine if quantitative or qualitative differences in mucosal cellularity are of prognostic significance. A weighted score for the severity of presenting clinical signs was assigned to each case based upon the presence or absence of specific signs. A response to treatment score was assigned based upon the initial response to drug therapy, the ability to successfully discontinue treatment and the incidence of clinical relapses. Quantitative cell counts were performed on gastric biopsies for CD3+ and CD79+ cells, eosinophils and plasma cells and an objective scoring system based upon the number and proportion of inflammatory cells was assigned to each sample. Intestinal and colonic samples were graded using previously published criteria. Cases were classified as eosinophilic or lymphocytic based upon the proportion of each cell type present in the most severe lesions. The means of the severity of clinical signs scores were: eosinophilic – 3.7, lymphocytic-3.1 (P = 0.29). The mean scores for response to treatment were: eosinophilic - 2.5, lymphocytic - 2.9 (P = 0.49). There were no significant differences in severity of presenting clinical signs or response to treatment between eosinophilic or lymphocytic IBD. These results indicate that, with current therapeutic protocols, there is no prognostic difference between eosinophilic and lymphocytic inflammatory bowel disease.
115: SPONTANEOUS COMPLEX ODONTOMA IN A SPRAQUE-DAWLEY RAT.
Complex odontoma from a female Sprague-Dawley rat is described histopathologically. Necropsy revealed a hard (bony), white mass (3.0 × 3.0 × 2.1 cm) on the left mandible. Microscopically, the mass consisted of islands or nests of epithelial and mesenchymal elements that formed abortive tooth structures. In other areas, tooth formation consisted of a pulp cavity lined by layers of odontoblasts, dentin, enamel, and ameloblasts. Based upon the features of normal tooth formation that was differentiated and mineralized yet completely disorganized, the diagnosis of complex odontoma was recommended.
116: IMMUNOHISTOCHEMICAL CHARACTERIZATION OF SPINDLE CELL PREDOMINANT ADRENAL NEOPLASMS IN FERRETS.
INTRODUCTION: Adrenal tumors and hyperestrogenism are common in ferrets. A variant tumor type with prominent stromal cell proliferation has been identified but poorly characterized to date. We wished to determine if the secretion of estrogen by the adrenal cortical tumor cells played a role in proliferation of the spindle cell component and if this was mediated by the presence of estrogen receptor (ER).
HYPOTHESIS: Similar to hormone-induced uterine leiomyomas in women, the spindle cell component of ferret adrenal cortical tumors is smooth muscle in origin and its proliferation is correlated to ER expression. MATERIALS AND METHODS: We characterized the spindle cell component of 24 ferret adrenal cortical masses (hyperplastic nodules, adenomas and adenocarcinomas) with varying amounts of stromal proliferation (mild, moderate, severe) obtained from the AMC and AFIP databases. Tumors were immunohistochemically stained for smooth muscle actin and ER expression.
RESULTS: Of 24 cases of spindle cell predominant adrenal tumors, 1 was a hyperplastic nodule, 13 were adenomas and 10 were adenocarcinomas. Hyperplastic nodules and adenomas were associated with minimal spindle cell component, while an equal number of adenomas and adenocarcinomas had moderate spindle cell proliferation. All proliferative spindle cells were smooth muscle actin positive. Estrogen receptor positivity was seen in nine of twenty-four cases.
CONCLUSIONS: The stromal cell component of these variant adrenal tumors is smooth muscle in origin and expresses ER in 37.5% of cases, the majority of which were adenocarcinomas. Additional studies are ongoing to delineate other hormonal mechanisms of stromal cell proliferation.
117: TRANSCRIPTION FACTOR GATA-4 AS A MARKER OF ANAPLASIA IN ADRENOCORTICAL CARCINOMAS OF THE FERRET (MUSTELA PUTORIUS FURO).
Adrenocortical carcinoma is a common cause of morbidity and in some cases mortality in ferrets. We have recently described and characterized myxoid differentiation, a more aggressive phenotype of adrenocortical carcinomas of ferrets. The goal of this study was to evaluate expression of the transcription factor, GATA-4, in the adrenal cortex of ferrets. GATA-4 is a highly conserved protein and a member of the GATA-binding protein family of zinc finger transcription factors. GATA-4 is expressed in the hypothalamus, pituitary, gonads, and adrenal glands of humans and mice. GATA-4 is important in fetal adrenal gene regulation in mice, and present only in fetal and neoplastic adrenal cortex. Fourteen adrenocortical carcinomas were evaluated immunohistochemically. Several neoplasms contained residual normal cortex, and four had direct hepatic invasion or metastasis. There was prominent nuclear immunoreactivity for GATA-4 in cells of adrenocortical carcinomas. The GATA-4 immunoreactivity varied from 1+ in well-differentiated adrenocortical carcinoma cells to 3+ in more anaplastic cells, and areas of the myxoid component. Normal adrenal cortex was negative for GATA-4 staining, and minimal to no nuclear staining was present in areas of spindle-cell component. In individual neoplasms, cell types with positive GATA-4 staining also had robust alpha-inhibin immunoreactivity (2+ to 3+), and a significantly elevated PCNA labeling index compared to normal zona reticularis cells (p<0.05). Prominent GATA-4 staining was present in hepatic metastases of the adrenocortical carcinomas. In summary, GATA-4 immunoreactivity was strongest in anaplastic adrenocortical carcinoma cells and areas of the poorly-differentiated myxoid component. This indicates that GATA-4 can function as a marker for anaplasia in adrenocortical carcinomas of ferrets.
118: COMPARISON OF BURSAL HISTOLOGIC LESIONS CAUSED BY DIFFERENT INFECTIOUS BURSAL DISEASE VIRUS STRAINS.
