Abstract
1: DETECTION OF FELINE IMMUNODEFICIENCY VIRUS IN MILK AND MAMMARY GLAND TISSUE BY PCR AND ISH.
Blood and milk samples were obtained at parturition (n = 7) and at 2-week intervals throughout lactation (n = 5) from queens infected with FIV-B-2542. Semi-quantitative DNA-PCR was performed on peripheral blood mononuclear cells (PBMC) and milk cells, and QC-RT-PCR was performed on plasma and milk supernatant. In situ hybridization (ISH) for viral RNA using digoxigenin-labeled riboprobes was performed on mammary gland biopsies obtained within 48 hours of parturition from 4 of the queens. Provirus detected in PBMC ranged from 102 to 104 copies/μg DNA. Queen plasma viral loads at parturition ranged from 104 to 2.5 × 106 copies/μg. In 6 of 7 cats, proviral levels were lower in milk cells than in PBMC, ranging from 0 to 102 copies/ μg DNA. In contrast, 6 of 7 cats had higher viral loads in milk than in plasma at parturition, ranging from 2 × 105 to 4 × 108 copies/mL. ISH performed on formalin-fixed mammary gland sections revealed moderate numbers of FIV-infected cells, many adjacent to alveolar lumina and morphologically compatible with epithelial cells. Phenotypic identification of infected cells is being pursued using combined ISH and immunohistochemistry. These data suggest that FIV RNA is concentrated in milk early in lactation, and that infected cells in mammary gland as well as in milk are contributing to the milk viral load. These observations have significance for the mechanisms of perinatal transmission of FIV, HIV, and other lentiviruses. (Abstract citation: Vet Clin Pathol
2: EVALUATION OF CYSTATIN C AS A MARKER OF DECREASED GLOMERULAR FILTRATION RATE IN DOGS. F.
Serum cystatin C (cysC) is a 14 kD protein produced by all nucleated cells at a constant rate. Its serum concentration is dependent on GFR and catabolism by renal tubules. CysC is a sensitive indicator of decreased GFR in humans and has been proposed as a replacement for serum creatinine (Cr) for detecting abnormal GFR. Serum levels of cysC are not affected by non-renal factors and it is not secreted by the renal tubules. The aim of this study was to evaluate serum cysC as a marker of decreased GFR in dogs. Anti-human cysC antibody (Dako Corporation, Carpinteria, CA) and Western blot analysis were used to demonstrate significant cross-species reactivity in canine serum. A commercially developed automated particle enhanced immunoturbidometric assay for humans (Dako Corp) and a Hitachi 912 analyzer were used to measure cysC concentrations in 20 dogs with renal failure for which a gold standard measurement of GFR was available. Serum Cr concentration was determined by an enzymatic method. CysC and Cr concentrations also were measured in 20 healthy dogs. Mean cysC concentration in dogs with renal failure was 4.42 ± 1.05 mg/L and creatinine was 3.72 ± 1.35 mg/dL. In dogs with renal failure, cysC was correlated better with exogenous creatinine clearance than Cr (r = 0.77, P < .0001 and r = 0.54, P < .0001, respectively). CysC was more sensitive than Cr (67% and 50%, respectively) in detecting decreased GFR. Intra-assay precision (n = 20) on serum samples with low, moderate, and high cysC concentrations showed CVs of 2.43%, 0.83%, and 0.85%, respectively. Inter-assay precision (n = 20) showed a CV of 1.75%. The assay was linear to 7.5 mg/L with r = 0.97, P < .0001. Serum cysC concentration appears to be a more sensitive indicator of decreased GFR in dogs than serum creatinine. (Abstract citation: Vet Clin Pathol
3: GREEN IGUANA (IGUANA IGUANA) BLOOD CELL MORPHOLOGY, CYTOCHEMISTRY, AND HEMATOLOGIC AND PLASMA BIOCHEMICAL REFERENCE RANGES.
This study determined the hematologic and plasma biochemical reference ranges for healthy green iguanas (Iguana iguana) housed in a warm, outdoor environment similar to their natural habitat. Plasma and whole blood samples were collected from 56 clinically healthy iguanas (23 males, 25 females, and 8 juveniles) from 3 locations in Florida. When compared to male iguanas, females had significantly (Student's t-test, P < .05) elevated levels of sodium, calcium, phosphorus, cholesterol, total protein, globulins, anion gap, and alkaline phosphatase. Calcium and phosphorus values in clinically normal, gravid females reached levels that have previously been considered an indication for renal disease. Hematologic ranges were higher in this study than previously reported in iguanas housed indoors. Cytochemical and morphologic evaluation of blood cells confirmed lymphocyte and thrombocyte morphology, peroxidase activity in heterophils, and distinct differences between iguana monocytes, and azurophils found in snakes. Geometrically-shaped crystalline inclusions and protozoal hemoparasites were seen within the erythrocytes of some healthy iguanas. These data provide the necessary baseline information required for the hematologic and biochemical evaluation of clinically healthy and diseased animals of this species. (Abstract citation: Vet Clin Pathol
4: CLONING AND EXPRESSION OF THE GENE ENCODING THE MAJOR ANTIGENIC PROTEIN 2 HOMOLOG OF EHRLICHIA CANIS AND POTENTIAL APPLICATION FOR SERODIAGNOSIS.
Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis (CME). The major antigenic protein 2 (MAP2) homolog of E. canis was cloned and expressed in Escherichia coli. The recombinant protein (rMAP2) with its fused C-terminal polyhistidine tag had a molecular mass of approximately 26 kDa. The antigen was identified by Western immunoblotting using both anti-histidine antibodies and serum from E. caws-infected dogs. The rMAP2 was tested in an ELISA format for potential application in the serodiagnosis of E. canis infection in dogs. Serum samples from 18 experimentally-infected and 34 naturally-infected dogs were evaluated for antibodies to the rMAP2. All dogs had demonstrable indirect immunofluorescence assay (IFA) titers against E. canis of >1:80. The rMAP2 was also tested using 55 serum samples from animals negative for E. can-is by IFA, using IFA as the “gold standard”. The MAP2 ELISA had a test sensitivity of 100% when tested with sera from experimentally-infected animals and a sensitivity of 93.9% when tested using sera from naturally-infected animals. When tested against sera from IFA-negative, noninfected animals, the MAP2 ELISA had a specificity of 92.6%. These data demonstrate the potential of the rMAP2 homolog of E. canis as a test antigen for the serodiagnosis of CME. (Abstract citation: Vet Clin Pathol
5: PERMETHRIN-INDUCED THYMIC HYPOCELLULARITY: ARE ANTIPROLIFERATIVE EFFECTS INVOLVED?
Permethrin is a topical pyrethroid insecticide that is used commonly in the treatment of head lice or scabies, and is impregnated in some military and hunting clothing for prophylaxis against ectoparasites. Chronic exposure to permethrin causes systemic immunomodulatory effects such as thymic atrophy, decreased thymic cellularity, diminished contact hypersensitivity, and decreased splenic leukocyte function in mice. The present study evaluates the cytologic, cytometric, and functional effects of acute topical permethrin in 6-week-old female C57BL/6 mice. A single topical dose of 25 μL (28 mg) permethrin resulted in decreased thymic organ weight and cellularity 48 hours after treatment. Evaluation of thymocyte surface markers demonstrated a drop in CD4+CD8+ thymocytes, which suggests either thymocyte apoptosis or inhibition of thymocyte proliferation. Cytologic examination and 7-aminoactinomycin D (7-AAD) staining were used to assess apoptosis of thymocytes. Apoptosis was not found following either in vivo or in vitro exposure of thymocytes to permethrin. However, thymocyte proliferation was significantly inhibited following a single in vivo dose of permethrin. The combination of decreased thymic to body weight ratio and diminished proliferation has a pair-wise predictive value of 0.83 for immunosuppression. This evidence suggests that mice exposed to a single topical dose of permethrin may have an impaired immune response if challenged. (Abstract citation: Vet Clin Pathol
6: A COMPARISON OF THREE POINT-OF-CARE COAGULATION ANALYZERS.
Three point-of-care coagulation analyzers, the ACT II (Medtronic, Parker, CO), the Rapidpoint™ Coag Analyzer (Bayer Corp, Tarrytown, NY), and the SCA2000™ (Synbiotics, San Diego, CA), were evaluated for after-hours testing of veterinary patients. Ease of use, sample volume and type, interferences, data management, and cost per test were compared. Prothrombin time (PT) and activated partial thromboplastin time (APTT) tests were performed on citrated whole blood (CWB), citrated plasma (CP) or both. Results were compared by regression analysis and bias plots to those obtained using CP and a fibrometer (Becton Dickinson, Cockeysville, MD) in the core lab. A total of 39 CWB and 34 CP samples from 34 dogs, 25 cats, and 6 horses were evaluated. Results from the ACT II correlated significantly with fibrometer results, with correlation coefficients of 0.97 for PT-CWB, 0.95 for PT-CP, 0.88 for APTT-CP, and 0.75 for APTT-CWB. ACT II results had a small positive bias with CWB and a negative bias using CP. Both the Rapidpoint and SCA2000 correlation coefficients were <0.8 for all tests and samples (range 0.75–0.53). Results from the Rapidpoint and the SCA2000 analyzers also showed significant positive bias compared to fibrometer results. Agreement ranged from 86% to 97% for tests on ACT II and the SCA2000. Agreement data were not calculated for the Rapidpoint Coag Analyzer due to lack of veterinary reference ranges. Overall, the ACT II was most economical, ran duplicate tests, and correlated best with the fibrometer method. The SCA2000 was very portable and had good agreement, however, correlation was variable and values were significantly higher than those obtained by fibrometer. The Rapidpoint analyzer had excellent data management and was easy to use, but requires further validation for veterinary use. (Abstract citation: Vet Clin Pathol
7: COMPARISON OF DIRECT ANTIGLOBULIN TEST AND FLOW CYTOMETRIC ERYTHROCYTE IMMUNOFLUORESCENCE ASSAY IN DOGS WITH IMMUNE-MEDIATED HEMOLYTIC ANEMIA.
A flow cytometric erythrocyte immunofluorescence assay (FC) was compared to the direct antiglobulin test (DAT) for detection of erthrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The DAT and FC yielded negative results for all healthy nonanemic dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 7 were positive using the DAT and 11 were positive using the FC. Sensitivity for detection of erythrocyte-bound IgG in the 26 dogs was 53% for DAT and 85% for FC. Specificity for DAT and FC was 100%, as IgG was not detected in any healthy dog. The addition of IgM and/or C3 did not increase the sensitivity of DAT or FC. There was good agreement between DAT and FC methods for detection of IgG, and poor agreement for detection of IgM and C3. Disagreement between tests was due to increased detection of immunoglobulin and complement using FC compared to DAT. These results suggest that, in dogs with IMHA, FC provides a rapid, cost effective, more sensitive method to quantitate erythrocyte-bound immunoglobulin and/or complement, compared to DAT. (Abstract citation: Vet Clin Pathol
8: PROTEIN ADDUCT FORMATION BY NORCOCAINE NITROXIDE, AN N-OXIDATIVE METABOLITE OF COCAINE.
It was demonstrated over 20 years ago that cocaine N-oxidative metabolism leads to formation of a reactive metabolite that binds irreversibly to proteins, and this binding correlates with cocaine-induced liver injury. The identity of the reactive metabolite has not been established with certainty. Early reports suggested that the stable nitroxide metabolite of cocaine (norcocaine nitroxide; NNX) was not sufficiently reactive to be responsible for protein binding. To test this directly, 14C-labeled NNX was synthesized and incubated with mouse hepatic microsomes. Rapid, irreversible binding of the radio-label to protein was observed. The addition of NADPH to the incubation produced only a modest increase in radiolabel binding, indicating that oxidation of NNX was not required for protein-adduct formation. In subsequent experiments, incubation of unlabeled NNX with mouse whole liver homogenate was found to result in selective adduction of specific proteins as determined by Western blot analysis. Many of these proteins appeared to have similar molecular masses as proteins adducted during cocaine hepatotoxicity in vivo. Two of the proteins adducted during cocaine hepatotoxicity in vivo have been identified recently as hsp60 and transferrin. Incubation of NNX with these proteins in vitro resulted in protein adduct formation. Incubation with cocaine under these conditions, in comparison, produced little or no protein adduct formation. These observations suggest that NNX formed from N-oxidative metabolism of cocaine is spontaneously reactive with proteins and that it could be responsible for much, if not all, of the protein adduct formation associated with cocaine hepatotoxicity. (Supported by National Institute on Drug Abuse, DA-06601.) (Abstract citation: Vet Clin Pathol
9: COMPARATIVE SEQUENCE OF FELINE ATRIAL NATRIURETIC PEPTIDE.
Natriuretic peptides have potential value as both therapeutic and diagnostic aids in various forms of heart failure. Our laboratory is investigating the role played by natriuretic peptides in heart disease in cats. In this study, we report on the comparative sequence of feline atrial natriuretic peptide (ANP). Total RNA was extracted from heart atrial tissue from a clinically healthy cat and complementary DNA (cDNA) prepared by reverse transcription using oligo-T primer. Genomic DNA (gDNA) was extracted from feline blood. Consensus ANP gene sequences of known species were identified and used as primers (5'ACGACGCCAG CATGAGCTCCTTC3’ and 5'CGGAAGCTGTTGCAGCCCAG3’) in PCR to amplify cat ANP. DNA fragments of the approximate predicted length were obtained from gDNA and cDNA, and sequenced. The ANP sequence was further extended using new primers designed from this sequence data and highly conserved regions at the 3’ and 5’ ends of the ANP gene. The cat ANP gene is approximately 1.2 kb in length and consists of three exons and two short introns. It contains many features of a typical eukaryotic gene, such as TATAA box, a cap site initiation, and splice signals flanking each intervening sequence. The final coding sequence from feline ANP (pre-pro-ANP) is 153 amino acids. The active C-terminal portion of ANP is likely 30 amino acids (SLRRSSCFGG RMDRIGAQSGLGCNSFRYRR), and contains a 17 amino acid ring formed by a cysteine-cysteine double bound, characteristic of natriuretic peptides. The ANP sequence is highly conserved among species with the feline C-terminal peptide identical to horse, cow, and sheep ANP, and only differing from human, dog, and pig by an extra RR at the C-terminal end. This fact strongly supports that ANP immunoassay kits from other species can be used for measurement of cat ANP. Regions of pre-pro-ANP also contain short segments of homology to other species, which may allow N-terminal ANP assay kits also to be used in cats. (Supported by the Winn Feline Foundation.) (Abstract citation: Vet Clin Pathol
10: EVALUATION OF BLOOD COLLECTION FROM THE SUBLINGUAL VEIN IN RATS. F.
Collection of blood from the sublingual vein offers an alternative method of blood collection in rats. Several parameters (clinical pathology, food consumption, body weight, and histopathology) were examined in male Sprague Dawley rats that were bled from either the sublingual vein or the retro-orbital sinus. In comparison to the retro-orbital method, the only consistent significant changes in clinical pathology analytes in sublingual blood samples included increases in basophils, potassium, chloride and amylase and decreases in creatine kinase, sorbital dehydrogenase, and calcium. Prolonged coagulation (oral dose of 0.2 mg/kg warfarin x 3 days) or increased salivation (single SC dose of 10 mg/kg pilocarpine) did not have any major adverse effects on the collection and/or evaluation of blood from the sublingual vein. Focal inflammation of the tongue was observed in 70% of rats bled by the sublingual method; however, no significant changes in body weight or food consumption were observed in this group. In conclusion, some minor changes were observed between the sublingual and retro-orbital methods; however, these changes should not prevent the use of sublingual blood collection method on rats in toxicology studies. (Abstract citation: Vet Clin Pathol
11: ESTROGEN-ASSOCIATED APLASTIC ANEMIA IN AN ADULT NZW RABBIT.