Infectious bursal disease virus (IBDV) is an important immunosuppressive virus of chickens. Six different strains of IBDV (STC, GLS, Variant E, Variant A, D78, and Bursine) were compared in order to establish an association between lesions and viral antigen distribution in the bursa. One day old SPF broilers were infected with the different IBDV strains and microscopic lesions in the bursa examined at 4 and 6 days post exposure. Immunohistochemical detection of IBDV antigen was carried out using a monoclonal antibody that bound all IBDV strains. Apoptosis, known to be induced by IBDV, was also detected in the bursas using the TUNEL method. The presence and severity of microscopic lesions in the bursa correlated with the location and number of positive IBDV-infected cells. Apoptosis was observed in all IBDV-infected bursas, but its distribution in the bursal follicles did not correlate with the location of IBDV antigen, indicating that IBDV-induced apoptosis also occurs in non-infected cells. There was great variation between the different IBDV strains in regard to the lesions present in the bursa. STC, GLS, and the variant strains, produced widespread lymphoid necrosis and high levels of staining of IBDV antigen. The pattern of distribution of the viral antigen and the apoptosis produced varied between the strains. Vaccine strains (D78 and Bursine) induced minimal microscopic lesions and resulted in fewer cells positive with stainable viral antigen, but more staining for apoptosis.
119: CAUSES OF MORTALITY IN CAPTIVE PUERTO RICAN AND HISPANOLIAN AMAZON PARROTS.
Approximately 170 endangered Puerto Rican parrots currently exist, increased from only 13 birds in 1975. Most are in a captive breeding program. The common, closely related Hispanolian amazon is also propagated as part of the program to provide foster parents and evaluate program strategies. Maintaining both species under similar captive conditions allows evaluation of the impact of limited genetic diversity (inbreeding depression) on fecundity and mortality. Inbreeding depression in the Puerto Rican parrot (PRP) is associated with lower fecundity compared to the common Hispanolian amazon (HA). Causes of mortality for adult PRP (n = 48) and HA (n = 26) parrots were categorized and compared to determine if inbreeding depression impacts cause. Causes of mortality were trauma and hemorrhage for 29% of PRP and 19% of HA, degenerative, dysplastic, and neoplastic lesions for 23% of PRP and 12% of HA, respiratory disease for 17% of PRP and 23% of HA, renal disease for 8% of PRP and 4% of HA, and reproductive disease for 4% of PRP and 12% of HA. A higher prevalence of degenerative, dysplastic, and neoplastic lesions in PRP was due to atherosclerosis and unusual dysplastic thyroid lesions not seen in HA. Some dysplastic thyroid lesions occurred in closely related birds. These lesions may have a genetic basis and indicate inbreeding depression in PRP. However, differences in numbers of PRP and HA in each category were not statistically significant. Mean age at death was also similar (PRP 9.2 years; HA 8.3 years).
120: INHIBITION OF TUMOR ANGIOGENESIS BY CATIONIC LIPOSOME-DNA COMPLEXES.
Cationic liposome-plasmid DNA complexes (CLDC) have been used for both local and systemic gene delivery. In a previous study, we observed that intravenous delivery of CLDC elicited potent activation of innate immunity and antitumor effects in mice with a variety of tumors. In a recently completed randomized trial evaluating intravenous endostatin gene therapy in dogs with soft tissue sarcoma, significant antitumor activity was observed in dogs treated with irrelevant plasmid DNA when compared to dogs treated with plasmid DNA encoding the canine endostatin cDNA. We speculated that cationic liposome-DNA complexes might inhibit tumor growth, in part, through inhibition of tumor angiogenesis. To assess this theory as a possible mechanism, the effect of CLDC treatment on tumor angiogenesis was evaluated using tumor biopsies from dogs in the study. Quantitative microvessel density analysis (QVDA) was performed utilizing immunohistochemistry for the endothelial cell marker CD146. Photomicrographs of immunohistochemically stained sections were acquired, digitized, and analyzed using imaging software. The results of our quantitative microvessel density analyses correlated positively with clinical observations of tumor responses (change in tumor volume) and demonstrated a significant decrease in tumor vessel density in the majority of post-treatment biopsy samples. From this study we have concluded that 1) intravenous administration of CLDC can inhibit tumor growth in the dogs with soft tissue sarcomas 2) the effects of CLDC administration are independent of the gene encoded by the plasmid DNA and 3) CLDC appear to elicit antitumor effects, at least in part, through the inhibition of tumor angiogenesis. We believe that CLDC-induced antiangiogenic effects are likely mediated by activation of the innate immune system with subsequent release of antiangiogenic cytokines such as type I and II interferons.
121: FUNCTIONAL RENAL ANATOMY.
The kidney is particularly susceptible to xenobiotics because of high blood flow to mass ratio, metabolizing enzyme activity and unique ability to concentrate urine. In industry animal R&D the kidney is the most frequent target tissue for adverse effects. In humans nearly 20% of all cases of acute renal failure are drug related. Refinement of the renal safety assessment requires comprehensive knowledge of the molecular, biochemical, and structural effects of the xenobiotic. Determination of the cellular and subcellular target represents the first step in understanding the functional consequences of a lesion. Nephrotoxins have predilection sites dependent on the molecular/biochemical target. Hence understanding the link between biochemical and molecular pathways with structural subtopography becomes requisite for effectiveness of the renal toxicologic pathologist. The second prerequisite for effectiveness must be the recognition of species differences. For example, expression of even minimal tissue injury, acute or chronic, can differ between rats and higher mammalian species despite common mechanisms of injury. Through determination of the earliest and most sensitive structural alteration or functional perturbation associated with xenobiotic exposure, the toxicologic pathologist with a sound fundamental understanding of the normal physiology of the affected anatomic site can readily identify probable causal mechanisms. For example, PPAR gamma agonists such as rosiglitazone cause plasma volume expansion. With the knowledge that the kidney is structurally normal after rosiglitazone exposure and that PPAR gamma receptors exist in glomerular and renal microvasculature, a likely influence of PPAR gamma on intrarenal hemodynamics can be hypothesized. Indeed PPAR gamma agonists cause sustained decrease in glomerular filtration rate without blood pressure effect. Key structural and physiologic features and response to nephrotoxins that target specific segments will be discussed in order to illustrate the linkage of molecular/biochemical attributes and renal substructure.