A 2-year-old, 4.4 kg female New Zealand White rabbit used for antibody production developed hematuria. Voided urine had fresh blood (4+ protein, 4+ RBC). CBC showed leukopenia (4100/μL), and decreased RBC, hemoglobin, hematocrit, and MCV with anisocytosis and polychromasia. These changes became more severe over a month. The only clinical chemistry change was high alkaline phosphatase (25 to 26 IU/L). Differentials included leukemia (myelophthistic), lymphosarcoma, and aplastic anemia. No neoplastic cells were observed on CBC. Other than these abnormal parameters, the rabbit was within normal limits and ate and behaved normally. No bruising or dermal hemorrhages were noted. Based on clinical signs of hemorrhage (hematuria) with leukopenia and anemia, serum estradiol was assayed which at 48.8 pg/mL was >2 fold higher than normal (17–20 pg/mL). Since this was a mature female rabbit, we suspected pituitary tumor, uterine tumor and/or mammary tumor. After the final antibody production blood collection, the rabbit was sacrificed with intravenous euthanasia solution and we performed a complete necropsy. No gross or microscopic evidence of neoplasia was seen. The bone marrow was markedly hypocellular. The uterus was the source of hemorrhage and had numerous endometrial vessel ulcers and erosions with thromboses. The pituitary gland was slightly enlarged and histologically had mild diffuse hyperplasia. Immunohistochemical staining for FSH, LH, and ACTH showed increased numbers of positive cells throughout the pituitary gland with no focus suggestive of a tumor. Serum LH, FSH, and estradiol were all >2 fold normal. We concluded that diffuse adenohypophysis hyperplasia with disregulated feedback inhibition resulted in excess production of FSH and LH that led to excess estradiol and aplastic anemia. (Abstract citation: Vet Clin Pathol
12: SERUM LIPID PEROXIDE AND α-TOCOPHEROL CONCENTRATIONS IN CAPTIVE BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS).
Nutritional husbandry plays a crucial role in the management of captive marine mammals. To clarify the relationship between oxidative and anti-oxidative physiological states in captive bottlenose dolphins, the serum lipid peroxide (LPO) and α-tocopherol concentrations and serum superoxide dismutase (SOD) activities were determined in dolphins from 2 aquariums (A and B). LPO levels (mean ± SD) of bottlenose dolphins from group A (9.0 ± 2.5 nmol/mL, n = 8) were significantly (P < .001) higher than those of group B (4.2 ± 1.0 nmol/mL, n = 8). Serum α-tocopherol concentrations were 22.3 ± 3.6 μg/mL (A) and 29.9 ± 9.2 μg/mL (B), and the difference was statistically significant (P < .05). Serum SOD activities of the captive dolphins in group B (10.2% ± 2.6%) were higher (P < .05) than those of group A (8.6% ± 1.9%). A significantly negative correlation was found between serum LPO and α-tocopherol concentrations (r = -0.62, P < .001) in all 16 dolphins. LPO levels in mackerel meat being fed to dolphins of groups A and B and in herring meat being fed to dolphins of group A were higher than those of arabesque greenling, banded blue-sprat, and shishamo smelt fed to dolphins of groups A and B. LPO concentrations were dramatically (P < .001) increased in frozen herring meat during Storage, compared with that before freezing. Higher (P < .05) concentrations of LPO were found in herring meat stored for 3 and 6 months at -20°C compared with that before freezing and with herring meat stored for 1 month. The monitoring for serum LPO, α-tocopherol concentrations and SOD activities appears to be valuable for managing captive marine mammals. (Abstract citation: Vet Clin Pathol
13: DELAYED APOPTOSIS OF CD18-DEFICIENT NEUTROPHILS FROM CALVES WITH LEUKOCYTE ADHESION DEFICIENCY.
The major mechanism of in vivo neutrophil (PMN) clearance is apoptosis, followed by phagocytosis of the apoptotic PMNs by macrophages. To evaluate the apoptosis of normal and CD18-deficient bovine PMNs, viability, nuclear fragmentation, intracellular calcium concentration ([Ca2+]i), expression of annexin V, and caspase 3 activity of PMNs from 5 normal calves (N) and 3 calves with leukocyte adhesion deficiency (BLAD) were evaluated. Decreased viability was found in N-PMNs compared to BLAD-PMNs at 6 and 24 hours incubation. The viability of heat-aggregated bovine immunoglobulin G (Agg-IgG)-stimulated N- and BLAD-PMNs was significantly decreased when compared to that of unstimulated PMNs at 24 hours incubation. Agg-IgG–induced apoptosis of BLAD-PMNs was significantly lower compared to that of N-PMNs. The expression of annexin V on Agg-IgG–stimulated N-PMNs was apparently increased, compared to that of BLAD-PMNs at 6 hours stimulation. The hydrolysis of DEVD-MCA substrate was increased in N-PMNs compared to BLAD-PMNs. The delay in apoptosis was demonstrated in BLAD-PMNs and this appeared to be closely associated with a decreased rate of necrosis, lowered signaling via [Ca2+]i and decreased annexin V expression and caspase 3 activity of PMNs. (Abstract citation: Vet Clin Pathol
14: ERYTHROCYTE GHOST CELLS AND PSEUDOTHROM-BOCYTOSIS WITH THE ADVIA 120.
The Bayer Advia 120 hematology system has a very advanced platelet detection system, which counts and measures the size of platelets up to 60 fL. It is the only system to use both volume and refractive index of cells to separate platelets and erythrocytes of similar volume. With this improvement, however, has been detected an occasional error in the counting of erythrocyte ghost cells as platelets. This error has been noted after the storage of equine blood 48 hours at refrigerated temperature but not at room temperature. This error is easily detected by evaluating the platelet cytogram, which displays a cluster of the RBC ghost cells. The error is also flagged by a very large MPV which increased from a mean ± SD of 6.9 ± 1.1 fL to 22 ± 3.5 fL in 5 blood samples, and from 8.6 fL to 34.2 fL at 48 hours of storage in another equine blood sample. The false increase in platelet count may not exceed the upper reference limit or may give a pseudothrombocytosis. In one horse the “platelet” count increased to 545 × 109/L at 48 hours of refrigerated storage from 136 × 109/L. The error is also noted on the RBC cytogram by the formation of a distinct second population of hypochromic and slightly macrocytic RBC. Bayer has made a software adjustment to correct this error in equine samples. The error has also been noted in freshly obtained canine blood samples. The formation of RBC ghost cells may be associated with lipemia. (Abstract citation: Vet Clin Pathol
15: EXPRESSION OF A GENE ENCODING THE MAJOR ANTIGENIC PROTEIN 2 HOMOLOG OF EHRLICHIA CHAFFEENSIS AND POTENTIAL APPLICATION FOR SERODIAGNOSIS.
The major antigenic protein 2 (MAP2) homolog of Ehrlichia chaffeensis was cloned and expressed. The recombinant was characterized and tested in an ELISA format for potential application in the serodiagnosis of human monocytic ehrlichiosis. The recombinant protein (rMAP2), which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblots using anti-histidine antibody. However, immune sera failed to react with the recombinant on immunoblots when the antigen was denatured by heat or reduced using 2-mercaptoethanol. The rMAP2 was used in an ELISA format with 40 blinded serum samples. Twenty of the serum samples were previously demonstrated to contain antibodies reactive with E. chaffeensis by indirect immunofluorescence assays. The remaining 20 samples were seronegative. All samples negative by IFA were also found to be negative for antibodies against the rMAP2 of E. chaffeensis using the ELISA. One of 20 IFA-positive samples tested negative on the rMAP2 ELISA. There was 100% agreement using IFA-negative samples and 95% agreement using IFA-positive samples, resulting in a 97.5% overall agreement between the two assays. These data suggest the rMAP2 homolog of E. chaffeensis has potential as a test antigen for the serodiagnosis of human onocytic ehrlichiosis. (Abstract citation: Vet Clin Pathol
16: MARROW AND ERYTHROCYTE CHANGES ASSOCIATED WITH CANINE NON-REGENERATIVE IMMUNE-MEDIATED ANEMIA: 44 CASES (1996–2000).
Forty-four canine cases of non-regenerative immune-mediated anemia (NIMA) were seen over a 5-year period at the Veterinary Hospital of the University of Pennsylvania. Peripheral blood smears from these patients were reviewed and compared with blood smears from dogs with regenerative IMHA. In addition, marrow smears were compared with marrow from cases of effective erythroid hyperplasia, either secondary to IMHA or hemorrhage. Peripheral blood stomatospherocytosis was a common finding in NIMA patients but was rarely observed in regenerative IMHA. Stomatospherocytes were identified by their small diameter (< 5 μm) and small stoma-shaped central pallor. Marrow macrophages were increased in NIMA dogs, with marked macrophage granulation frequently noted. Macrophage granules were numerous, coarse, very dark blue-stained, and variably sized. Erythroblast phagocytosis was often evident within granulated macrophages; therefore, granules were interpreted as nuclear debris post-erythroblast phagocytosis. Increased macrophage cytoplasmic granularity was rarely detected in association with effective erythroid hyperplasia, either secondary to IMHA or hemorrhage, despite an assumed increase in phagocytosis of nuclei extruded from maturing erythrocytes. If noted, granulation tended to be scant. Furthermore, the two NIMA changes were linked in that marked peripheral stomatospherocytosis strongly correlated with detection of changes in macrophage morphology and frequency. In conclusion, observation of peripheral blood stomatospherocytosis, coupled with detection of granulated macrophages in marrow, suggests immune targeting of erythroid precursors as a cause for non-regenerative anemia. (Abstract citation: Vet Clin Pathol
17: QUANTITATIVE ANALYSIS OF CANINE MULTIDRUG RESISTANCE GENE EXPRESSION USING A COMPETITIVE REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION ASSAY.
An over-expression of P-glycoprotein (P-gp) has been implicated as an important factor in the development of multidrug resistance (MDR) by tumor cells and treatment failure. The plasma membrane protein P-gp is a product of the multidrug resistance family of genes. Both mdrl and mdr3 P-gp genes have been associated with the development of a MDR phenotype. We have developed a quantitative competitive reverse transcriptase polymerase chain reaction (qcRT-PCR) assay for the quantification of canine mdr1 and mdr3 mRNA. A drug sensitive cell line derived from human KB epidermal carcinoma cells and canine lymphoma cell lines were used to develop the assay. The expression of mdr1 and mdr3 were quantified by simultaneous RT-PCR of cellular RNA with decreasing amounts of heterologous competitor RNA, which shared primer sequences with the cellular mdr1 and mdr3 mRNA, but yielded PCR products that were approximately 10% smaller in size than the parent templates. This allowed for resolution of the amplified cDNA fragments. The amounts of mdr1 and mdr3 mRNA measured by the assay were accurate and reproducible over a wide range. This study demonstrates that the qcRT-PCR using a heterologous competitor RNA is a rapid, reliable, and non-radioactive method for evaluation of mdr1 and mdr3 expression in clinical samples. (Abstract citation: Vet Clin Pathol
18: CANINE HYPERLIPASEMIA IN ASSOCIATION WITH PANCREATIC AND/OR HEPATIC NEOPLASIA: A CASE SERIES.
Increased serum pancreatic lipase (PL) levels (5,410–42,900 U/L; mean of 23,750 U/L) without significant increases of serum α-amylase were observed in 6 dogs diagnosed with pancreatic and/or hepatic neoplasms. The purposes of this study were 1) to describe clinicopathologic findings and 2) to investigate possible production of PL by neoplastic tissue, in this group of dogs. These objectives were met by reviewing clinicopathologic data and evaluating formalin-fixed, paraffin-embedded tumor tissue from affected dogs and normal canine pancreatic tissue. Tissue sections were stained cytochemically with diastase periodic acid Schiff and immunohistochemically with human monoclonal PL antibody, to demonstrate zymogen granule content and PL, respectively. Normal canine pancreatic tissue stained positively for zymogen granule content and PL. Five of the 6 tumors stained positively for zymogen granule content and 2 of the 6 tumors stained positively for PL. Together, the histochemical and immunohistochemical staining patterns suggested production of PL in 5 of the 6 cases investigated. The mean age for affected dogs was 10 years. Four of the 6 dogs were anorexic and no dogs had cranial abdominal pain. Serum pancreatic lipase appears to be a useful biochemical marker for pancreatic/hepatic neoplasia in middle-aged to older dogs. (Abstract citation: Vet Clin Pathol
19: IMMUNOLOCALIZATION OF ENDOTHELEN-1 IN END-STAGE KIDNEYS OF DOGS WITH HEREDITARY NEPHROPATHY.
Endothelin-1 (ET-1) is a 21 amino acid peptide with potent vasoconstrictive, proinflammatory, and mitogenic properties. The vasoactive properties of ET-1 have encouraged studies of the pathophysiologic role of this peptide in cardiovascular and renal diseases. We have established a colony of mongrel dogs with X-linked hereditary nephropathy (HN) that is a model of human Alport syndrome. Affected dogs predictably develop juvenile-onset proteinuria and progress to end-stage renal failure by 18 months of age with microscopic renal changes similar to those seen with many chronic progressive renal diseases of diverse etiology. This commonality of end-stage changes has prompted us to investigate these dogs as a model of chronic progressive nephropathy. A commercially-available monoclonal antibody to human ET-1 was used in an avidin-biotin complex immunohistochemical assay. Formalin-fixed, paraffin-embedded sections of kidney from dogs with end-stage HN, age-matched normal dogs, and dogs with etiologically-diverse chronic renal disease were labeled for ET-1. Discrete ET-1 labeling was consistently identified in hypertrophied parietal epithelial cells lining thickened Bowman's capsules, as well as variably within epithelial cells lining cortical and medullary tubules of sections of kidney from HN dogs and dogs with morphologically-similar renal changes without HN. To our knowledge, the in vivo distribution of ET-1 in kidneys with chronic progressive disease has not been previously described. Secretion of ET-1 by parietal epithelial cells might contribute to the pathophysiology of glomerular ischemia, atrophy, and sclerosis associated with the progression to end-stage renal disease.
20: FELINE LEUKEMIA VIRUS-ASSOCIATED MYELOPATHY—NOVEL OR JUST RARE?
Feline Leukemia virus (FeLV) is a retrovirus with a complex life cycle associated with proliferative and degenerative diseases in cats. We have recently identified a neurological syndrome in long-term FeLV-infected cats consisting of abnormal vocalization, hyperesthesia, and paresis that progressed to paralysis. Affected cats were FeLV antigenemic for more than 3 years but did not have tumors or hematological abnormalities. Cats were invariably euthanized due to progressive neurological dysfunction. Necropsy of eight affected cats did not identify gross central nervous system abnormalities; however, microscopically there were profound lesions in the spinal cord and brain stem. Lesions consisting of diffuse white matter degeneration characterized by dilated myelin sheaths and swollen axons were present in the absence of mononuclear cell infiltrates. Immunohistochemical examination revealed expression of FeLV antigens in neurons and glial cells in spinal cords of affected animals. Furthermore, proviral DNA was amplifiable from sections of spinal cord as well as intestine, spleen, and lymph nodes. These findings suggest that in a proportion of chronically FeLV-infected cats, a virus has evolved with an expanded host cell range enabling infection of neurons and glial cells and resulting in axonal degeneration.
21: FATAL NON-NEUROLOGICAL VASCULOTROPIC MULTI-SYSTEMIC EHV-1 INFECTION IN EQUIDAE.
A fatal non-neurological, multisystemic, prominently vasculotropic Equine Herpesvirus 1 (EHV-1) infection was identified in two young adult female horses, one- (case 1) and two-years-old (case 2) respectively, and in an adult zebra stallion (case 3). All three animals had short clinical courses, severe hydrothorax with pulmonary edema, vasculitis with EHV-1 antigen (EHV-1 Ag) within endothelial cells, and circulating monocytes. In case 1 there was vasculitis, hemorrhage and edema in the lungs and dorsal third ventricle choroid plexus, as well as mild intestinal crypt necrosis. EHV-1 antigen was also identified within dendritic-like cells of the pharyngeal lymphoid follicles, pharyngeal glandular epithelium, and crypt enterocytes. In case 2, the vascular involvement was particularly prominent within the liver. In Case 3 there was also multifocal necrotizing rhinitis, with EHV-1 Ag in epithelial cells, endothelial cells and intravascular leucocytes of the nasal mucosa, lung, and reproductive tract with infected Leydig cells and germinal epithelium. EHV-1 infection was also identified with virus isolation PCR and electron microscopy. This novel fatal form of EHV-1 infection always should be considered and included in the possible EHV-1 manifestations.
22: PHYSEAL DYSPLASIA WITH SLIPPED CAPITAL FEMORAL EPIPHYSIS IN 13 CATS.
Separation of the femoral capital epiphysis is associated with severe trauma in most species. This report describes 13 cats with atraumatic slipped capital femoral epiphysis characterized by a distinctive lesion in the physeal cartilage. The lesion consists of irregular clusters of chondrocytes separated by abundant matrix on both the epiphyseal and metaphyseal side of the cleavage site. Abnormalities in the extracellular matrix are demonstrated by histochemical staining. The affected population in this study is 85% male, 90% overweight, 23% Siamese, and 4.5 to 24-months-old. The histopathology and demographics are similar to slipped capital femoral epiphysis in humans which most often affects overweight adolescent boys.
23: FELINE CAPITAL PHYSEAL DYSPLASIA SYNDROME. A.