122: COMBINED CLINICAL CHEMISTRY AND QUANTITATIVE HISTOMORPHOMETRY IMPROVES CLASSIFICATION OF TOXICOLOGICAL RESPONSES.
Toxic responses to compounds involve a variety of global tissue and clinical changes as well as individual responses. Some changes may be captured by clinical chemistry, while others by tissue histomorphological changes. An investigative toxicology study, sponsored by Tissue-Informatics and InforMax, analyzed the effects in rats of hepatotoxins methapyrilene and N-diethylnitrosamine, the negative hepatotoxin pyrilamine maleate and the renal toxin gentamicin. We demonstrate an improved ability to classify toxicology responses through the combined use of histomorphometric and clinical chemistry data. The study utilized 6 treatment groups (n = 24/group) of male Sprague-Dawley rats. Each group was treated with either vehicle or test compounds with a daily injection for 3 days. Animals were sacrificed after a 24-hour fast at 8-, 24-, 48- and 72-hour intervals. Serum chemistries were analyzed and a complete panel of 27 electrolytes, metabolites and enzymes was reported. Using an automated slide imaging and histomorphometric software (He-patf(x)) system, H&E stained liver sections were analyzed for microscopic anatomical features and fine cellular structures in a hyper-quantitative fashion. Individually, the clinical chemistry or hyper-quantitative liver histomorphometric data could be used to separate the 6 different treatment groups through Fisher Discriminant Analysis. However there was significant overlap of individual animals, and the hepatotoxin groups were not clearly separated. In contrast, when the combined dataset of histomorphometric and clinical chemistry data was used, a markedly improved ability to classify individual toxic responses and to more clearly separate the hepatotoxins was observed. The results are consistent with our previously reported findings using a combination of gene expression and histomorphometric data. The ability to assess co-variant data provides a more rapid, sensitive and predictable evaluation of predictive toxicology study data.
123: COPPER AND ZINC SUBTOXICOSES IN QUEBEC DAIRY HERDS STEMMING FROM NUTRITIONAL HERD SURVEILLANCE BY BIOCHEMICAL PROFILE ANALYSIS.
In this study, serum concentration of copper and zinc, and GGT and GPX activity were evaluated in 680 herds (581 in early lactation and 99 dry) to survey nutritional status in dairy cattle. The observations were based on 1997–1998 data of metabolic profile analysis with most testing performed in the October to January period. Using commercial kits, colorimetric and enzymatic methods were performed to determine Cu and Zn serum concentrations and GGT and GPX activities. The mean values ranging between the 5th and 95th percentiles were considered normal and the mean values lower than the 5th percentile and higher than the 95th percentile were considered respectively as deficiency or subtoxicosis. Results: the 5th and 95th percentiles of Cu (μmol/L), Zn (μmol/L), GGT (U/L) and GPX (U/L) were respectively (10.2–17.4), (13.8–21.5), (145–316), and (18–33). Copper serum concentrations were significantly different according to the lactational stage (early lactation vs dry, p < 0.0003), the season (winter, springtime and summer-fall, p < 0.006), and the reason for the analysis request (surveillance, metabolic disease or reproductive problems, p < 0.006). Zinc serum concentrations were significantly different according to milk production (< 8500 kg vs > 8500 kg, p < 0.005) or the request (p < 0.002). GGT serum activity was significantly different according to the lactational stage (p < 0.05) or to the request (p < 0.05). Conclusion: Serum concentrations of Cu and Zn and serum activities of GGT and GPX reflect some cases of subtoxicosis in Quebec dairy herds. Mean values of Cu, Zn and GGT in herds with reproductive problems were significantly different from herds for which other reasons were cited in the analysis request. This study approach allowed to establish the occurrence of 7 % and 6 % Cu and Zn sub-toxicoses in Quebec dairy herds, and highlighted a significant association between the high serum activity of GGT, GPX and Cu subtoxicosis.
124: VALIDATION OF POTENTIAL THERAPEUTIC TARGETS USING LASER CAPTURE MICRODISSECTION AND REAL-TIME RT-PCR ANALYSIS.
In situ gene expression profiling is a valuable tool for validating and localizing potential therapeutic targets. Until recently, non-iso-topic in situ hybridization (ISH) was the primary method for localizing target mRNA expression in selected tissues. While useful, ISH is limited to single gene analysis, does not allow quantitative assessment of mRNA expression levels and in some instances, is hampered by lack of sensitivity for low abundance transcripts. An alternative approach without these limitations is laser capture microdissection (LCM) of specific cellular elements from tissue sections followed by Real-Time RT-PCR analysis. To assess the utility of this technique, we applied LCM and Real-Time RT-PCR in combination with ISH to qualitatively and quantitatively evaluate mRNA expression of two potential cardiovascular targets, neuromedin U1 receptor (AXOR13) and Rho kinase (ROCK-1/ROCK-2), in selected tissue elements of atherosclerotic human arteries. Evaluation of mRNA expression of AXOR13 and ROCK-1 and ROCK-2 demonstrated that LCM/Real-Time RT-PCR provided a more sensitive method for detection of low abundant transcripts. For AXOR13 (a relatively highly expressed target), ISH correlated closely with LCM/Real-Time RT-PCR mRNA expression. However, ROCK-1 and ROCK-2, mRNA expression was only detected by LCM/Real-Time RT-PCR as quantities that were well below the limits of detection by ISH. LCM followed by Real-Time RT-PCR analysis is a powerful adjunct and/or alternative to ISH for target validation when quantitative evaluation of expression is needed or when target mRNA is a low abundance transcript at the cellular level.
125: EXPRESSION PROFILING OF DRUG-INDUCED VASCULAR INJURY: METHODS DEVELOPMENT.
Expression profiling of a cells transcriptional landscape may provide insight into pathogenic mechanisms and potential biomarkers of vascular injury. Data obtained from isolates of entire vessels however, represent a composite profile in which the signals are confounded by regional heterogeneity of the vasculature. To address this, we tested the feasibility of utilizing laser capture microdissection (LCM), RNA linear amplification (LA) and GeneChip expression analysis to profile regional and cell-specific transcriptional changes in drug-induced vascular injury. Regions of rat mesenteric vasculature (endothelium, vascular smooth muscle and adventitia) were microdissected from frozen-OCT-embedded rat mesentery and the RNA isolated. Fifteen nanograms total cellular RNA from each sample was subjected to two-cycles LA. Following LA, samples were biotinylated, fragmented and hybridized on rat GeneChip arrays. Quantity and quality of resultant samples were evaluated. Spectrofluorometric analysis of LCM material revealed yields on average of 90ng total cellular RNA/tissue element. RNA quality assessed by 18S and 28S ribosomal bands indicated RNA preparations were optimal for LA. Two-cycles of LA generated approximately 90 μg labeled cRNA, sufficient to hybridize to GeneChips. As anticipated, electrophoretic analysis following two-cycles LA showed transcript sizes between 500 to 1000 bases. Evaluation of standard array quality metrics (Raw Q = 1.9, Scaling Factor = 0.46, Percent Calls = 66%, and GAPDH 3/M signal intensity ratios = 2.8) indicated array performance from LCM material was optimal and comparable to non-LCM material. Additionally, genes known to be differentially expressed across tissue elements were also identified. Endothelin-1, von Willebrand factor, VEGF and VCAM were expressed only in endothelial cells. These data demonstrate the feasibility of utilizing this approach to evaluate injury-mediated transcriptome changes in select regions of rat mesenteric vasculature.
126: PEPSINOGEN I AS A MARKER OF GASTRIC CHIEF CELL INJURY IN CYNOMOLGUS MONKEYS.
Measurement of pepsinogen I and II is frequently used for noninvasive monitoring of upper gastrointestinal diseases in man. Recently, gastric mucosal chief cell degeneration was identified in 4-day and 1 -month studies in cynomolgus monkeys given pharmacophores with different molecular targets. The purpose of this study was to validate the use of pepsinogen I as a biomarker for monitoring gastric chief cell injury in cynomolgus monkeys. Linearity and reproducibility of the human Biohit Pepsinogen I ELISA kit were verified, and parallelism and reproducibility for cynomolgus monkey serum were confirmed. Control ranges (male and female) for serum pepsinogen I values were determined using serum from fasted, healthy monkeys. Pepsinogen I values for normal male and female monkeys were 23.9 ± 1.85μg/L and 19.2 ± 1.60 μg/L, respectively. Pepsinogen I values were obtained at varying time points pre-dose and on days 4, 5, 29 or 60 in four toxicology studies of three different compounds which caused fundic mucosal injury characterized by either chief cell, parietal cell or mixed parietal and chief cell degeneration. In monkeys with chief cell or mixed chief and parietal cell injury, pepsinogen I values on day 4 or 5 were mildly (< 3.5 fold) to markedly (< 36 fold) increased relative to pre-treatment and vehicle control values, however only minimal (< 1.6 fold) increases in pepsinogen I values were observed on day 5 in monkeys in which there was selective parietal cell injury (depletion). Pepsinogen I values were more markedly increased on day 4 or 5 than on day 30, and generally correlated well with severity of chief cell injury. These findings suggest that pepsinogen I is a sensitive marker of gastric chief cell injury in cynomolgus monkeys.
127: B CELL HYPERPLASIA ASSOCIATED WITH IMMUNOSUPPRESSION IN CYNOMOLGUS MONKEYS.
Post-transplant lymphoproliferative disease (PTLPD) has been well characterized in human transplant patients treated with immunosuppressive drugs. B-lymphocyte proliferation in PTLPD is associated with latent infection of B cells by an endemic gammaherpesvirus, Epstein-Barr Virus (EBV). Discreet histomorphologic stages of PTLPD carry prognostic significance and include plasmacytoid hyperplasia, polymorphic B cell hyperplasia and polymorphic B cell lymphoma. Withdrawal of immunosuppressive therapy may result in reversal of PTLPD and full recovery. Non-human primates (NHPs) are endemically infected with a gammaherpesvirus homologous to EBV and PTLPD has occurred in NHPs in transplant studies wherein monkeys were exposed to clinical immunosuppressive regimens. The histomorphologic stages of PTLPD in these animals were markedly similar to those described in humans. This is a report of B cell hyperplasia in NHPs subjected to levels of a T lymphocyte depleting biopharmaceutical. Lymphoma occurred in one animal from a group (n = 24) of cynomolgus monkeys undergoing long term supraclinical dosing with this agent. Informed scrutiny of lymphoid tissue from these and other NHP's exposed to the biopharmaceutical allowed recognition of probable gammaherpesvirus mediated B cell hyperplasia that was very similar in important biologic, pathologic and molecular characteristics to EBV-driven PTLPD of humans. As in human PTLPD, gammaherpesviral-encoded RNA was demonstrated to be abundantly present in affected lymphocytes in lymphomatous tissue. Additional animals were identified with occult plasmacytoid hyperplasia and polymorphic B cell hyperplasia. Since many novel immunomodulatory therapeutics are currently in development and are being studied in NHP's, it is necessary to recognize this syndrome and incorporate the relevant specific diagnostic terminology into the lexicon of toxicologic pathology.