In recent years, a higher incidence of capital physeal fractures have been noted in cats, unrelated to any major traumatic injury. Nine cats were evaluated clinically and radiographically, and histopathology of the surgically removed femoral head and neck was evaluated. The intent of the present study was to evaluate the histopathologic features of these tissues. The cats ranged from 15 to 27 months-of-age (mean = 21.5 months). All were housed indoors and had no history of trauma. The age at neutering was as follows: 2–3 months (3 cats), 5 months (2 cats), 6 months or older (4 cats). Duration before surgical treatment ranged from 0.5 to 104 days. Histopathologic examination revealed variable features dependent on interval from diagnosis to surgical removal of the detached femoral head. Generally, the proximal femoral growth plate was histologically deranged with no identifiable resting zone and absence of any zonal delineation. Often, there were large clusters of chondrocytes. Frequently, foci of endochondral ossification and fibrosis occurred with chronicity. Normally, capital physeal closure occurs in cats at 210–280 days and precedes the earliest clinical presentation of any of these cats. Gonadectomy is known to delay the physeal closure in cat radii and it may also delay the closure of the femoral capital physis. One hypothesis is that delayed physeal closure may predispose cats to capital physeal fracture with minor trauma.
24: SCRAPIE PRION (PrP-Sc) IS LOCALIZED TO THE UTERINE ENDOMETRIUM AND PLACENTAL CHORION OF EWES INFECTED WITH SCRAPIE.
Scrapie is a prion (PrP) disease causing fatal neurodegenerative disorders in sheep. The primary mechanism of natural disease transmission remains unclear. It is hypothesized that scrapie is transmitted through reproductive processes because scrapie infectivity and the disease-associated PrP (PrP-Sc) are detected in ovine placenta. However, the cellular localization of PrP-Sc in uterus and placenta has not been reported. The objective of the present study was to determine the distribution of PrP-Sc and the presence of PrP-Sc at the fetal-maternal interface with immunohistochemistry (IHC) and Western blotting (WB). The scrapie status of infected ewes (n = 8) used in this study was confirmed by IHC and WB. Results showed that PrP-Sc was located in a patchy distribution within both the fetal placental villous tree and maternal septal tissue of placentomes of 3 of the 8 scrapie-infected ewes. No staining was found in myometrium, uterine glands, allantois, and blood vessels. PK resistant PrP-Sc in dissected caruncles and cotyledons was confirmed with WB. No staining was seen in uterine and placental tissues from 50 uninfected ewes and 5 of the 8 infected ewes. Although the mechanism of PrP-Sc production in the uterine endometrium and placental chorion during pregnancy is presently unclear, the accumulation of PrP-Sc at the fetal-maternal interface may be a critical step in natural transmission of scrapie.
25: PRECLINICAL DIAGNOSIS OF SCRAPIE USING AUTOMATED MONOCLONAL ANTIBODY IMMUNOHISTOCHEMISTRY.
Scrapie of domestic sheep and goats is a fatal neurologic disorder associated with deposition of an abnormal form (PrP-Sc) of a normal host protein (PrP-C). Immunohistochemistry (IHC) assay for PrP-Sc in brain is an approved diagnostic test for scrapie. Because PrP-Sc accumulates in certain lymphoid tissues well before accumulation in the central nervous system, IHC of lymphoid tissue in tonsil and third eyelid has been proposed as a preclinical test for scrapie. Preliminary characterization of a monoclonal antibody IHC assay of these tissues showed concordance between test results and true infection status to be greater than 95%. Optimized conditions for PrP-Sc detection by automated IHC of lymphoid tissue were determined. A cocktail of two monoclonal antibodies (F89/160.1.5 and F99/97.6.1) with antigen retrieval by autoclaving in citrate buffer, formic acid treatment, and mild protease digestion produced sensitive, specific staining of ovine lymphoid tissues suitable for testing sheep of varying genetic backgrounds. Availability of a standard, automated, monoclonal antibody based immunoassay for preclinical antemortem and postmortem testing of transmissable spongiform encephalopathies based on lymphoid tissue immunohistochemistry will facilitate diagnostic assay validation and scrapie control programs by regulatory groups.
26: LYMPHOCYTE RESPONSES AND IMMUNOTYPES IN HORSES WITH NATURAL SARCOCYSTIS NEURONA INFECTION.
Sarcocystis neurona is a protozoan that can cause debilitating neurologic disease in horses. Although the prevalence of serum antibody to S. neurona is relatively high, the incidence of clinical disease is much lower. Individual horse immune responses probably play an important role in clinical outcome of infection. Our objective was to determine whether there were differences in lymphocyte responses and subsets in seropositive asymptomatic horses and seropositive horses with clinical signs of equine protozoal myeloencephalitis (EPM). Peripheral blood mononuclear cells (PBMC) were collected from 15 asymptomatic seropositive horses and 9 seropositive horses with clinical signs of EPM. These cells were assayed for response to non-specific (Con A) and specific S. neurona antigens in lymphocyte blastogenesis assays. Percentages of CD4+T cells, CD8+T cells, B cells, and macrophages were determined by flow cytometry. PBMC from the symptomatic horses had a significantly lower response to non-specific stimulation than those from the asymptomatic group and were less responsive to stimulation with 5. neurona antigen. Asymptomatic horses had a significantly higher mean percentages of circulating CD4+ T lymphocytes than did symptomatic, non-treated horses with no significant differences detected in CD8+, macrophage, or B cell percentages between the 2 groups. The findings suggest that infected horses with neurologic signs may have T cells that are generally less responsive than those that remain asymptomatic. In addition, the finding that asymptomatic horses have significantly higher proportions of CD4+ or T helper cells suggests that increased T helper cell reactivity may be important in controlling S. neurona infection.
27: ENCEPHAUTOZOON CUNICULI PLACENTITIS AND ABORTION IN A QUARTERHORSE MARE.
There are few reports of placentitis due to Encephalitozoon cuniculi infection in any species. A late-term aborted fetus and placenta from a Quarterhorse were examined at the University of Kentucky. Gross placental changes were: thickening with blunted chorionic villi and scattered patches of a yellow viscous material over chorionic surfaces of the body and adjacent areas of the horns. Histopathologic changes in the placenta were; multifocal necrosis and partial loss of chorionic villi; moderate stromal infiltrates of macrophages, granulocytes, lymphocytes and occasional plasma cells within villi and the subjacent placental membrane; and distension of chorionic blood vessels by fibrin thrombi. Chorionic epithelial cells were expanded by 20–30 μm intracytoplasmic vacuoles containing 1–2 μm diameter, cigar-shaped basophilic organisms with eccentric nuclei and gram-positive walls. Ultra-structural features of the organisms included a thick wall, a single eccentric electron-dense nucleus, and bilateral transverse sections through a coiled filament. DNA was isolated from a paraffin block of placenta and a 672 bp band was amplified by PCR, using the DNA as a template and primers to rDNA of the genus Microsporidium. The sequence of this fragment has 97% homology with that of Encephalitozoon cuniculi. Fetal synovial joints were distended by brown semi-opaque fluid and there was a diffuse subacute to chronic proliferative synovitis. This is the first documented case of E. cuniculi placentitis and abortion in a horse.
28: BOVINE ADENOVIRUS ASSOCIATED ENDOMETRITIS IN DAIRY CATTLE.
Five cases of suppurative, ulcerative endometritis in postparturient dairy cows associated with adenoviral endometritis are described. Characteristic amphophilic intranuclear viral inclusion bodies were identified within degenerate endometrial lining epithelium by histopathology from 5 cows from 4 separate dairies. In contrast to typical cases of bovine adenovirus, these cows had no evidence of enteritis or pneumonia, and viral inclusion bodies could not be identified histologically in endothelium (although viral particles were demonstrated in uterine endothelial cells by electron microscopy). Adenoviral infection was confirmed by a combination of fluorescent antibody assays, viral isolation, and ultrastructural examination of the uterus. Isolates from 2 of the cases were found to react with anti-Adenovirus subgroup 2 antibodies but not with any known Adenovirus serotype-specific antibodies. Bovine Adenovirus-associated endometritis has not been previously reported in cattle, but may be an emerging cause of endometritis in dairy herds.
29: FIELD-BASED STUDY OF PULMONARY INJURY IN ACUTE INTERSTITIAL PNEUMONIA OF FED CATTLE. G.
The pathogenesis of acute bovine pulmonary emphysema and edema (fog fever, ABPEE) in pastured cattle includes pulmonary biotransformation of rumen-derived 3-methylindole (3-MI) to reactive species such as 3-methyleneindolenine (3-MEIN). Lesions of acute interstitial pneumonia (AIP) in feedlot cattle are similar to those of pastured cattle with ABPEE, however the mechanism of injury in fed cattle is poorly documented. This study was designed to determine if AIP shares pathogenic mechanisms with ABPEE. Lung and/or blood samples were collected from 186 cattle with suspected respiratory disease from 13 feed yards in the western Great Plains. Blood samples were also collected from normal pen-mates. Lung tissue was classified as AIP, normal, or infectious pneumonia using histopathology and standard pathogen detection methods. Blood and lung 3-MEIN concentrations were measured by ELISA using polyclonal antisera. Cattle with AIP had significantly higher blood and lung 3-MEIN concentrations than pen-mates with infectious respiratory disease or normal lungs. Blood 3-MEIN concentrations were also higher in cattle with AIP versus normal pen-mates. These data add to the growing evidence that feedlot AIP shares mechanistic features with 3-MI induced lung injury known to affect pastured cattle.
30: MALIGNANT SEMINOMA WITH ABDOMINAL DISSEMINATION IN AN ATLANTIC BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS).
In late November 1997, a 283-cm, adult, male Atlantic bottlenose dolphin (Tursiops truncatus) stranded and died on Topsail Island, in North Carolina. At postmortem examination, the animal was extremely thin, with protrusion of the ribs and scapulae and scant subcutaneous fat. On internal examination, a large, up to 30-cm diameter, lobulated, red-gray, and variably friable mass filled the caudal abdomen. The mass infiltrated and expanded the mesentery, entrapped segments of the intestines, and attached to the celomic wall blending with and expanding the cranial pole of the right testis. Multiple (up to 3-cm diameter) masses were attached to the peritoneum, liver capsule and mesentery. Histologic examination of the masses revealed sheets and rare packets of neoplastic cells supported by a fine to moderately coarse fibrous stroma. The neoplastic cells were round to polygonal with distinct cell borders and variable amounts of granular eosinophilic cytoplasm. Nuclei were round to oval, with finely stippled, occasionally coarsely clumped chromatin and 1 to 3, variably-sized and shaped magenta nucleoli. Anisokaryosis was moderate. The mitotic rate was high, with up to 3 per high-power-field. Necrosis was extensive. There are few reports of neoplasia in cetaceans and even fewer of malignancy. To our knowledge, this is the first report of a malignant seminoma in an Atlantic bottlenose dolphin.
31: THE UTILITY OF IMMUNOHISTOCHEMISTRY AS A TOOL FOR THE DIAGNOSIS AND DIFFERENTIATION OF MARINE MAMMAL MORBILLIVIRUSES.
In recent decades, morbilliviruses have been responsible for major epizootics in marine mammals. Recognition of disease due to morbilliviruses can be straightforward when based on histopathology; however, determination of the exact virus based on routine histopathologic findings alone is problematic. Often carcasses are too autolyzed for virus isolation to be rewarding, and it is known that many of these viruses can infect species other than the target species. Given these shortcomings, the development of specific immunohistochemical tests for the differentiation of morbilliviruses infecting marine mammals would provide diagnosticians with a distinct advantage. Four monoclonal antibodies made against phocine distemper virus (PDV) and three monoclonal antibodies against cetacean morbillivirus (CMV) were tested on formalin-fixed, paraffin embedded tissues from confirmed cases of PDV, CMV, and canine distemper virus (CDV). Of the four PDV monoclonal antibodies, all stained both PDV and CDV but not CMV. Of the three CMV antibodies, two were positive for CMV and negative for CDV and PDV. One CMV antibody was positive for CMV and CDV and negative for PDV. Application of these antibodies to histopathologic samples having lesions consistent with morbillivirus infection may provide a diagnostic means for distinguishing between infections of PDV and CMV.
32: CORONAVIRUS ASSOCIATEP EPIZOOTIC CATARRHAL ENTERITIS (ECE) OF FERRETS.
Since 1993, epizootics of a green mucous diarrhea have caused significant morbidity and variable mortality in ferret breeding colonies and rescue operations throughout the U.S. and Canada. Necropsy material from naturally infected ferrets from a wide range of sources was examined in order to identify an etiologic agent as well as to elucidate lesions associated with this disease. Characteristic microscopic lesions consistent with coronavirus infection in other species were consistently present in affected ferrets and included vacuolar degeneration and necrosis of villar enterocytes and goblet cell hyperplasia in acute cases, and villar atrophy, fusion, and blunting, and varying degrees of lymphocytic enteritis in more longstanding infections. Coronavirus particles were identified by transmission electron microscopy in both feces and jejunal enterocytes from affected animals. Immunohistochemical staining of jejunal sections from affected ferrets were positive for coronaviral antigen. Antigen staining was absent in clinically normal ferrets and in ferrets with other gastrointestinal syndromes. The pathogenesis and clinical progression of this condition in ferrets mirrors corona-viral enteritides in other species. The findings presented here strongly support a coronavirus as the etiologic agent of ECE.
33: AN EPIZOOTIC OF GASTRITIS ASSOCIATED WITH HELICOBACTER-LIKE ORGANISMS IN BABOONS.
Several species of the genus Helicobacter have been associated with subclinical and clinical gastritis in humans and animals. H. pylori and H. heilmannii-like organisms are the species most commonly identified in the gastric mucosa of nonhuman primates and humans. We report a high prevalence of subclinical gastritis in baboons (Papio spp.) from a toxicity study in a research facility. The lesions were similar in xenobiotic-treated and control animals, suggesting a spontaneous rather than chemical-induced disease. Histologic examination of the antral mucosa revealed a lymphoplasmacytic gastritis with minor neutrophilic infiltrates within lumens of occasional gastric glands. Numerous argyrophilic bacteria morphologically resembling H. pylori were present in antral gastric pits and glands. The fundic mucosa contained minor scattered aggregates of lymphocytes and plasma cells. Numerous argyrophilic bacteria morphologically consistent with H. heilmannii-like organisms were present in fundic gastric pits and glands. Further characterisation of both organisms is under way, using transmission electron microscopy and sequence analysis of 16S rRNA and urease genes following PCR amplification. This is the first report of gastritis associated with Helicobacter-like organisms in baboons. This infection has the potential to complicate toxicity studies and other experiments using baboons.
34: INTRAABDOMINAL PSEUDOMYCETOMA IN A PERSIAN CAT.
Mycetoma is an infectious, noncontagious disease caused by free-living bacteria (actinomycetoma) or fungi (eumycetoma) that are traumatically implanted into the body. A similar lesion may rarely be caused by dermatophytes and is called “pseudomycetoma”. Dermal dermatophytic pseudomycetomas in Persian cats occur as raised cutaneous nodules that are typically on the back or base of the tail. A 7-yr-old spayed female Persian cat was presented for chronic weight loss. Previous medical problems included dermatophytosis, vaginal prolapse, and ovariohysterectomy during the first 6 months of life. An intraabdominal mass was identified by palpation. Laparatomy was performed, and masses were excised from the omentum. The masses were formalin-fixed, routinely processed for histopathology, and examined microscopically. The masses consisted of pyogranulomatous inflammation and fibrosis with grain-like clusters of nonpigmented hyphae suggestive of white grain eumycetoma; however, the fungus were determined to be a dermatophyte by immunohistochemical staining of paraffin tissue sections. This is the first report of intraabdominal pseudomycetoma in a cat. The infection most likely was established 6.5 years previously during surgical entry into the abdomen at the time of ovariohysterectomy.
35: PCR-BASED CLONALITY ANALYSIS OF CANINE B- AND T-CELL LYMPHOSARCOMA USING PARAFFIN-EMBEDDED TISSUES.
PCR-based clonality analysis of lymphocyte populations is an emerging diagnostic test to aid in the diagnosis of B- and T-cell lymphosarcoma (LSA) in the dog. The diagnosis of LSA is usually uncomplicated, however for a minority of cases, LSA is difficult to distinguish from benign hyperplasia. Since the vast majority of veterinary biopsy submissions are fixed in neutral buffered formalin, it would be beneficial to have a molecular diagnostic test for LSA that could be applied to paraffin-embedded, formalin fixed tissue (PET). Our objective was to determine the reliability of a PCR-based test for lymphocyte monoclonality to aid in the diagnosis of B- and T-cell LSA in dogs using PET. Clonality was determined by PCR-amplifying unique gene rearrangements of the immunoglobulin gene for B lymphocytes and T cell receptor gene gamma for T lymphocytes. Genomic DNA served as the PCR template and was extracted from biopsy specimens of 31 dogs with previously confirmed LSA (20 B-cell and 11 T-cell) and from 18 norma] dog lymph nodes. We found the assay was sensivitive and specific for detecting monoclonal lymphocyte populations in B-cell LSA [85%,17/20] and T-cell LSA [64%,7/11]. As expected, none (0/18) of the normal lymph nodes had PCR evidence of monoclonal populations of T- or B-cell lymphocytes. The PCR clonality assay appears to be a useful ancillary test to aid in the diagnosis and lineage assignment of canine LSA using PET.