128: INHIBITION OF HISTONE DEACETYLASE ATTENUATES AIRWAY INFLAMMATION IN A MOUSE ASTHMA MODEL.
Histone acetylation in chromatin, using a specific histone deacetylase (HDAC) inhibitor, has been demonstrated to regulate the expression of a restricted set of cellular genes. In recent studies, treatment with HDAC inhibitors suppressed T cell proliferation, reduced IL-2 secretion and reduced CD40 expression; all measures of T cell suppression. The purpose of this study was to determine whether HDAC inhibition influences allergen-induced airway inflammation in a mouse asthma model. BALB/c mice were immunized to ovalbumin (OVA) and challenged with an OVA aerosol. Trichostatin A (TSA), a specific HDAC inhibitor, was injected into the mice at a dose of 1 mg/kg of body weight. Treatment with TSA significantly attenuated the airway hyperesponsiveness and decreased absolute eosinophil and lymphocyte numbers in bronchoalveolar lavage (BAL) fluid. Treatment with TSA significantly decreased cellular infiltrates and mucus occlusion in the lung, as well as levels of IL-4, IL-5 and IgE in the BAL. In addition, the TSA-treated mice also had reduced pulmonary infiltration of CD4+ cells. These results suggest that histone acetylation plays an important role in the development of allergic airway inflammation and that the HDAC inhibitor may prevent airway inflammation in asthma patients.
129: CHEMOPREVENTION OF HELICOBACTER PYLORI-ASSOCIATED GASTRIC CANCER BY NIMESULIDE IN MICE.
H. pylori is known to be involved in the genesis of several gastric diseases, including gastric cancer. WHO/IARC classified H. pylori as a group 1 carcinogen and a definite cause of gastric cancer in humans. COX-2 over expression has been noted in human gastric cancer. The aim of this study was to evaluate the chemopreventive potential of nimesulide, a selective COX-2 inhibitor, against H. pylori-associated gastric carcinogenesis. To induce gastric cancer, C57BL/6 mice were treated with N-methyl-N-nitrosourea (MNU) and H. pylori. To determine its effectiveness as a chemopreventive, nimesulide (200 ppm in CRF basal diet) was fed throughout the experiment. All mice were sacrificed at week 50 and histopathology, immunohistochemistry and Western blotting for COX-2 were performed using tissues taken from the stomach. The incidence of gastric cancer was 68.8% in control mice (MNU + Hp), and was 38.9% in nimesulide treated mice, consistent with a significant preventive effect of nimesulide. The expression of COX-2 protein was significantly decreased in nimesulide treated mice, as compared to control mice. Nimesulide showed intense suppression of H. pylori-associated gastric cancer in mice, which suggests that COX-2 may be the target for the prevention or treatment of gastric cancer in humans.
130: PREDICTING HEPATIC EFFECTS FROM GENE EXPRESSION CHANGES IN MICE.
Previous gene expression profiles for 10 hepatotoxins in mice revealed 4 clusters of compounds with similar profiles; compounds within 2 clusters had similar mechanisms of toxicity within each cluster, suggesting that these profiles identified certain mechanisms. The present study tested whether gene expression could predict the cluster, and possibly the mechanism of toxicity, of unknown hepatotoxins. Groups of 3 male CD-1 mice received oral acetaminophen, bromobenzene, alpha-naphthylthiocyanate (ANIT) or phenobarbital, each at 2 dose levels, for 3 days. Acetaminophen was previously evaluated; other compounds had reported mechanisms of toxicity similar to those evaluated previously. On Day 3, blood was collected for serum chemistry evaluation and samples of liver were taken for microscopic evaluation and gene expression analysis. Gene expression was performed on hepatic RNA using Mouse U47a Affymatrix GeneChips in a blinded fashion. Higher group mean AST, ALT, and LDH values occurred in the high-dose acetaminophen (500 mg/kg) and ANIT (40 mg/kg) groups; ALK, direct bilirubin and cholesterol were also higher in mice receiving this dose of ANIT. Microscopic findings in the high-dose acetaminophen group included centrilobular necrosis and hemorrhage; while findings in the high-dose ANIT group included biliary epithelial hypertrophy and cytoplasmic basophilia, peribiliary cellular infiltrates, edema and fibrosis. Principal component analysis (PCA) of 48 probe sets that changed at least 2 fold with a p< 0.05 for each compound successfully identified the high-dose acetaminophen group. Neither PCA nor hierarchical clustering enabled predictions on other test articles or dose levels. These data suggest that changes in a small group of liver gene transcripts can be used for a given hepatotoxin but larger training sets are required for transcriptional profiling to make mechanistic predictions.
131: PRELIMINARY SUBACUTE TOXICITY OF AN ONCOLYTIC ADENOVIRAL VECTOR IN MACAQUES.