36: FELINE PRIMARY OCULAR MALIGNANT LYMPHOMA: IMMUNOPHENOTYPING OF LEUKOCYTES, FELV STATUS AND RELATIONSHIP TO IDIOPATHIC LYMPHOCYTIC UVEITIS. H.
This study was conducted to examine the natural history of primary feline ocular lymphoma. Sixty-one cases were retrieved from submissions between 1987–1999. Age, sex, laterality and tumor extension, serologic history, and survival was determined. Fifty-seven tumors were examined for tumor cell phenotype, FeLV antigen and genome. Males outnumbered females (38/20); mean age was 10.8 years and no breed predilection existed. 25 cats had lymphoma alone; 36 had concurrent uveitis (10 trauma-associated). Occurrence was slightly greater OD (28/23). 19% were FeLV (+) and 33% FIV (+). Cats having malignant lymphoma with uveitis expressed CD11/CD18, TCR/CD3, BCR/CD79 or no markers in 73%, 41%, 18% and 14%, respectively. Evidence of FeLV occurred in 36% of these cases. Tumor distribution was nodular anterior uveal in 57%. In cats with only malignant lymphoma, 72% were diffuse anterior uveal in distribution and tumor antigen expression was 56% BCR/CD79, 40% CD11/CD18, 32% none, and 20% TCR/CD3. FeLV was present in only 16%. In tumors from 10 cats with traumatic uveitis: 20% exhibited FeLV infection, 60% exhibited no antigen expression, 20% BCR/CD79 and 20% CD11/CD18 expression. 80% of tumors were not anterior uveal. Cats with lymphoma and uveitis have an average survival of 110 days and 4 cats are still alive. Cats with pure lymphoma survive a mean of 30 days; six are still alive. FeLV is infrequently a factor in development of neoplasia. There are differences in tumor distribution, cellular phenotype, and survival between cats with lymphoma with or without concurrent uveitis.
37: IMMUNOHISTOCHEMICAL CHARACTERIZATION OF CANINE ORAL MELANOMAS WITH MELANOCYTIC DIFFERENTIATION ANTIGEN MELAN A.
The diagnosis of oral melanomas may be challenging because of variation in the degree of pigmentation and microscopic resemblance to carcinomas, sarcomas or round cell tumors. Immunohistochemistry for a melanocytic differentiation antigen, Melan A, was done on 129 formalin-fixed, paraffin-embedded canine oral and metastatic melanomas and 165 non-melanocytic tumors. Heat-induced epitope retrieval by steaming the slides in EDTA buffer (pH 8.0) was necessary for optimal results. Melan A was detected in 113/122 oral (92.6%) and 5/7 (71.9%) metastatic melanomas. Only 4/163 nonmelanocytic tumors (2/8 salivary carcinomas, and 2/10 transitional cell carcinomas) were focally and weakly positive for Melan A. The stain was cytoplasmic, and diffuse or punctate. All morphologic types of melanomas were positive for Melan A. Other markers tested (vimentin, S100 protein and neuron-specific enolase) stained 129 (100%), 98 (76%), and 115 (89.1%) of 129 melanomas, respectively but their specificity was much lower than that of Melan A. The metastases of 6/6 oral melanomas had immunohistochemical features similar to those of the primary tumors. It is concluded that Melan A is a highly specific and sensitive marker of canine melanotic or amelanotic oral melanomas in routinely processed tissues.
38: FELINE CUTANEOUS FIBROPAPILLOMAS (SARCOID): CLINICOPATHOLOGIC FINDINGS AND ASSOCIATION WITH PAPILLOMAVIRUS INFECTION.
Twenty-three feline cutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by dermal fibroblastic proliferation with overlying, often ulcerated hyperplastic epidermis. Electron microscopy supported the fibroblastic nature of the neoplastic cells. The 23 tumors came from 20 cats and were submitted from veterinary clinics in Wisconsin and Minnesota. The tumors occurred most commonly in young cats and were found primarily on the head, neck, and digits. Fifteen of the 17 cats for which breed was reported were domestic shorthair cats. In 11/20 cases, there was known exposure to cattle. Local recurrence of the tumor following surgical excision was reported in 7 of the 18 cats for which follow up information was available. Metastasis was not documented in any of the cases. Two of the 19 tumors tested by polymerase chain reaction (PCR) had no amplifable DNA. The remaining 17 were positive for papillomavirus by PCR. No papillomavirus DNA was detected in 3 other feline skin tumors (cutaneous mast cell tumor, lymphoma, and fibrosarcoma) that served as controls. This is the first report of detection of papillomavirus in feline tumors that have clinicopathologic features similar to equine sarcoid.
39: VACCINE-ASSOCIATED FELINE SARCOMAS: INVESTIGATION OF POTENTIAL VIRAL ETIOLOGIES.
The pathogenesis of vaccine-associated feline sarcomas (VAFS) remains obscure. The low prevalence of VAFS suggests that congenital or acquired genetic factors within individual susceptible cats may act as initiators of oncogenesis. The purpose of our study was to investigate potential viral candidates in the etiopathogenesis of VAFS. Retroviruses investigated included feline immunodeficiency virus (FIV), feline foamy virus (FeFV) and endogenous feline leukemia virus (enFeLV). Vaccination could increase replication and/or expression of latent retroviruses or endogenous retroviruses within proliferating fibroblasts and/or inflammatory cells. Malignant transformation could then result from insertional activation or inactivation of cell cycle regulatory genes. DNA viruses evaluated included papillomavirus, herpesvirus and polyomavirus. Members of these three viral families have oncogenic potential and could be introduced either as vaccine contaminants or as skin contaminants transported to the subcutis by vaccination. Also, non-pathogenic or latent viruses pre-existing in host tissues could become oncogenic within the local milieu created by vaccination. Fifty formalin-fixed, paraffin-embedded VAFS were negative for the five exogenous viruses using polymerase chain reaction (PCR) and/or immunohistochemistry. The level of enFeLV RNA in VAFS was not significantly different compared to that in non-vaccine site associated feline fibrosarcomas using semi-quantitative reverse transcriptase PCR. The retroviruses FIV, FeFV and enFeLV, and the DNA viruses papillomavirus, polyomavirus and herpesvirus do not appear to be directly involved in the etiopathogenesis of VAFS.
40: HISTOPATHOLOGY AND BIOLOGICAL BEHAVIOR OF SUBCUTANEOUS ANGIOLIPOMAS IN DOGS AND A CAT.
Variants of lipoma are uncommon. Fibrolipoma and infiltrative lipoma have been reported. This presentation describes several cases of angiolipoma in dogs and a cat and a single case of infiltrative angiolipoma in a dog. The unencapsulated masses are composed of mature adipose tissue mixed with variable numbers of blood vessels. The infiltrative angiolipoma extended into and disrupted bundles of striated muscle with degeneration and necrosis of myofibers. The tumors were solitary, located on the thorax and occurred in adult animals. A recent patient survey revealed no history of recurrence or additional tumors in patients with pure angiolipomas. A subcutaneous mass in the general area and a dermal mass at a distant site have been found in the patient with the infiltrating angiolipoma. The patient is in good health three years postoperatively and there are no plans to biopsy the lesions.
41: NITRIC OXIDE ISOFORMS IN GASTRIC DILATATION-VOLVULUS SYNDROME: IMMUNOHISTOCHEMICAL LOCALIZATION AND DIFFERENTIAL EXPRESSION IN CANINE PYLORUS AND DUODENUM.
Necropsy specimens of gastric pylorus and duodenum from a military working dog (MWD) having died from complications associated with GDV, a MWD having undergone surgical correction of GDV (gastropexy) and having died of unrelated causes, a MWD without clinical history of GDV, a civilian-owned at-risk breed dog, and a dog breed not considered to be at-risk for GDV were evaluated immunohistochemically for expression of nNOS and iNOS and for presence of fibrinogen. Slides were evaluated for distribution of immunoreactivity including cell type and number and for intensity of reaction. Distribution and intensity of reactivity for each antibody were compared between dogs. Anti-iNOS and anti-fibrinogen immunoreactivity were compared for each dog. Gastrointestinal tissues from the MWDs and the at-risk breed dog, regardless of GDV status, had reduced nNOS expression compared to those from the non-at-risk breed dog. Fibrinogen and iNOS immunoreactivity were greatest in the MWD that died from GDV. Expression of nNOS appears to be reduced in dog breeds considered to be at-risk for GDV. Expression of iNOS appears to be elevated in gastrointestinal tissues in fatal GDV and concurs with the release of the acute phase protein, fibrinogen.
42: FELINE INFECTIOUS PERITONITIS: REPORT OF THE FIRST NATURAL CASE IN KOREA.
An 8-month-old, domestic shorthaired cat exhibiting lacrimation, uveitis, anorexia and coughing was examined. Gross lesions were characterized by ascites, grayish-white membrane adhesion on the serosa of visceral organs, grayish-white foci in liver and kidney. Histopathologically characteristic lesions were fibrinous serositis, multifocal granuloma and necrosis, vasculitis, and perivasculitis in various organs. FIPV antigens were detected in the cytoplasm of the infiltrating macrophages of the granuloma by immunohistochemistry. The purpose of this report is to describe the lesions of the first FIP case in Korea.
43: IMMUNOPHENOTYPING OF PRIMARY CENTRAL NERVOUS SYSTEM (CNS) NEOPLASMS IN THE DOG.
This study determined immunohistochemical staining patterns for primary canine CNS neoplasms. Formalin-fixed, paraffin-embedded tissue from 27 tumors was obtained. The selected tumor groups included: 7 astrocytomas, 3 oligodendrogliomas, 2 choroid plexus papillomas (CPP) and 15 meningiomas. Tumors were evaluated for reactivity to 6 immunohistochemical markers: vimentin, pancytokeratin, glial fibrillary acidic protein (GFAP), S-100, neuron-specific enolase (NSE), and synaptophysin. The sections were stained using a DAB-horseradish peroxidase technique. Vimentin expression was detected in 15/15 meningiomas, 6/7 astrocytomas, and 1/3 oligodendrogliomas. Meningiomas (11/15) and CPP (2/3) expressed pancytokeratin. GFAP was positive in 7/7 astrocytomas and 1/15 meningiomas. S-100 produced variable results. NSE was positive in 2/2 CPP and variable in other tumors. Synaptophysin was detected in 2/3 oligodendrogliomas. Specific trends for several tumor types were identified. Astrocytomas consistently expressed GFAP and vimentin but failed to express pancytokeratin. All meningiomas expressed vimentin, and dual positivity for pancytokeratin was common. One meningioma had focal GFAP positivity. CPP were negative for vimentin and positive for pancytokeratin and NSE. Synaptophysin expression in 2/3 oligodendrogliomas was unexpected, and no consistent patterns were identified for these tumors. These results will help to establish immunohistochemical profiles that improve our ability to obtain a correct diagnosis for CNS neoplasms.
44: MULTIPLE ENDOCRINE NEOPLASIA IN A RHESUS MONKEY (MACACA MULATTA).
A 36-year-old, male, rhesus monkey was euthanized at the end of a research study evaluating the chronic effects of space radiation. This animal had been exposed to a one-time, whole body dose of 2300 MeV proton radiation at approximately age 3, and was allowed to live out the remainder of its life without further manipulation other than routine health checks. At necropsy, a large pituitary mass was noted to have destroyed the sella turcica with suprasellar extension into the brain through the area of the interthalamic adhesion and extending into the lateral ventricle. Histologically, the pituitary adenoma was unencapsulated, well-demarcated and expansile, and composed of polygonal cells arranged in small nests and packets. The neoplastic cells were multifocally immunohistochemically positive for ACTH, but not for LH, GH, FSH, TSH, and prolactin. Two other neoplasms, a thyroid C-cell adenoma and a pancreatic islet cell tumor were found during the microscopic examination. The C-cell tumor was well-circumscribed, encapsulated, and expansile, and composed of polygonal to columnar cells arranged in cords and packets; the neoplastic cells were positive for calcitonin. The islet cell tumor was fairly well-circumscribed, expansile, and composed of polygonal cells arranged in small packets and cords; the neoplastic cells were diffusely positive for glucagon, and multifocally positive for insulin. To our knowledge, mis is the first report of an invasive pituitary adenoma and of multiple endocrine neoplasia in a rhesus monkey.
45: PATHOLOGY OF C57BL/6-Cybb tm1 MICE—A MOUSE MODEL OF HUMAN X-LINKED CHRONIC GRANULOMATOUS DISEASE.
A colony of knock out mice gene designation C57BL/6-Cybb tm1 (subunit of NADPH oxidase) has been maintained at this institution for 5 years. These mice have been shown to be susceptible to experimental infection with Aspergillus fumigatus but there is no documentation of naturally occurring diseases. Lesions were documented in 44 mice that were submitted to necropsy. All mice had an acidophilic macrophage pneumonia. In addition, 16 had lobar suppurative and necrotizing pneumonias caused by Paecilomyces sp. (11), Aspergillus fumigatus (3), Rhizopus sp. (1) and Candida guilliermondii (1). 21 mice had severe bacterial suppurative and necrotizing to pyogranulomatous pneumonias. Bacteria cultured from lung abscesses included Pseudomonas aeruginosa (2), Enterococcus (6), coagulase negative staph (3), gram negative enteric bacilli (4), Klebsiella pneumoniae (1) and Proteus mirabilis (2). Seven mice had a necrotizing and suppurative adenitis of the cervical lymph nodes caused by coagulase negative staph and Staphylococcus equorum was recovered from abscesses on the head and extremities of 4 mice. The pneumonias were the leading cause of death along with the cutaneous abscesses involving the cervical lymph nodes, head and extremities. Splenomegaly was found in 25 and lymphadenopathy in 10 mice. The array of spontaneously occurring lesions and infectious agents in these mice is similar to that occurring in human chronic granulomatous disease patients.
46: A CONCISE SURVEY OF NEOPLASIA IN DOMESTIC CATS BY TUMOR TYPE AND BODY LOCATION.
The incidence of disease within a species, infectious or otherwise, may vary over time. The purpose of this study was to provide a contemporary assessment of the incidence of neoplasia in domestic cats (Felis domesticus) by tumor type and body/ organ location for use by clinicians and diagnostic pathologists, and for comparison with data cited in previous veterinary literature. 728 cases of feline neoplasia were identified in records of the diagnostic pathology service at UTCVM for a period spanning 1995–98. The most frequently diagnosed tumors were sarcomas* at vaccination sites (16.6% of all cases), squamous cell carcinomas (13.9%), and lymphosarcoma (9.6%), followed by mast cell tumors (7.7%), basal cell tumors of haired skin (6.9%), and mammary tumors (5.2%). Not surprisingly, subcutaneous sarcomas at vaccination sites were the most common neoplasm of haired skin/subcutaneous tissue, and comprised over one-third (36.1%) of all tumors at this location. With the exception of basal cell tumors and mast cell tumors, most of which are benign in cats, the majority of the most commonly encountered tumors of domestic cats were histologically aggressive/malignant. (*Fibrosarcoma, myxofibrosarcoma, malignant fibrous histiocytoma, giant cell sarcoma, and poorly-differentiated sarcoma.)
47: CHARACTERIZATION OF THE INFLAMMATORY INFILTRATES IN CANINE CHRONIC ACTIVE HEPATITIS. J.
Canine chronic active hepatitis (CCAH) is a progressive inflammatory disease of unknown cause. In order to characterize the inflammatory infiltrates in CCAH, formalin-fixed, paraffin-embedded liver tissue from 15 cases were reviewed and classified into 3 groups based on the degree of fibrosis. The inflammatory infiltrate of each liver section was immunohistochemically characterized and semi-quantitatively evaluated. Preliminary results indicate that the number of CD3+ lymphocytes was inversely proportional to the degree of fibrosis. Apoptotic hepatocytes were occasionally surrounded by CD3+ lymphocytes. No correlation was present between the number of lysozyme-positive cells (Kupffer cells) and the degree of fibrosis. Number of alpha-smooth muscle actin-positive cells (myofibroblasts) was positively correlated to the degree of fibrosis observed with Mas-son's trichrome stain. Neutrophils appeared to be numerous when acute necrosis was observed. These results suggest that CD3+ lymphocytes are involved in the early phase of CCAH.
48: ACUTE RESPIRATORY DISTRESS SYNDROME IN A NEONATAL FOAL.