Previously, a single injection of a replication-defective adenoviral vector in cynomolgus monkeys (M. fascicularis) produced hepatotoxicity without mortality. In the present 2-week study, 1 male and 1 female monkey/dose group received a single intravenous dose of 0, 1x1011, 5x1011, 1x1012 or 3x1012 particles/kg of a tumor-selective replication-competent adenoviral vector. The lowest dose was well tolerated whereas the two highest doses were toxic with mortality; the intermediate dose of 5x1011 particles/kg caused decreased platelet counts, increased serum transaminases and morphological changes of liver, and vascular and perivascular infiltrates in the pancreas and lung. Mortality was associated with clinical laboratory and morphological evidence of disseminated intravascular coagulation (PIC), including thrombocytopenia, increased PT and aPTT, and fibrin thrombi. Periportal leukocyte infiltrates, prominent sinusoidal lining cells and apoptosis were present in the liver at doses ≥ 5x1011 particles/kg, while only periportal infiltrates were seen at 1x1011 particles/kg. Intranuclear inclusions were present in hepatocytes and pulmonary endothelial cells at 3x1012 particles/kg. Transfection of hepatocytes, sinusoidal lining cells, marginal zone cells of the spleen and endothelial cells of several organs (gall bladder, lung, kidney, spleen, pancreas) was indicated by immunoreactivity to Ela at doses ≥ 5x1011 particles/kg. Vector replication was suggested by the presence of hexon positive cells in the pulmonary vasculature of 1 animal at 3x1012 particles/kg. Ultrastructurally, the hexon-negative hepatic inclusions contained electron-dense granular material measuring 18 to 26 nm. In conclusion, this study indicated that endothelial cells may be targets of adenoviral vectors and that non-transformed cells may allow partial replication of tumor selective vectors at doses ≥ 5x1011 particles/kg in macaques.
132: THE MOUSE TUMOR BIOLOGY DATABASE: A PUBLIC RESOURCE FOR IMMUNOMARKERS FOR PARAFFIN-EMBEDDED MOUSE TISSUES.
Histology and immunohistochemistry are the primary tools used to classify human cancer for comparison with respective animal models. The study of cancer in mice has been hampered by the absence of a large set of readily available antibodies adapted to paraffin-embedded tissues. We tested 189 antibodies on paraffin-embedded mouse tissues preserved in five different fixatives (Fekete's acid-alcohol-formalin, 10% neutral buffered formalin, 4% paraformaldehyde, IHC Zinc Fixative, and Bouin's fixative). A total of 84 (44%) antibodies provided an adequate signal with minimal or no background, and 10 (5%) provided a weak signal with at least one fixative. Of the antibodies providing adequate labeling, heat-mediated antigen retrieval or enzymatic digestion was required for 38 and 8 antibodies, respectively. Antigen identification was best for tissues fixed with Fekete's acid-alcohol-formalin (n = 75) and Bouin's solution (n = 44). However, 7 antigens could be detected adequately only in IHC Zinc Fixative, establishing that there is no single universal fixative suitable for the preservation of all epitopes. Regularly updated and expanded data and images are accessible online in the Mouse Tumor Biology Database (MTB) at http://tumor.informatics.jax.org. Each image panel includes descriptive information on the tissues and staining patterns observed, methods used, and links to the antibody suppliers’ web sites. This work was supported in part by grants from NIH/NCI (CA89713 and CA34196).
133: GSTP AND TGF-ALPHA EXPRESSION IN HEPATIC PRENEO-PLASIA INDUCED BY PCB126 AND ARSENIC.
Polychlorinated biphenyls (PCBs) and arsenic are common pollutants and are likely to be found in environmental mixtures. The coplanar PCB126 (3,3,4,4,5-pentachlorobiphenyl) exhibits dioxin-like promotional effects on hepatocarcinogenesis. Arsenic is a clastogen that has been suggested to act at the progression stage of carcinogenesis or as a cocarcinogen. This study evaluated mixtures of these two chemicals to determine potential interactions at the promotion and progression stages of carcinogenesis. Staining for glutathione-S-transferase (placental form) (GST-P) identifies promoted foci, while staining for transforming growth factor alpha (TGF<=UNICODE:F061/>) is presumed to represent progression. Rats were injected with an initiating dose of diethylnitrosamine (DEN) and received a partial hepatectomy three weeks after DEN injection. Gavage with corn oil or 10ug/kg PCB126 in corn oil was started at two weeks after DEN administration. Sodium metaarsenite (75ppm) in drinking water was started in two groups at two weeks and in two groups at eight weeks after DEN. Rats were sacrificed at 8, 16, and 24 weeks. Our data indicate an increase in GST-P preneoplastic foci in livers of rats exposed to PCB126 alone and in combination with arsenic. Arsenic as a single agent did not increase GST-P positive foci over DEN controls. The addition of arsenic at two and eight weeks post-DEN to animals with PCB exposure reduced the relative area of GST-P positive foci at 16 weeks. At 24 weeks, this inhibitory effect was absent. Approximately ten percent of foci in the PCB & PCB/arsenic-treated groups, particularly those foci largest in size, also stained for TGF<=UNICODE:F061/>. This suggests complex interactions between arsenic and PCB126 in hepatic promotion and progression, with potential time-dependent effects.
This research is supported by NIEHS Grant #5 K08 ES00380.
134: INCREASED HEPATOCYTE DIVISION LEADS TO TRANSIENT DECREASE IN LIVER INJURY FOLLOWING CHRONIC ETHANOL CONSUMPTION.