A 24-hour-old male Quarter horse was presented as an emergency because of progressive weakness and difficulty nursing since birth. The foal had received colostrum via nasogastric tube and also penicillin and tetanus antitoxin IM. Passive transfer was adequate. At physical exam, the signs included injected mucous membranes, tachycardia (132 beats/minute), dyspnea and tachypnea (90 breaths/minute), and harsh, loud lung sounds. CBC showed mild leukopenia with left shift. Clinical chemistry showed azotemia, hyperglycemia, acidemia, hypercapnia, hypoxemia, and low O2 saturation. On radiographs, lungs had a marked alveolar pattern and air bronchograms consistent with acute severe pneumonia or neonatal respiratory distress syndrome (NRDS). The owners elected euthanasia due to a poor prognosis. Grossly, the lungs were diffusely edematous and failed to collapse. Microscopically, the lungs had markedly diffuse interstitial and alveolar edema with prominent homogeneous eosinophilic material especially in the bronchioles and terminal airways typical of hyaline membranes. Some alveolar macrophages had foamy cytoplasm. There was scattered alveolar necrosis. Immunohistochemistry (IHC) for inducible nitric oxide synthase was positive but not different from normal equine lung. IHC for endothelin-1 was diffusely positive and much more prominent than normal equine lungs or those with chronic lung disease processes. We conclude that RDS in this foal is associated with increased expression of endothelin-1 in pulmonary.
49: PNEUMONIA OF CAPTIVE, WILD-CAUGHT PRONGHORN ANTELOPE.
A group of Pronghorn Antelope, Antilocapra americana, were captured in Wyoming and transported to Texas for a Brucella vaccine trial. Animals were vaccinated as they were released into a secluded, screened paddock. Animals began to die immediately with an acute necrotizing pneumonia. Bacterial isolates were primarily coliforms (mostly E. coli) and Corynebacterium spp. Histologic lesions consisted of necrosis, hemorrhage and limited exudate in alveoli and airways and fibrinous pleuritis. By day 28, 39 pronghorns had died with pneumonia. Lesions were always acute without fibrosis or abscesses. Aspiration pneumonia was a primary differential, and supplementation with trace minerals including selenium was instituted. Only three more antelope died with pneumonia in the next 10 days. At that time, although the pneumonia problem ceased, animals began to succumb to necrobacillosis. This outbreak was not typical of shipping fever of domestic ungulates; rather, it was the product of environmental changes, crowding, and increased susceptibility of these wild ungulates to opportunistic pathogens. Similar pneumonia outbreaks have been reported in other wild-caught, captive ungulates, and this disease problem poses a serious management problem in situations where wild ungulates may be crowded for study or by urban encroachment. The prophylactic role of mineral supplementation needs to be studied.
50: ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL EVALUATION OF A MUCINOUS OLIGODENDROGLIOMA FROM A DOG.
Ultrastructural characteristics of canine oligodendrogliomas have not been described and their immunohistochemical characteristics are only rudimentarily defined. Some canine oligodendrogliomas have accumulations of mucinous material of unknown origin that can be of help in making the diagnosis. Our objectives were to describe the ultrastructural characteristics of a canine oligodendroglioma, to elucidate the origin of the mucinous material found in some canine oligodendrogliomas, and to expand the immunohistochemical database for oligodendrogliomas. We examined a well-differentiated mucinous oligodendroglioma located in the left cerebral hemisphere of a Labrador Retriever by transmission electron microscopy and for a variety of antigens. Ultrastructurally, the cells had round to oval nuclei and moderate amounts of cytoplasm with moderate numbers of mitochondria. Occasional concentric laminations surrounded attenuated cytoplasmic extensions. The mucinous material appeared extracellular and electron-lucent and clear. Some tumor cells contained intracytoplasmic vacuoles, possibly the origin of the mucinous material. Tumor cells were positive for synaptophysin and vimentin, and negative for S-100, cytokeratin, neurofilaments and GFAP. These ultrastructural and immunohistochemical findings, while probably characteristic of an oligodendroglioma, are relatively non-specific and might not lead to a definitive diagnosis. In a recent pilot study, one author (KPC) has noted that some canine oligodendrogliomas, like some human oligodendrogliomas, stain positive for galactocerebroside.
51: SEVERE COMBINED IMMUNODEFICIENCY IN JAPANESE BLACK BEEF CATTLE.
Severe combined immunodeficiency (SCID) was diagnosed in 6 Japanese black calves (5 male, 1 female). Affected calves became ill from 10 to 40 days-of-age. All calves had diarrhea, increased pulmonary rate, and mild inspiratory dyspnea. All affected calves became progressively worse over a period of 2–4 weeks. Total protein concentration was 3.0–4.0 g/dl. The calves had lymphopenia and extremely low concentrations of serum IgG, IgM, and IgA. At necropsy, lymph nodes, thymus, and Peyer's patches were appreciably smaller than normal. The spleen appeared to be small and splenic follicles were not visible. Disseminated pulmonary atelectasis and diffuse hemorrhagic rumenitis and abomasitis were present. Microscopically, there was depletion of lymphocytes in the cortex and medulla of the thymus with little or no corticomedullary differentiation. Hassall's corpuscles were usually present but absent in some cases. In the spleen and lymph nodes, lymphoid follicles were extremely small or absent and devoid of germinal centers. Peyer's patches were deficient in lymphocytes and lacked follicles. In the lung, multifocal granulomas containing fungi were present. Diffuse hemorrhagic necrosis with hyphal proliferation and thrombosis were marked in the mucosa and submucosa of the forestomach and abomasum. The SCID syndrome in Japanese Black calves is very similar to that in humans and Arabian horses.
52: USEFULNESS OF IMMUNOHISTOCHEMISTRY IN THE DIAGNOSIS OF PROVENTRICULAR DILATATION DISEASE.
Proventricular dilatation disease (PDD) is a devastating disease of psittacine birds that is characterized by mononuclear cell infiltrations in ganglia and nerves of the autonomic nervous system in the gastrointestinal tract, heart, lung, and adrenal. Although suspected to have a viral cause, the agent has not been identified. Currently, antemortem diagnosis is based on finding typical inflammation in ganglia and nerves in crop biopsy samples. Unfortunately, this method can be challenging due to minimal inflammation in some cases and difficulty in identifying nerves in small, contracted crop samples. The objective of this study was to determine if immunohistochemistry could be used to help identify inflammatory cells in ganglia and nerves. We examined crop biopsies from birds with and without PDD with markers for B (BLA.36) and T (CD3) lymphocytes, cytokeratin, and S-100. S-100, but not cytokeratin, was useful for identifying ganglia and nerves. Both BLA.36 and CD3 positive cells were demonstrated in ganglia and nerves of most affected birds. Both affected and unaffected birds had BLA.36 and CD3 positive lymphocytes in the submucosa, perivascular spaces and tunica muscularis, but birds with PDD had greater numbers in these locations. We concluded that immunohistochemistry for S-100 and lymphocytes is sometimes a helpful ancilliary test for diagnosing PDD from the crop.
53: OMENTAL MESOTHELIOMA WITH PULMONARY METASTASIS IN A CAT.
An eight-year-old male castrated domestic shorthaired cat presented with respiratory distress. At necropsy, the lungs were dull red, wet, and rubbery. The omentum contained numerous focal to coalescing, 2–4 mm diameter, cauliflower-like, red to tan, friable, glistening, nodules diffusely throughout the serosal surface. Histologic examination of the omentum revealed a densely cellular monomorphic population of round to cuboidal cells arranged in papillary fronds, separated by adipose tissue and resting on a thin fibrovascular core. The cells contained a moderate to abundant amount of eosinophilic cytoplasm with plump round basophilic, frequently basally located nuclei and prominent nucleoli. Histologic examination of the lung revealed a diffuse neoplastic cellular infiltration with obliteration of the normal pulmonary parenchyma and marked congestion. This cellular population was morphologically similar to the neoplastic cells described in the omentum. Neoplastic cells in the omentum and lungs stained positive for cytokeratin and vimentin. Mesotheliomas are rare tumors which arise from the serosa. They are considered to be malignant; however, they disseminate by implantation rather than metastasis. No reported cases of abdominal mesothelioma with pulmonary metastasis in cats are known. Co-expression of vimentin and cytokeratin in neoplastic mesothelial cells has been reported in the human literature.
54: IMMUNOHISTOCHEMISTRY OF BENIGN CANINE CUTANEOUS VASCULAR LESIONS.
Considerable controversy exists in the classification of benign canine vascular lesions. It is often difficult to separate hemangiomas from congenital vascular malformations such as hamartomas. A 1.5-year-old Siberian husky presented with a poorly circumscribed mass that extended from the base of the left ear to the thoracic inlet. Histologic features were consistent with a vascular hamartoma, with numerous markedly dilated well differentiated vessels lined by flattened endothelium in the dermis, subcutis and in the sternomastoideus, cleidomastoideus and platysma muscles. Vessels ranged from capillary-sized to 1.5 mm diameter, and in the dermis and subcutis were surrounded by loose connective tissue. Many of the vessel walls contained one to several layers of smooth muscle cells. The concept has arisen in the veterinary literature that hamartomas contain smooth muscle in the walls of vessels, whereas hemangiomas lack smooth muscle. In addition to the hamartoma, six cavernous hemangiomas and one capillary hemangioma from dogs ranging in age from 5 to 15 years old were stained by alpha-smooth muscle actin (SMA). The hamartoma and all seven hemangiomas stained strongly positive for smooth muscle actin. Results from this study indicate that hemangiomas contain smooth muscle within the lining stroma of vascular channels and that immunohistochemistry for SMA cannot reliably distinguish hemangiomas from hamartomas or other congenital vascular malformations.
55: MALIGNANT RHABDOID TUMOR IN A FERRET (MUSTELA PUTORIUS FURO).
A 6-month-old female ferret (Mustela putorius furo) that presented with lethargy, inappetance, pyrexia, and poor condition had a rapidly growing infiltrative mass in the medial proximal region of the right quadriceps muscle. Histologically, the mass was composed of round cells with a moderate amount of cytoplasm and often a distinct eosinophilic cytoplasmic droplet. Nuclei were central to eccentric, irregularly oval to reniform and vesicular with marginated chromatin. There were multifocal binucleate and trinucleate cells, and moderate anisocytosis and anisokaryosis. Mitoses averaged 3 per 10 high powered fields. The neoplastic cells induced a scirrhous reaction. Immunohistochemically, the neoplastic cells were diffusely and strongly positive for vimentin and rare cells were strongly positive for keratin. They were negative for desmin, muscle-specific actin, smooth muscle actin, S100 protein, epithelial membrane antigen, glial fibrillary acidic protein, neurofilament protein, and myoglobin. Malignant rhabdoid tumor has not been previously reported in the ferret.
56: STUDIES ON THE PATHOLOGY OF THE EQUINE PROXIMAL SESAMOID BONE.
Fracture of the proximal sesamoid bones (PSB) is the most common fracture in catastrophic musculoskeletal injury in racehorses. In a study of Colorado racehorses, 9 PSB fractures were found in 49 cases. In addition, 3 cases of distal sesamoid ligament rupture and/or avulsion from the insertion site on the proximal first phalanx (P1) were found. The cause of sesamoid bone fracture is unclear but mechanical factors are involved. To see if microdamage accumulation plays a role, morphometric studies were performed on 2-year-old horses with and without 6 month treadmill exercise. Subchondral bone of exercised horses had trends to increased bone density associated with increased fluochrome labeling of bone formation. On preliminary evaluation, there also was increased density of matrix microcracks in the distal subchondral zone. These results suggest increased bone formation in response to exercise is associated with increased microdamage and may play a role in PSB failure. In the 3 cases with distal sesamoid ligament avulsion, erosion or increased porosity of bone at the P1 insertion site was found on both the affected and contralateral sides in the 2 cases for which samples were available. Similar lesions were also seen bilaterally in 3 of the sesamoid bone fracture cases but only unilaterally in 1 of 3 age and sex matched controls. This may indicate that loosening of the sesamoid apparatus is a factor in fracture of the PSB.
57: DISCRIMINATION BETWEEN VACCINE STRAINS AND KOREAN FIELD ISOLATE OF CLASSICAL SWINE FEVER VIRUS (CSFV) BY RESTRICTION ENDONUCLEASE CLEAVAGE OF RT-PCR AMPLICONS AND DETECTION OF THE VIRUS BY IN SITU HYBRIDIZATION.
The aim of this study was to develop practical techniques for discrimination between vaccine strains and field isolates of CSFV in Korea. Considering the genetic differences between vaccine and field isolates, a technique was conducted, which is based on the amplification of the 5’ non-coding region of CSFV using reverse transcriptase-polymerase chain reaction (RT-PCR), followed by Xho I cleavage of the amplified DNA fragments. In situ hybridization (ISH) was performed to detect CSFV in paraffin-embedded tissue sections using a homologous probe made of the purified PCR product labeled with biotin. Five vaccine strains, originating from the same Korean K-LOM strain and a field isolate, which was recovered from pigs with typical CSF signs, were tested. The results showed that the expected amplified size of 312 bp was observed in all CSFV strains. The product of the field isolates was cut into 226 and 87 bp with Xho I, while vaccine strains were not. The CSFV positive signals were detected by ISH in tissue sections of infected pigs but not of vaccinated or normal pigs. The data indicate that endonuclease cleavage of RT-PCR amplicons is useful to discriminate between vaccine and field isolates of CSFV and ISH is a fast and sensitive assay to detect CSFV infection.
58: EXPRESSION OF CYCLOOXYGENASE-2 IN CANINE SQUAMOUS CELL CARCINOMAS.
Squamous cell carcinoma is one of the most common neoplasms encountered in dogs. It is a malignant tumor that can arise from the epidermis or from a mucosal surface, and presents a wide variety of clinical forms. Cyclooxygenase-2 (COX-2) is upregulated in human squamous cell carcinomas, and is believed to play a role in its oncogenesis. However, the implication of COX-2 in canine squamous cell carcinomas has thus far remained uncharacterized. The objective of the present study was to determine whether COX isoenzymes were expressed in canine squamous cell carcinomas. Normal skin (n = 4) and canine squamous cell carcinomas (n = 40) from different anatomical locations (skin, mouth, digits) were studied by immunohistochemistry and by immunoblot analysis using polyclonal antibodies selective for COX-1 or COX-2. Small amounts of COX-1 were found in normal and cancerous tissues (in stromal fibroblasts, blood vessels and neoplastic keratinocytes). COX-2 expression in normal skin was faint or absent, but all squamous cell carcinomas (40/40, 100%) displayed strong immunoreactivity for COX-2 (P < 0.01). COX-2 immunostaining was predominantly localized in the cytoplasm of neoplastic keratinocytes, often being concentrated around the nuclear membrane. Immunoblot analysis confirmed the restricted expression of COX-2 (Mr = 72 000) in squamous cell carcinomas. These results demonstrate for the first time the expression of COX-2 in canine squamous cell carcinomas, and suggest that COX-2 could be involved in the oncogenesis of this common neoplasm in dogs.
59: RETROSPECTIVE STUDY OF 338 CANINE ORAL MELANOMAS WITH CLINICAL AND HISTOLOGIC REVIEW OF 129 CASES.
Diagnostic records from 338 dogs with oral melanomas received at the Veterinary Medical Diagnostic Laboratory (1992–1999) were reviewed. Of these tumors, 122 plus an additional 7 metastatic melanomas of unknown origin were selected for clinical follow-up and histologic review. Chow Chow, Golden Retriever, and Pekingese/Poodle mix breeds were over-represented whereas Boxer and German Shepherd breeds were under-represented. There was no gender predisposition. The average age at presentation was 11.4 years. Forty-nine dogs were euthanized due to recurrence or metastasis. The average post-surgical survival time was 173 days. Melanoma was the second most common oral tumor following squamous cell carcinoma and more common than acanthomatous epulis. The gingiva and the labial mucosa were the most common sites. Most tumors were composed of either polygonal cells (27 cases, 20.9%), spindle cells (44 cases, 34.1%) or a mixture of the two (polygonal and spindle) (54 cases, 41.9%). Clear cell (3 cases, 2.3%) and adenoid/ papillary (1 case, 0.8%) patterns were uncommon. Morphologic features of six metastatic melanomas were similar to those found in the primary tumors.
60: GONADOSTROMAL TUMOR IN A NEW ZEALAND WHITE RABBIT (ORYCTOLAGUS CUNICULUS).
A 2-year-old male New Zealand white rabbit in an experimental protocol developed a diffuse enlargement of the left testis and was humanely euthanized. At necropsy, the left testis was approximately three times larger than the right testis. No other gross lesions were noted. Histologically, the left testis was effaced by polygonal to spindle neoplastic cells that filled and replaced seminiferous tubules and expanded the interstitium. Multifocally, there were cystic spaces that contained proteinaceous fluid and individual polygonal neoplastic cells. The neoplastic cells had histomorphologic features of both Sertoli cell and granulosa cell origin. Mitoses averaged one per high power field. There were areas of coagulative necrosis within the neoplasm, and remaining seminiferous tubules were atrophied. The right testis was histologically normal. Testicular neoplasms are infrequently reported in rabbits. Most reports of testicular neoplasms in rabbits have been interstitial cell tumor and seminoma. This is the first report of gonadostromal tumor in a rabbit.
61: DETECTION OF CHLAMYDIAL ANTIGEN AND DNAs IN CANINE ATHEROSCLEROTIC LESIONS.
Chlamydia pneumoniae has been demonstrated in human atherosclerotic lesions. We attempted to detect chlamydial antigen and DNAs in the arteries from dogs with systemic atherosclerosis. Materials and Methods: Aorta, heart (coronary artery) and spleen from 7 dogs with systemic atherosclerosis were examined histopathologically, and immunohistochemical detection of chlamydial antigen was also done on those tissues. PCR was performed for detection of chlamydial DNAs in the heart, spleen and kidney from one dog. Results: Histology of the vascular lesions revealed atheromas with deposition of sudanophilic and hyaline substance and infiltration of macrophages and foam cells. Immunopositivity against canine-ApoB-100, -albumin, -IgG, -IgM, and -IgA antibodies were found in extracellular matrices and macrophage and foam cell cytoplasm. Immunopositive reactions against C. pneumoniae and C. psittacii antibodies were found in the endothelial cells, infiltration of macrophages, foam cells and smooth muscle cells. Detection of C. pneumoniae DNAs was demonstrated in the heart, spleen and kidney by PCR. Conclusion: This study provides direct evidence of the presence of chlamydial antigen and DNAs in atheromatous lesions. These results support that the organism may be a factor in the pathogenesis of canine atherosclerosis as well as human atherosclerosis.
62: MUTATION ANALYSIS OF FELINE NIEMANN-PICK TYPE C DISEASE.
Niemann-Pick type C disease (NPC) is a neurovisceral lysosomal storage disorder resulting from defective intracellular transport of cholesterol. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically identical to human NPC1. Complementation studies using cultured fibroblasts support that the gene responsible for NPC in the feline model is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. Utilizing these sequences, feline specific PCR primers were generated and designed to amplify six overlapping bands which span the entire feline NPC open reading frame. To date, 97% of the cDNA sequence has been determined. A single base substitution has been identified in all NPC affected cats evaluated. Carriers are heterozygous for the same allele and a PCR-based assay has been developed to identify heterozygotes. The point mutation results in a conserved cysteine to serine substitution. Several of the mutations seen in people involve the same area of the NPC1 gene. Karyotyping revealed that mutations resolvable at the cytogenetic level were not evident, supporting a small mutation. The predicted feline amino acid sequence shows 90% homology to the human NPC1 protein. The human NPC1 cDNA sequence predicts a 1278 aa protein with a lysosomal-targeting sequence, several trans-membrane domains and extensive sequence homology to other known mediators of cholesterol homeostasis.
63: COMPARISON OF NEUROANATOMICAL PATTERNS OF SPONGIFORM ENCEPHALOPATHY AND IMMUNOHISTOCHEMICAL STAINING OF PRION PROTEIN IN BRAIN AND LYMPHOID TISSUES OF FREE-RANGING, RESEARCH, AND RANCH-RAISED ELK.
A spongiform encephalopathy compatible with chronic wasting disease has been found in ranch-raised elk in at least 5 states in the US and one province in Canada. There is a tremendous concern in regard to the origin of this disease, as to whether it is the same disease as chronic wasting disease of free-ranging elk. There is also an effort to develop methods to diagnose early cases especially in live animals. A comparison of the histological lesions and immunohistochemical staining of brain and lymphoid tissues were examined in 5 free-ranging elk and 26 captive (1 research and 25 ranch-raised) elk with a spongiform encephalopathy compatible with chronic wasting disease and in 10 control elk (5 free-ranging and 5 ranch-raised). The immunohistochemical staining was done with monoclonal antibody 160.1.5 using the Ventana automated system. The neuroanatomical locations of spongiform encephalopathy and immunohistochemical staining of brain in free-ranging elk were identical to those of captive elk. The prevalence of positive immunohistochemical staining of lymphoid tissues was also identical, however, the immunohistochemical staining was not consistently present in the lymphoid tissues of brain-positive elk. The demonstration that the spongiform encephalopathy and immunohistochemical staining of brain and lymphoid tissues of free-ranging elk was indistinguishable from those of captive elk may suggest that the spongiform encephalopathy as seen in free-ranging elk of Colorado is the same as observed in these ranch-raised elk. Since the occurrence of positive lymphoid staining was not consistently found in brain positive elk, the use of immunohistochemical staining of lymphoid tissues may be of limited value as a live-animal test in elk.
64: SEVERE ASCARIDIASIS IN CAPTIVE PSITTACINES IN THE SOUTHEASTERN UNITED STATES.
An Ascaridia not matching any currently described species has been reported as a cause of mortality in captive psittacines from an aviary in the southeastern United States. The present report describes severe, fatal ascaridiasis with the same nematode in three macaws (Ara chloroptera, Ara militaris, and Anodorhynchus hyacinthinus) from different aviaries in the same geographic location. All three birds were adult breeding females with no antemortem clinical signs. One macaw (A. hyacinthinus) had severe feather loss on its torso. Gross lesions included ascarid impaction in the small intestine (A. hyacinthinus), large intestine (A. chloroptera), or both (A. militaris). One case had crop distension (A. hyacinthinus). Hepatic heterophilic granulomas were present in all three cases with an intralesional larva in one case (A. chloroptera) and Escherichia coli in the other two cases (A. militaris and A. hyacinthinus). Widespread intravascular bacteria were also present in one case (A. militaris). The recovered ascarids have some features in common with both Ascaridia columbae and Ascaridia hermaphrodita. These latter species differ mostly in their cervical alae and spicule length. The ascarids in these macaws had lengths intermediate to those published for these parameters in A. columbae and A. hermaphrodita. Males also had variable numbers of caudal papillae (ten to thirteen). The native host for this ascarid is unknown, but could be a native bird or psittacine species common to all three aviaries. The hepatic granulomas are suggestive of aberrant liver migration, similar to Ascaridia dissimilis migration in turkeys. Intestinal impaction, secondary bacterial hepatitis, or sepsis could contribute to death in psittacines infected with this ascarid.
65: ANATRICHOSOMA DERMATITIS IN A FERRET.
An adult, neutered male ferret was presented for treatment of alopecia and multifocal cutaneous lesions. Lesions extended from the dorsal cervical-scapular region to the perineum, laterally over the thorax, and multifocally on the hind feet. Grossly, lesions consisted of raised firm nodules, pustules, and crusts with associated mild erythema. No ectoparasites were observed. Scrapings and impressions of skin lesions revealed no etiologic agent. Flea antigen testing was negative. Fecal examination was negative for intestinal parasites. Histologic examination of punch biopsy specimens of skin revealed multifocal ulceration of the epidermis with adherent serocellular crusts. A moderate to marked dermal accumulation of macrophages and neutrophils with scattered dense accumlulations of eosinophils extended from ulcerated regions into the dermis. Within the dermis and occasionally within the superficial exudate, numerous unstained to basophilic larvated, bi-operculate ova and nematode larvae were observed. The ova and larvae were morphologically consistent with those of Anatrichosoma sp. Lesions resolved following treatment with ivermectin. To our knowledge, this is the first published report of Anatrichosoma sp. dermatitis in a ferret.
66: CANINE MODEL OF GLYCOGEN STORAGE DISEASE TYPE Ia.
A canine model of glycogen storage disease type Ia (GSP Ia) has been established by cross breeding Maltese carrying a mutated glucose-6-phosphatase (G6Pase) gene with Beagles. Ten crossbred, homozygous recessive puppies have been born. Two were stillborn, one is still alive at 17 months, and the others have survived for periods ranging from <1 day to 76 days-of-age. Surviving affected puppies have been weak, lethargic, and slow growing with fasting hypoglycemia, hyperlipidemia, lactic acidosis, and elevated uric acid. Hepatic G6Pase levels were 10-fold less in affected puppies than in non-affected puppies. At necropsy, livers were markedly enlarged and pale. Kidneys were slightly enlarged and occasionally pale. Microscopically, livers from all affected puppies had marked hepatocellular vacuolation with abundant glycogen accumulation (7% to 9% glycogen in affected puppies vs. 0.6% in non-affected puppies). Proximal convoluted tubules of affected puppies were mildly to moderately vacuolated suggesting glycogen accumulation. Segmental to diffuse glomerulosclerosis was present in affected puppies that died between 32 and 76 days-of-age. The clinical signs, biochemical abnormalities and pathological changes in affected dogs closely resembles the human disease. Gene therapy of affected dogs is being attempted using an adeno-associated virus vector encoding G6Pase and driven by the mouse albumin promoter/enhancer.
67: GUINEA PIGS AS AN EXPERIMENTAL MODEL OF HENDRA VIRUS ENCEPHALITIS.
In September 1994, in Hendra, a suburb of Brisbane, Australia infection with a virus designated as equine morbillivirus caused the deaths of 13 horses and 1 human. Subsequent studies have renamed the virus Hendra virus (HeV). Experimental studies, have found horses, fruit bats, guinea pigs and cats to be susceptible to HeV infection. Fourteen of fifteen guinea pigs inoculated with HeV developed vascular disease with immunoreactivity in a range of tissues. Eight guinea pigs had histological lesions of encephalitis with positive immunohistochemical staining. Lesions were observed in the medulla, cerebellum and the thalamus. Hendra virus was isolated from five of the encephalitic cases. Severe vascular degeneration in the center of encephalitic lesions in six of the eight encephalitic guinea pigs and positive immunostaining in the choroid plexus of another guinea pig indicated that the virus appears to enter the brain following virally induced vascular injury and choroid plexus invasion. Guinea pigs could therefore be a suitable model in which to study HeV encephalitis.
68: INTRATRACHEAL INSTALLATION OF IL-9 INDUCES AN ASTHMATIC PHENOTYPE IN MICE.
IL-9 is a T-cell derived cytokine that has recently been implicated in the pathogenesis of asthma. C57B16 mice were intratracheally (IT) instilled with 5mg of recombinant murine IL-9 either daily, every 2nd or 3rd day for up to 9 days. Mice were sacrificed on days 4, 7 or 10 after bronchoalveolar lavage (BAL) was performed. Additionally, in the group receiving 9 daily doses, airway hyperresponsiveness (AHR) was assessed by administration of a single i.v. dose of 5-hydroxytryptamine (30 mg/ kg). Mice given IL-9 IT had increased (p = 0.009) AHR (APTI = 398 ± 63.3) compared to controls (APTI = 161 ± 8.4). Histopathology at day 10 demonstrated perivascular and peribronchiolar lymphocytic and eosinophilic inflammation with bronchiolar mucus cell metaplasia. Serum total IgE levels were elevated (720 ng/ml ± 234) compared to controls (62 ng/ml ± 0). The increase in WBC in the BAL with IL-9 administration was both dose and time dependent. Substantial numbers of WBC did not appear in the BAL until day 10 of the study and required at least every 2nd day administration of IL-9 (data not in table).
These results indicate that IL-9 can induce many of the features of an asthmatic phenotype. However, because the development of the inflammatory cell influx is protracted, the effect of IL-9 on the induction of allergic inflammation is probably dependent on up-regulation of other mediators.
69: CHROMOSOMAL MAPPING OF QUANTITATIVE TRAIT LOCI (QTLs) CONTRIBUTING TO VIRUS-INDUCED ASTHMA-LIKE DISEASE IN RATS.
Sendai virus infection and recurrent ovalbumin exposure in BN rats induces airway lesions and pulmonary dysfunction that serves as a model of asthma. Asthmatic phenotypes induced in BN rats include chronic bronchiolar inflammation with airway fibrosis, mucous cell hyperplasia, increased airway resistance with hyperresponsiveness and elevated serum IgE. F344 rats are resistant to these asthma-like phenotypes. Our objective was to identify genetic loci (QTLs) controlling susceptibility to this virus-induced and allergen-accentuated airway disease. The linkage analysis study was based on analysis of 176 backcross (FBNF1 × BN) rats using 187 PCR genetic markers covering 21 chromosomes at approximately 10 cM intervals. Initial linkage analysis with MAPMAKER and MAPMAKER-QTL programs identified QTL candidates on chromosomes 6, 7, 17 and 20. The strongest linkages to elevated IgE and bronchiolar mucous cell hyperplasia in these initial studies were on chromosome 20 in the D20UW1 to D20Rat1 interval (highest lod = 6.60). Candidate genes in this interval include RT1 and TNF-α that could be controlling asthmatic phenotype. (Supported by NIH HL61018.)
70: MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS (M.ptb) INFECTION IN EUTHYMYIC BALB/C AND ATHYMIC NUDE BALB/C MICE.
Three- to eight-day-old-euthymic BALB/c and athymic nude BALB/c mice were infected IP with 3 × 107 M.ptb ATCC strain 19698 diluted in 0.1 ml saline, and necropsied five months post inoculation. Only nude mice had gross lesions, which consisted of hepatomegaly, splenomegaly, mesenteric lymphadenopathy, and duodenal wall thickening. Histologically, nude mice had granulomatous lesions with acid-fast bacteria in the intestine, mesenteric lymph nodes, liver, spleen, and pancreas. The euthymic mice had granulomatous lesions with acid-fast bacteria in the small intestine, liver and spleen. Lesions were more severe in the nude mice as measured by surface area image analysis of the liver, and subjective scoring of the spleen and duodenum. The duodenum was consistently the most severely affected portion of intestine. Ultrastructural examination showed that duodenal lesions of nude mice had macrophages and rarely neutrophils, within the lamina propria, which were enlarged by numerous bacilli-laden membrane bound vacuoles (phagosomes). Bacterial ultrastructure was consistent with Mycobacterium sp. Euthymic mouse hepatic macrophages had fewer bacteria, a higher percentage of degraded bacteria, increased numbers of secondary lysosomes, and increased numbers of primary lysosomes, compared to nude mice. In both strains of mice, intact bacteria were typically located individually in phagosomes with no evidence of lysosomal fusion. Conclusions: macrophage activation is markedly reduced in athymic nude mice infected with M.ptb as compared with euthymic mice. The T-cell deficiency in the nude mouse appears responsible for the large accumulation of M.ptb in the lamina propria of the intestine, which is similar to natural disease of ruminants.
71: AXONAL AMYLOID PRECURSOR PROTEIN ACCUMULATION IN SIV-INFECTED MACAQUES.
Axonal accumulation of amyloid precursor protein (APP) reflecting disturbances in axonal transport has been identified in HIV-infected individuals including pre-AIDS cases. To study the underlying mechanisms of APP accumulation in disease progression, the amount of APP immunostaining in the corpus callosum of 24 SIV-infected macaques and 3 control macaques was measured by computerized image analysis. The amounts of APP accumulation were compared with time post-inoculation, extent and character of inflammation, and viral load in the CNS. Significant increases over control values were present in 10 of 24 SIV-infected animals. Eight animals with elevated APP had developed SIV encephalitis at 3 months PI; 2 SIV-infected animals without encephalitis also had elevated APP levels at 3 weeks and 8 weeks PI. Increases in APP correlated most strongly with CNS viral load (P = 0.005) but significant associations with macrophage infiltration and microglial activation (P = 0.04) and infiltration by cytotoxic lymphocytes (P = 0.05) were also identified. These data demonstrate that APP accumulation in the white matter of SIV-infected macaques develops during SIV infection in correlation with increases in CNS viral antigen, although myelin pallor is not a remarkable feature of the SIV/ macaque model of AIDS dementia.
72: EARLY VIRAL TISSUE AND CELL TROPISM IN MUCOSAL FIV INFECTION.
To identify the early target cells and tissues in transmucosal lentivirus infection, cats were exposed via oral-nasal and vaginal mucosa to clade B and C feline immunodeficiency virus (FIV) isolates and necropsied from days 1–12 post inoculation (PI). Mucosal tissues, regional and distant lymph nodes (LN), blood mononuclear cells (PBMC), and other tissues were examined by quantitative virus coculture, DNA PCR, tyramide signal amplified in situ hybridization (TSA-ISH) and immunohistochemistry (IHC) for cell phenotype. FIV RNA was first detected in rare subepithelial cells in the oral, vaginal, and intestinal mucosae as early as day 1 and in regional (retropharyngeal, tracheobronchial, and/or iliac) LN germinal centers at day 2 PI. From days 2–6 FIV RNA and DNA was detected sporadically in regional and some distant lymph nodes and/or spleen. During this interval viral DNA also was detected occasionally in PBMC by PCR and TSA-ISH. By day 8 PI, viral RNA, DNA, and coculturable virus were detected consistently in regional and distant lymphoid tissues, indicating that systemic infection had occurred. The earliest FIV-bearing cells demonstrated by TSA-ISH had phenotypic features consistent with dendritic cells and T cells. These findings, likely representative of mucosal lentivirus infections in general, extend our knowledge of the initial events in lentivirus pathogenesis.