Alcoholic liver disease (ALD) accounts for more than 100,000 deaths and public health costs are more than $116 billion per year in the US. ALD is characterized by four progressive pathological stages namely steatosis, steatohepatitis, fibrosis and cirrhosis. This study was designed to investigate the temporal pattern of liver injury during alcoholic steatosis. Steatosis was induced in male Sprague-Dawley rats by feeding an ethanol-containing Lieber-DeCarli diet (36% of calories from ethanol) for 5 weeks. The control rats received isocaloric maltose-dextrin diet. Microvesicular steatosis was evident in H&E sections by three weeks in the ethanol-treated rats, which further developed into panlobular macrovesicular steatosis by 5 weeks. Liver injury, measured by plasma transaminase activities, indicated progressive increase in liver injury up to 3 weeks with a transient and significant decrease at 4 and 5 weeks. The mechanism behind the transient decrease in liver injury was hypothesized to be enhanced hepatocyte division rather than decrease in hepatic CYP2E1, a microsomal P450 enzyme involved in ethanol-mediated liver injury. CYP2E1 protein and activity were evaluated in livers of ethanol-treated and control rats and indicated significant induction in CYP2E1 protein levels, which remained consistently higher until the 5th week in EtOH-treated rats. PCNA analysis of liver sections was conducted to assess liver cell division. Ethanol-treated rats had a significantly higher number of cells in S and G2 phases of cell division at weeks 1 (3.20±0.19), 2 (7.03±0.92), and 3 (4.23 + 1.41) following EtOH treatment as compared to controls (1.5+0.22). In summary, these data indicate that stimulation of liver regeneration rather than CYP2E1 induction following ethanol treatment may be responsible for the transient decrease in liver injury following chronic ethanol administration.
135: PRECLINICAL PROFILING OF THE ALPHA-7 NICOTINIC ACETYLCHOLINE RECEPTOR (ALPHA-7 nAChR) IN RAT USING NON-ISOTOPIC IN SITU HYBRIDIZATION (nISH) AND LASER CAPTURE MICRODISSECTION-QUANTITATIVE PCR (LCM-qPCR).
Evaluating the mRNA expression profile pattern at the cellular level for putative therapeutic targets is a critical part of drug discovery and development. Expression patterns for the target can elucidate potential novel approaches for therapeutic intervention and alert the toxicologic pathologist to potential sites for exaggerated pharmacology or target-related toxicities. The <=UNICODE:F061/ >7 nAChR is a calcium ion channel expressed in areas of the brain important for learning and memory and preclinical animal experiments have established that compounds that activate the <=UNI-CODE:F061/>7 nAChR improve learning and memory. A full-length rat <=UNICODE:F061/>7 nAChR antisense mRNA probe with incorporated digoxigenin-labeled UTP in addition to PCR primers selective for the <=UNICODE:F061/>7 receptor subtype were used to characterize the <=UNICODE:F061/>7 nAChR expression pattern using nISH and LCM-qPCR, respectively. Antisense nISH probes identified expression in several tissues and cell types including neurons in the retinal ganglion cell layer, hippocampus, and cerebral cortex. Sense and nonsense nISH probes were negative in these tissues. LCM-qPCR of several positive tissues or cell types confirmed the specificity of the nISH. The pharmacological and toxicological implications of these results will be discussed.
136: TRANSCRIPTIONAL PHENOTYPING OF CIRCULATING ENDOTHELIAL CELLS FROM SORTED RAT WHOLE BLOOD.
Vascular toxicity in pre-clinical toxicology species presents a hurdle for progressing new chemical entities into clinical development. Further, monitoring of drug-induced vascular toxicity is hampered by lack of sensitive and predictive biomarkers of vascular injury. Circulating endothelial cells (CEC) have recently been identified as possible biomarkers for vascular injury. In normal rat peripheral blood, small subpopulations of putative circulating endothelial cells show variable degrees of Dil-Ac-LDL uptake and vWF and CD31 cell surface expression. To assess the utility of CEC as potential biomarkers of vascular injury, further characterization of these cell populations was performed using immunophenotypic labeling, followed by flow cytometric cell sorting and transcriptional profiling of unique subpopulations. Unsorted, LDL+/CD11b-, LDL+/ CD11b-/vWF+/CD31-, LDL+/CD11b-/vWF-/CD31 +, rat blood nucleated cells were analyzed for expression of Endothelin-1 (ET-1), FLK-1, and VEGF by real-time RT-PCR. Cell surface and transcriptional expression of CD3 was included as both a negative and positive control. Unsorted blood nucleated cells were negative for ET-1 and VEGF, weakly positive for FLK-1 and positive for CD3. Sorted CD3 cells, were negative for all genes except the T-cell receptor, where expression was very high. LDL+/CD11b-cells were positive for ET-1, FLK-1, and VEGF but not CD3. LDL+/CD11b-/vWF+/ CD31- cells were positive for ET-1, VEGF, weakly positive for FLK-1, but negative for CD3. Sorted LDL+/CD11b-/vWF-/CD31+ cells did not express ET-1, VEGF or CD3 but were weakly positive for FLK-1. Transcriptional analysis of sorted CEC provides a means to further characterize these cells using on previously described endothelial cell markers and demonstrates the heterogeneity of CEC based on cell surface and transcriptional expression profiles.
137: CIRCULATING ENDOTHELIAL CELLS AND VWF AS BIO-MARKERS OF FENOLDOPAM-INDUCED ARTERIAL INJURY IN RATS.