73: THE PROLINE-RICH MOTIF OF BOVINE LEUKEMIA VIRUS TRANSMEMBRANE PROTEIN GP30 IS REQUIRED FOR MAINTENANCE OF HIGH VIRAL LOAD IN VIVO.
The cytoplasmic tail of the bovine leukemia virus (BLV) transmembrane protein, gp30, has multiple amino acid motifs that mimic motifs in signaling proteins associated with B cell and T cell receptors. We have hypothesized that these signaling motifs allow the virus to interact with host signaling pathways. One motif of gp30, the proline-rich motif, is often the recognition site of SH3 and WW domains of signaling molecules. Here, we tested in vivo the functional significance of the proline-rich motif. Using PCR-based site-directed mutagenesis of an infectious molecular clone of BLV, we introduced point mutations which changed three of the prolines to alanines. The influence of these mutations on the pathogenicity of BLV was studied in three groups of 5 sheep which received either (a) plasmid DNA with proline to alanine mutations of the provirus (pppBLV), (b) plasmid DNA with wild type provirus (wtBLV), or (c) transfection reagent alone (negative control). Although all of the BLV-transfected animals seroconverted at approximately the same time, viral expression at later time points was much higher in the wtBLV group than in the pppBLV group (as determined by semiquantitative PCR, p24 ELISA, and serologic titers). These data indicate that the proline-rich motif of gp30 is required for high viral load in vivo, but is not required for viral infectivity. The necessity of this specific motif suggests that SH3- or WW-mediated gp30 interactions are critical for viral propagation following infection.
74: IN SITU DETECTION OF APOPTOTIC CELLS IN BOVINE ILEAL LIGATED LOOPS INFECTED WITH SALMONELLA TYPHIMURIUM.
We demonstrated Salmonella typhimurium causes apoptosis in bovine monocyte-derived macrophages in vitro by two distinct mechanisms of cell death, early cell death that is dependent on the expression of the sipB gene and a delayed sipB-independent mechanism of cell death. In the mouse, SipB binds and activates caspase-1 which cleaves interleukin-1. To address the role of apoptosis in inflammation of S. typhimurium infected ligated loops, 8 Holstein calves had twelve 6 to 9 cm loops prepared in the terminal ileum. Four calves received intra-lumen injection of LB broth as a control in 12 loops. Four other calves were inoculated with 2 × 109 CFU of S. typhimurium (strain IR715) per loop in 12 loops. Samples for histopathology, ultrastructure and bacteriologic culture were collected at 5, 15, and 30 minutes and at 1, 2, 3, 4, 5, 6, 8, 10, and 12 hours post infection (PI). The volume of fluid per unit length (ml/cm) was measured in each loop. H&E stained sections were scored for inflammation, ranging from 1 (absence of inflammation) to 5 (severe inflammatory changes). Apoptotic cells were detected in situ by TUNEL. Images from 20 randomly selected microscopic fields (36,500 μm2 each, 10 from the mucosa and 10 from the lymphoid nodules) were captured from each TUNEL-stained slide and analyzed using NIH Image 1.60 software. Fluid accumulation started at 3 hours PI. Inflammatory changes were detected in all infected loops at 1 hour PI. The area of TUNEL-labeled cells in the infected samples was significantly higher than the controls at 12 hours PI in both mucosa and lymphoid nodules. Our results indicate that there is an increase in the number of apoptotic cells in S. typhimurium-infected ileal ligated loops. Additional experiments employing caspase inhibitors and selected mutants of S. typhimurium will define the role that apoptotic cell death plays in triggering the inflammatory response during S. typhimurium infection.
75: INTERLEUKIN-4 EXACERBATES COLITIS IN MICE INDUCED BY CD45RB high CD4+ T CELLS.
Transfer of the CD45RBhigh subset of CD4+ T cells from wild type (WT) or IL-10-/- mice into immunodeficient mice results in a Th1-mediated colitis. Interleukin (IL)-4 promotes Th2-like responses and can suppress Th1 responses. In the present study, we show that daily intraperitoneal administration of 20 ug of IL-4 was unable to prevent the Th1-like colitis induced by transfer of WT or IL-10-/- CD45RBhigh cells into Rag2-/- mice, and, in fact, the IL-4 treatment exacerbated the intestinal inflammation. The incidence and severity of colitis was greatly reduced in mice when IL-4 was administered with IL-10 or anti-IL-12 (which block Th1-mediated inflammation), and most of these mice had no colitis. These results suggest that IL-4 acted by exacerbating Th1-mediated inflammation, rather than by promoting a Th2-like inflammatory process. To further evaluate the role of IL-4 in this model, IL-4 was administered to Rag2-/-mice reconstituted with CD45RBhigh CD4+ T cells from IL-4 receptor alpha-deficient mice. IL-4 was also able to exacerbate colitis in this experiment, suggesting that IL-4 indirectly promoted Th1-type inflammation through activity on non-T cells.
76: ESTABLISHMENT OF MTA RAT MAMMARY TUMOR CELL LINE AS A NONRESPONDER TO RISEDRONATE THERAPY.
Bisphosphonates, potent osteoclast inhibitors, have shown promise in reducing the destructiveness and incidence of bone metastatic lesions. However, not all tumors have shown favorable responses to bisphosphonate therapy. Human and animal trials have identified individuals that have not responded to treatment. In some cases there was no decrease in number or size of lytic lesions while other patients had persistent hypercalcemia. Specific properties that render tumor cells responsive or nonresponsive to bisphosphonates are not well characterized. The objective of this study is to establish the MTA rat mammary tumor cell line as a nonresponder to bisphosphonate, specifically risedronate, therapy. In in vivo metastasis assays, 35–45-day-old BDIV rats were injected with 105 monodispersed MTA (rat mammary tumor) cells into the left cardiac ventricle. Rats were divided into treated and untreated groups. Treated animals received 0.2 mg/kg risedronate/day via subcutaneous injection. Untreated animals received daily subcutaneous injections of physiologic buffered saline. Animals were sacrificed and necropsied on day 16 post tumor cell injections. Femora and cervical, thoracic, and lumbar vertebrae were collected, decalcified, and processed routinely to produce hematoxylin and eosin-stained histologic sections. Bone metastatic lesions were counted and tumor areas measured from digital images. There was no significant difference in mean tumor number or mean tumor size between treated and untreated animals using the Student's t-test. This establishes the MTA rat mammary tumor cell line as a nonresponder to risedronate treatment and a possible model for studying the mechanisms of action of non-responding tumors.
77: EVALUATION OF A ROLE OF INFLAMMATORY CYTOKINES IN DEVELOPMENT OF HELICOBACTER PYLORI-ASSOCIATED GASTRIC PROLIFERATION.
The objective of this study was to evaluate the effect of IFN-γ, TNF-α, IL-1α, and conditioned media from H. pylori-stimulated splenocytes on gastric epithelial cells. This laboratory has shown a strong correlation between CD4+ cells in chronic H. pylori gastritis and gastric epithelial proliferation, and that IFN-γ is secreted in high levels from murine splenocytes stimulated with H. pylori antigen. Also TNF-α and IL-1 have been reported to be found in high concentration in H. pylori gastritis. GSM06 (murine) and NCI-N87 (human) cells were used. GSM06 cells were treated with IFN-γ (1–10 ng/ml), TNF-α (1–10 ng/ml), and conditioned media from H. pylori antigen-stimulated splenocytes. NCI-N87 cells were treated with IFN-γ (1–10 ng/ml) and IL-1α (0.1–1 ng/ml). Proliferation was measured using the Alamar-blue proliferation assay. An in vitro TUNEL assay was used to measure apoptosis after IFN-γ treatment in both cell lines. GSM06 cells showed an anti-proliferative dose response with increasing concentration of IFN-γ and TNF-α. GSM06 cells treated with conditioned media from stimulated splenocytes showed a significant increase in proliferation over untreated control cells. NCI-N87 cells showed an anti-proliferative dose response with increasing concentration of IFN-γ and IL-1α. Both cell types showed a significant increase in apoptosis over untreated controls after treatment with 10 ng/ml IFN-γ. We conclude that IFN-γ, TNF-α and IL-1α are anti-proliferative and IFN-γ is pro-apoptotic to gastric epithelial cells. The proliferation induced in GSM06 cells by conditioned media from stimulated splenocytes is due to another mediator. A compensatory gastric epithelial proliferative response in response to increased apoptosis, might contribute to overall gastric proliferation.
78: THE ROLE OF GRANULOCYTE COLONY-STIMULATING FACTOR IN THE DEVELOPMENT OF IMMUNOPATHOLOGIC CHANGES IN VIABLE MOTHEATEN MICE.
The viable motheaten (hcphmev) mutation, (abbreviated as mev), disrupts the structural gene for hematopoietic cell phosphatase, which encodes for the protein-tyrosine phosphatase SHP-1. Mice homozygous for this mutation are immunodeficient, develop severe inflammatory lesions in the lung, skin and elsewhere and die as a result of acidophilic macrophage pneumonia. SHP-1 functions as a negative regulator for signaling through a number of hematopoietic growth factor receptors including the receptor for granulocyte colony-stimulating factor (G-CSF). To determine the role of G-CSF in the pathology of mev/mev mice, we carried out genetic crosses between C57BL/6J- mev/mev and 1
79: YOUNG BALB/c MIN MICE WITH CUTANEOUS CYSTIC EPIDERMAL TUMORS OF THE HEAD AND NECK RESEMBLING GARDNER SYNDROME IN HUMANS.
The “min” or multiple intestinal neoplasia mouse is a murine model of human familial adenomatous polyposis (FAP). Min mice have an adenomatous polyposis coli (APC) gene mutation resulting in truncation of a protein involved in the regulation of B-catenin. The min mutation was originally placed on a C57/BL6 background, however, the present findings were observed on a BALB/c background. Seven of 76 (9.21%) of the BALB/c Min mice developed a solitary 5–20 mm diameter cystic, nodular, subcutaneous mass on the face or neck compared to a background incidence of less than 0.1%. Histologically, the masses had sheets and islands of variably stratified epithelium with squamous differentiation and prominent keratin production including keratin pearl formation, and lacked a connection or opening to the overlying haired skin. The combination of FAP and nodular, cystic, epidermal lesions involving the craniofacial region has been observed in humans and referred to as Gardner's Syndrome. Affected families have intestinal polyposis progressing to fatal carcinoma in combination with nodular craniofacial lesions diagnosed as epidermal/sebaceous cystic masses or pilomatricomas within the early years of life. These may be due to impaired DNA repair. Cutaneous tumors in juvenile BALB/c Min mice may reflect a unique modulating effect of the host background or a novel genetic mutation possibly involving the APC gene. The combination of head and neck cystic epidermal tumors and bowel lesions make this an attractive murine model for studying the dermatologic manifestations associated with human FAP.
80: SUBTILISIN-LIKE CONVERTASES ARE RESPONSIBLE FOR POST-TRANSLATIONAL PROCESSING OF CADHERINS.
Intercellular adhesion between keratinocytes is conferred by classical and desmosomal cadherins assembled in the higher ordered structure of either adherens junctions or desmosomes. Disturbed cadherin-mediated adhesion has been implicated in invasive and metastatic behavior of epithelial tumors. One limiting step in the establishment of functional intercellular adhesiveness is correct post-translational processing of the inactive cadherin precursors. Here, we investigated the proteases which are responsible for cadherin maturation to occur. Using a baculovirus co-expression system, we demonstrate that a member of the subtilisin-like convertase family, furin, efficiently and specifically cleaves E-cadherin, desmoglein 1 and desmoglein 3. Taking advantage of a furin-deficient cell line, we show that E-cadherin can alternatively be processed by other members of this convertase family. We demonstrate that primary mouse keratinocytes express a set of four different convertases, which are furin, PACE4, PC6 and PC7 and that they correctly process cadherins. As theses proteases may cooperate in the maturation of intercellular adhesion molecules we currently investigate the yet unexplored interplay between these convertases during the maturation of cadherins in keratinocytes.
81: INFECTIVITY OF A MOLECULAR CLONE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE-1 FOLLOWING ORAL MUCOSAL EXPOSURE.
Human T-lymphotropic virus type-1 (HTLV-1) is a complex retrovirus associated with both a chronic, debilitating neurologic disease and an aggressive T-cell neoplasm. HTLV-1 infection has a worldwide distribution with endemic areas occurring in the Caribbean basin, Africa and Japan and among at-risk groups in the United States. The virus infection is transmitted by the exchange of whole cell blood products, by breast milk, and through sexual contact. Using the rabbit animal model, we have previously shown that an HTLV-1 infectious molecular clone, designated ACH, is equally infectious as wild-type virus via the intravenous route. The oral cavity is an important route of exposure particularly within endemic areas where the passage of HTLV-1 infected lymphocytes through the breast milk of infected mothers to their infants is common. We hypothesize that ACH will be equally infectious in rabbits when transmitted by exposure to the oropharyngeal mucosa. Using our previously published viral parameters, including detection of HTLV-1 specific antibodies by immunoblot assay, viral p19 antigen by exvivo culture of rabbit PBMC, and real-time QC-PCR for proviral load, data is presented indicating the relative oral infectivity of ACH-immortalized human lymphocytes when compared to wild-type virus. The efficient intravenous infectivity of the ACH molecular clone in rabbits has already provided our laboratory the opportunity to define certain molecular determinants of HTLV-1. The addition of an efficient oral transmission balances this important animal model to study both viral genetic determinants as well as potential antiviral therapies against HTLV-1.
82: DIFFERENTIAL PROTEIN NITRATION IN HYPOTENSION RESULTING FROM HEAT STROKE OR HEMORRHAGE.
Circulatory shock induced by heat stroke from environmental heating (EH) or by hemorrhage (HEM) produces protein nitration in lung and liver. The purpose of this study was to determine differences in the nitration patterns induced by these conditions for use as potential biomarkers. Urethane-anesthetized rats were subjected to EH (40 C ambient temperature until colonic temperature > 41.5 C) or HEM (to blood pressure of 50 or 75 mmHg; maintained for 1 hr). Tissues were evaluated for 3-nitrotyrosine (3-NT) accumulation using a 3-NT specific polyclonal antibody. Immunohistochemistry and confocal microscopy (CF) were used to determine overall 3-NT accumulation and organ distribution and Western blots to determine if specific proteins appeared to be targets of nitration. Urine and plasma were evaluated for systemic elevations in 3-NT. CF and image analysis revealed significant accumulation of 3-NT in the liver for all treatments; significant 3-NT in the lung was only seen in the EH group. In the liver, EH and HEM (at both levels) caused increased accumulation of a 31kD and a 45kD nitrated protein. In the lung, EH and HEM to 50 mmHg caused accumulation of a 77kD and a 154kD nitrated protein; this was not seen in controls or rats bled to 75 mmHg. Urine and plasma NO2/NO3 were increased by HEM but not by EH. Relatively mild hypotension produced by EH or HEM seems sufficient to produce tyrosine nitration in liver and lung and the pattern varies with the cause of the hypotension. Tyrosine nitration appears to target specific proteins suggesting it acts as a intermolecular signal.
83: PATHOLOGICAL CHARACTERIZATION OF TIGHTLY SPIRALED BACTERIA IN MICE AFTER INOCULATION OF STOMACH HOMOGENATE FROM BACTERIA-INFECTED PIGS.
Four of fifty domestic pigs showed tightly spiraled bacterial infection in the pyloric mucosa of stomach, as stained with carbol-fuscin and Steiner's silver. Twenty-two male SPF ICR mice were inoculated with the gastric homogenate of pigs infected with tightly spiraled bacteria. Two mice each were sacrificed at 3, 7, 10, 17, 21, 24, and 28 days postinoculation (PI) and 8, 12, 16, 20 weeks PI. Tightly spiral bacteria were mainly found in the mucosa surface, the gastric pits and the lumen of gastric glands. After 21 days PI, lymphoplasmocytic infiltration with lymphoid follicles, epithelial hyperplasia, cystic dilatation of gastric glands, and necrosis of surface epithelium were observed in the gastric mucosa. All the mice used in this study except one at 3 days PI were urease-positive. Isolation and culture of the organisms was unsuccessful. A common antigenicity was observed between the tightly spiraled bacteria and Helicobacter pylori with Western blot, ELISA, and immunohistochemistry. 16S rDNA fragment of 374 bp was amplified by PCR technique with a Helicobacter genus-specific primer. By electron microscopy, the tightly spiraled bacteria had two to six spiral turns, and was approximately 2-4 μm long and 0.5–0.8 μm wide. Two to five flagella with about 20 nm in diameter were seen arising from each pole. In this study, we established a suitable mouse model of chronic gastritis caused by tightly spiraled bacteria.