There are no widely accepted biomarkers of vascular injury in animals or humans. Circulating endothelial cells (CEC) might be useful as a biomarker of vascular injury/toxicity since they are known to increase in number after various physical, infectious and chemical insults to the vasculature. Plasma vWF was previously shown to increase after vascular injury. Fenoldopam is a selective dopaminergic DA1 agonist that consistently causes lesions in mesenteric arteries when administered to rats. To determine the temporal relationship between CEC, plasma vWF and morphology, a time course study in Sprague-Dawley rats given a single subcutaneous injection of 0 or 100 mg/kg fenoldopam was performed. Rats (n = 6/group) were euthanized at 0.08, 0.5, 1, 2, 4, 12, 24 or 48 hr after injection. Whole blood was collected at necropsy from the caudal vena cava for flow cytometric examination of CEC, and plasma was collected for vWF concentrations. The intestinal mesentery was collected, rolled and fixed in 10% buffered formalin, sectioned and stained with H&E. CEC were detected by uptake of DiI-acetylated LDL, lack of CD62L, and expression of surface vWF. Significant elevations in CEC were observed at 0.5, 2, 24 and 48 hr post-dose when measured as percent nucleated cells (peaking at 2 hr), but only at 4 hours when measured as absolute cell number. Plasma vWF (ELISA) was significantly elevated at 1,2, and 4 hr post-dose, peaking at 2 hr. Mesenteric arterial medial necrosis was first apparent at 12 hr post-dose and increased in severity by 24 hr. Perivascular inflammatory cell infiltrates were present by 24 hr and increased in severity at 48 hr. Increases in CEC and vWF preceded the onset of morphologic damage in rat mesenteric arteries after a single subcutaneous dose of fenoldopam, and might be useful biomarkers for vascular injury in rats.
138: APOPTOSIS OF ENDOTHELIAL CELLS IS NOT INVOLVED IN PATHOGENESIS OF FENOLDOPAM-INDUCED ARTERIAL INJURY IN RATS.
Fenoldopam is a selective dopaminergic DA1 agonist that consistently causes lesions in mesenteric arteries when administered to rats. A time course study in Sprague-Dawley rats given a single subcutaneous injection of 0 or 100 mg/kg fenoldopam was performed. Rats (n = 6/group) were euthanized at 0.08, 0.5, 1, 2, 4, 12, 24 or 48 hrs after injection, and the intestinal mesentery collected, rolled, fixed in 10% buffered formalin, sectioned and stained with H&E. Additional sections were analyzed by immunohistochemistry for activated caspase 3, and the TUNEL assay to detect apoptosis. Mesenteric arterial medial necrosis was present from 12 hr post-dose, and increased in severity at 24 hr post-dose. Perivascular inflammatory cell infiltrates were present by 24 hr and increased in severity by 48 hr post-dose. No endothelial cells were positive for both activated caspase 3 and TUNEL. Individual endothelial cells were either activated caspase 3 or TUNEL positive in 2/47 control rats (2 and 4 hr), and 3/48 rats given 100 mg/kg fenoldopam (24 and 48 hr). Individual smooth muscle cells in the media of mesenteric arteries from fenoldopam-treated rats were positive for both activated caspase 3 and TUNEL beginning at 4 hr (1/6) with increasing incidence at 24 hr (3/6) and 48 hr (5/6). While there was minor evidence of apoptosis in endothelial cells after administration of fenoldopam in rats, it was similar in incidence to control rats and did not appear until after morphologic evidence of vascular medial necrosis had already occurred. Apoptosis of medial smooth muscle cells appears to be a secondary event in the pathogenesis of fenoldopam-induced vascular damage.
139: IMMUNOPHENOTYPIC ANALYSIS OF RAT BONE MARROW BY FLOW CYTOMETRY.
A combination of four monoclonal antibodies was used to identify cells of the erythroid lineage and lymphoid lineage directly and cells of the myeloid lineage indirectly from rat bone marrow by three-color flow cytometric analysis based on differential expression of cell surface antigens on
subpopulations of rat bone marrow mononuclear cells. Cells of the erythroid lineage were identified by expression of the transferrin receptor, CD71. Lymphoid cells were identified by expression of either CD3 (T lymphocytes) or CD45R (B lymphocytes). Cells of the myeloid lineage were identified by subtraction of lymphoid cells from a population of bone marrow cells that expressed the leukocyte common antigen, CD45. Analyses by flow cytometry yielded differentials for erythroid (31.9%), lymphoid (26.9%) and myeloid (33.9%) subpopulations and myeloid:erythroid ratios (1:1) that were similar to manual differentials and published reference ranges. This new approach to bone marrow analysis affords an advantage over previously reported methods that rely heavily on single, two-color, and/or side and forward scatter profiles alone or in combination for analyses. This method should offer a more efficient alternative to the time-consuming and laborious currently utilized manual methods of bone marrow evaluation.
140: ACUTE TUMOR LYSIS SYNDROME IN P53± MICE.
During a study to determine if tumor incidence increased in p53 (±) mice following intravenous treatment with a test article given three times per week for 26 weeks, unexpected mortality was observed. Of 348 mice, 48 early deaths were reported. The highest incidence of early deaths/moribund sacrifices (16/58) occurred in mice receiving the highest dose of test article. Acute tumor lysis syndrome (ATLS) was identified as the likely cause of early death in four p53 ± mice in the high dose group. The affected mice typically had disseminated T cell lymphomas, with marked involvement of the liver and spleen. Histopathologic examination revealed widespread necrosis of lymphoblastic tumor cells in affected tissues and in circulation. Variably sized emboli composed of tumor cell derived nuclear and cytoplasmic cell debris were present in the vessels of the kidneys, lungs, heart, and brain, with the pulmonary arteries and capillaries being most severely affected. The obstruction of pulmonary and cerebral capillaries by DNA microemboli and of pulmonary arteries by embolic cell debris was the likely cause of early death in these mice, although metabolic derangements and electrolyte abnormalities associated with the massive lysis of tumor cells may also have been involved. The precipitating cause of ATLS in these mice is unknown, but the large tumor burden in the high-dose group and/or a direct toxic or the test article on lymphoma cells may have been involved. Our findings suggest that ATLS may be a significant cause of early death in p53 (±) mice used in carcinogenicity or toxicology studies.