84: MODEL OF OSTEOBLASTIC BONE METASTASIS: BONE INDUCTION IN NUDE MOUSE CALVARIA BY CANINE PROSTATE TISSUE.
Osteoblastic metastases are common in patients with advanced prostate cancer. The pathophysiology of the new bone formation at metastatic sites is not currently known. Most rodent models of prostate cancer metastasis to bone are osteolytic and not osteoblastic. Osteolysis by tumor cells may also lead to fractures or bone instability. Misinterpretation of new periosteal bone that is associated with bone stabilization as tumor-cell osteoinduction may occur with present models. We have developed a model system of new bone formation in the calvaria of nude mice stimulated by normal canine prostate tissue. Collagenase-digested normal canine prostate tissue was implanted adjacent to the calvaria of nude mice. Calvaria were examined at 2 weeks for changes in the bone microenvironment by histology, calcein uptake at sites of bone mineralization, and tartrate-resistant acid phosphatase staining of osteoclasts. The prostate tissue induced reactive stromal cell proliferation around the prostate acini and abundant new bone formation on the adjacent periosteal surface. In some cases new bone formation also was induced on the distant or concave calvarial periosteum. The new bone stained intensely with calcein, which demonstrated regions of bone matrix mineralization. Calvaria implanted with canine prostate tissue were significantly thicker than controls. New bone formation was not induced in calvaria of mice implanted with normal kidney, bladder, or muscle tissue. Osteolysis of calvarial bone was also present in addition to the new bone formation. Osteoclast numbers were not different between implanted and control calvaria. This animal model will be useful for investigating the roles of prostate-derived growth factors on new bone formation.
85: 42 kD mRNA BINDING PROTEIN MAY REGULATE PTHrP mRNA STABILITY AFTER TGFβ TREATMENT IN SQUAMOUS CELL CARCINOMA CELLS.
Humoral hypercalcemia of malignancy (HHM), a paraneoplastic syndrome associated with epithelial cancers, is due to expression and secretion of biologically active parathyroid hormone-related protein (PTHrP). PTHrP mRNA transcription rate and stability are increased in response to TGFβ, which is often produced by malignant neoplasms. Utilizing a cell-free mRNA degradation assay, we have demonstrated that TGFβ-mediated PTHrP mRNA stabilization appears to be predominately mediated by sequences in the coding region. This is unlike other rapidly degraded mRNAs, which are regulated by sequences in the 3′-untranslated region (3′-UTR). UV cross-linking of mRNA and cytoplasmic proteins from squamous carcinoma cells treated with either vehicle or TGFβ and [32P]-PTHrP RNA from coding and 3′-UTR regions, revealed a 42 kD binding protein that bound within a 176 nucleotide RNA comprised predominately of the terminal coding region with a small region (30 nucleotides) of the 3′-UTR. The protein-mRNA binding was present only in vehicle-treated cytoplasmic extracts and was completely inhibited by 100-fold excess of unlabeled mRNA. Protein binding to the mRNA constructs was inhibited by TGFβ. This protein is within the molecular weight range of a family of mRNA binding proteins involved in mRNA instability (i.e. AUF-1 family), and appears to bind to the last 100 bases of the coding region. TGFβ expression by cancer may play an important role in the pathogenesis of HHM by modulating PTHrP mRNA stability.
86: DETECTION OF METASTATIC MATLyLu PROSTATE TUMORS BY RETROVIRAL-MEDIATED GENE TRANSFER OF THE Na+/I− SYMPORTER USING WHOLE-BODY IMAGING IN RATS.
The human sodium iodide symporter (hNIS) mediates active iodide uptake in thyroid follicular cells. The symporter provides a mechanism for effective radioiodide treatment of residual, recurrent, and metastatic thyroid carcinomas. The objective of this study was to investigate whether retroviral-mediated gene transfer of hNIS to MATLyLu (MLL) rat prostatic adenocarcinoma cells permits the detection of metastatic disease following radionuclide administration in vivo. We have shown that iodide-concentrating activity is increased up to 72-fold in a mixed population of MLL cells transduced in vitro with retrovirus expressing hNIS. Within 30 days following subcutaneous inoculation of MLL-hNIS cells in rats, metastases developed in lymph nodes and lungs. Metastatic tumors were visualized by whole-body imaging of rats within 2.5 hours following administration of Tc-99m (2 mCi) and I-123 (300 μCi). hNIS expression in tumor tissues was also confirmed by Western Blot analysis and immunohistochemistry. In conclusion, retroviral-mediated expression of hNIS in MLL cells permits the detection of distant metastases in vivo. Additional studies in progress will investigate I-131 therapy to interrupt tumor progression, and vector-conferred prostate-specific expression of hNIS.
87: FELINE HEAD AND NECK SQUAMOUS CELL CARCINOMA LINE: CHARACTERIZATION, PRODUCTION OF PTHrP AND REGULATION BY TGF-β.
A useful natural animal model does not exist for human head and neck squamous cell carcinoma (H/N SCC). The cat develops a relatively high rate of H/N SCC, which is similar to the human disease in aggressiveness and incurability. We have developed a cell line from a laryngeal SCC of a cat and described its important features. This will permit (1) mechanistic experiments on genetic dysregulation in neoplastic keratinocytes of the feline oral cavity, and (2) development of an important natural model of the human disease. Neoplastic keratinocytes from a laryngeal SCC (F1) were maintained in culture for greater than 30 passages. Cells were characterized immunohistochemically with strong cytokeratin staining and absent vimentin and p53 staining. Electron microscopy demonstrated the presence of cytokeratin filaments and desmosomes, as well as features of anaplasia (irregular cytoplasmic and nuclear margins, surface microvilli, and abnormal intermediate filament production). Karyotype analysis revealed aneuploidy. The growth curve of the cells demonstrated logarithmic growth for six days until confluency. The cytoplasm stained positive for PTHrP. Secretion of the protein was demonstrated by measuring PTHrP in the medium with a two-site immunoradiometric assay. Treatment of SCCF1 cells with TGF-β at four levels of confluency (30% to postconfluence) demonstrated upregulation of PTHrP mRNA and protein secretion as compared to vehicle-treated cells. RT-PCR was used to amplify a 321-base pair region of feline PTHrP mRNA encoding the prepro and part of the coding region. The cDNA was cloned and sequenced. The cDNA and predicted amino acid sequences (PTHrP -29 to 78) were similar to human and canine (94–99% homology).
88: COMPARATIVE PATHOLOGY AND PATHOGENESIS OF A/CHICKEN/HONG KONG/220/97 (H5N1) AVIAN INFLUENZA VIRUS IN CHICKENS, GEESE, AND DUCKS.
In 1997, an outbreak of H5N1 highly pathogenic avian influenza virus (HPAIV) occurred in chickens in Hong Kong, and this virus subsequently infected and caused severe respiratory disease and mortality in humans. The objective of this study was to ascertain and compare the susceptibility of White Leghorn chickens (Gallus domesticus), Pekin ducks (Anas platyrhyncos), and Embden geese (Anser anser) to intranasal infection with the A/chicken/Hong Kong/ 220/97 (H5N1) HPAIV. In chickens, this virus proved highly pathogenic, causing 100% mortality within 2 days post-inoculation (d.p.i.). Clinically, the chickens had diarrhea to hematochezia and rapidly progressive depression to death. Conversely, there was no mortality within 10 d.p.i. in either the geese or ducks, and only the geese exhibited clinical signs of anorexia, diarrhea, and neurologic dysfunction. Gross lesions in the chickens included hemorrhages in multiple organs, splenomegaly, and severe pulmonary edema and congestion. In the geese, the predominant gross lesions were pancreatic necrosis, splenomegaly, and thymic and bursal atrophy. The only gross lesion observed in the ducks was moderate splenomegaly. Histologically, exudation, hemorrhage, and necrosis were the characteristic lesions in the chickens, with the lung, heart, adrenal glands, spleen, and brain being the most severely affected organs. The geese had severe necrotizing pancreatitis and meningoencephalitis most consistently. Mild splenic histiocytic hyperplasia and infrequent mild pneumonia were observed in the ducks. The presence of viral antigen, as determined by immunohistochemistry, was widely distributed in the chickens, and corresponded to the presence of histopathologic lesions in each species.
89: CHARACTERIZATION OF INTRACELLULAR SURVIVAL OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBER-CULOSIS (M. A. PTB) IN J774 CELLS BY LASER CONFOCAL MICROSCOPY.
The objective of this study was to evaluate intracellular survival of M. a. ptb by determining the extent of acidification and maturation of the bacterial phagosome in the murine macrophage cell line J774. Monolayers of J774 cells were grown on chambered glass slides and infected with either M. a. ptb, heat-killed M. a. ptb, the nonpathogenic species Mycobacterium smegmatis (M. smg), or Zymosan A. Intracellular acidified compartments were labeled by addition of the acidotrophic fluorigen Lysotracker Red to the monolayers. In separate studies the Transferrin Receptor (early phagosomal marker) and Lamp-1 (late phagosomal/lysosomal marker) were labeled by indirect immunofluorescence. Colocalization of labeled bacteria with pH or phagosomal membrane markers was detected by laser confocal microscopy. The results demonstrate that M. a. ptb has reduced colocalization with Lysotracker red and Lamp-1, and increased colocalization with the Transferrin receptor compared to killed M. a. ptb, M. smg, and Zymosan A. The conclusions of this study are that M. a. ptb survives intracellularly by residing in a phagosomal compartment with diminished acidity and that the phagosome fails to develop into a functional phagolysosome due to failure of maturation along the phagosomal/endosomal network.
90: GENETIC CONSERVATION OF A HUMAN T CELL LEUKEMIA VIRUS TYPE 1 CLONE DURING PROGRESSION TO CUTANEOUS T CELL LYMPHOMA AND AS TRANSMISSION THEREFROM RESULTS IN MYELOPATHIC NEURODEGENERATIVE DISEASE.
Studies comparing functional differences in human T cell leukemia virus type 1 (HTLV-1) clones that mediate distinct outcomes in experimentally infected rabbits, resulted in a dermatopathic smoldering adult T cell leukemia/lymphoma following chronic infection with HTLV-1 strain RH/K34. During the 3.5 years follow up, HTLV-1 skin disease progressed to cutaneous T cell lymphoma. When infection was passed to several naive rabbits, progressive paraparesis due to myelopathic neurodegeneration, analogous to HTLV-associated myelopathy, resulted in one of 4 transfusion recipients. Similar proviral loads were detected in the two diseases, regardless of stage of progression or tissue compartment of infection. Complete proviral sequences obtained from the donor and affected recipient aligned identically with each other, and with the inoculated virus clone. Existence of disparate pathogenic outcomes following infectious transmission further extends analogy of using rabbits to model human infection and disease. Although the experimental outcomes shown are limited by numbers of animals affected, they mimic the infrequency of HTLV-1 disease and authenticate epidemiological evidence of virus sequence stability regardless of disease phenotype.
91: CYTOMEGALOVIRUS AND SIMIAN IMMUNODEFICIENCY VIRUS (SIV) COINFECTION OF CELLS IN A RHESUS MACAQUE.
Cytomegalovirus (CMV) is a common opportunistic infection in humans infected with human immunodeficiency virus (HIV) and in macaques infected with SIV. It is reported that infection with the human CMV accelerates disease progression in HIV-1 infected individuals. This may be due to either direct effects via coinfection of the same cells and transactivation of HIV-1 gene expression by CMV gene products or indirect via immune activation. In humans, coinfection of individual cells with HIV and CMV is rare and thus indirect mechanisms for enhancement of HIV disease progression are apparently more important. Recently a case of disseminated CMV in a rhesus macaque experimentally inoculated with SIVmac251 was euthanized 118 days postinoculation and evaluated for the presence of SIV and CMV coinfected cells. Histologically, the animal had disseminated CMV infection as well as SIV encephalitis and pneumonia with multinucleated giant cells (MNGC). Double labeling was accomplished using in situ hybridization for SIV and immunohisto-chemical staining for CMV on sections of lung and stomach. These tissues had the most florid histologic evidence of both CMV (intranuclear inclusions) and SIV infection (MNGC). As seen in humans, there were scattered cells that were positive for both SIV and CMV, and typically these cells were MNGC. While, the number of coinfected cells does not explain the rapid disease course this case illustrates that under certain conditions SIV/CMV coinfection can occur at a fairly high frequency (∼1% of MNGC) and may play a minor role locally.
92: AN EXPERIMENTAL MODEL OF CANINE LEPTOSPIROSIS.
To define a model of canine leptospirosis seronegative 9-week-old female beagle dogs were inoculated with Leptospira kirschneri serovar grippotyphosa (principals, n = 12) or leptospire-free culture media (controls, n = 5) via the conjunctiva. Clinical signs, clinical pathology assays, renal immunohistochemistry, pre- and post-inoculation serologic titers, tissue culture, fluorescent antibody, and necropsies were done. Control dogs remained healthy. In infected dogs, clinical signs included conjunctivitis, lethargy, diarrhea, dehydration, vomiting, icterus, and anuria/oliguria. Consistent clinical pathology alterations were azotemia, hyperphosphatemia, increased anion gap, hyperbilirubinemia, increased alkaline phosphatase, and frequently, hyperkalemia. Gross lesions were multifocal pulmonary hemorrhage (9/12), perirenal edema (5/12), icterus (8/12), and pale, friable liver (7/12). Positive immunoreactivity was observed in kidney sections using 2 different antibodies. Leptospires were cultured from kidney (11/12), urine (6/9), aqueous humor (9/12), blood (12/12), and liver (12/12). Fluorescent antibody test demonstrated leptospiral antigen in kidney (10/12), liver (12/12), and urine (5/9). Histopathologic lesions included interstitial nephritis, renal tubular degeneration and necrosis, pulmonary hemorrhage, lymphoid depletion and erythrophagocytosis, and hepatic edema, vasculitis, and perivasculitis. This model is reproducible and will be used to further define canine leptospiral pathogenesis.
93: CHARACTERIZATION OF GROSS AND MICROSCOPIC LESIONS FROM BALB/C MICE EXPERIMENTALLY INFECTED WITH HERPESVIRUS SAIMIRI-1 (HVS1).
Accidental B virus infection of humans working with macaques is often fatal and the pathogenic potential of other simian alphaherpesviruses (HVS1, HVP2) is unknown. As part of an effort to develop a murine model for simian alphaherpesvirus infections, Balb/c mice were inoculated in the hindlimb (IM) with 101 to 106 PFU of HVS1, euthanized at 21 days, and tissues collected for histopathology. Clinically, mice receiving 103 to 106 PFU of HVS1 exhibited severe, pruritic, ulcerative skin lesions at the site of inoculation, unilateral or bilateral hindlimb paralysis and severe muscle atrophy. Microscopically, lesions consisted of a necrotizing dermatitis and a nonsuppurative myelitis affecting primarily the ipsilateral dorsal horn of the thoracolumbar spinal cord with occasional extension to ventral and contralateral spinal cord structures. Immunohistochemical stains confirmed the presence of viral antigen within the lesions and anti-HVS1 IgG levels correlated with the occurrence of lesions in viral dosage groups. We conclude that HVS1 will infect mice and cause clinical signs and lesions. Interestingly, the infection in mice appears to extend into the ventral and contralateral spinal cord resulting in bilateral hindlimb paralysis. Lastly, spinal cord lesions are resolved in most animals without extension to cranial nervous system structures, i.e. cervical cord or brain.
94: PATHOGENESIS OF FIVE DIFFERENT PIGEON-ORIGIN ISOLATES OF NEWCASTLE DISEASE VIRUS FOR DOMESTIC CHICKENS.
Five pigeon-origin isolates of Newcastle disease virus (NDV) were inoculated intraconjunctivally into five groups of White Leghorn chickens. Three isolates were defined by monoclonal antibody binding profiles using the hemagglutination-inhibition test as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1). Birds inoculated with PPMV-1 isolates were euthanized at 2, 5, and 10 days post-inoculation (dpi). Tissues were sampled and examined by histopathology, by immunohistochemistry (IHC) for presence of virus, and by a TUNEL assay for apoptosis. Birds inoculated with APMV-1 isolates were sampled at 2, 4, and 5 dpi. Histologically, birds inoculated with PPMV-1 isolates had prominent lesions in heart (at 5 and 10 dpi) and brain (at 10 dpi). Presence of virus in the lesions was confirmed by IHC using an anti-peptide antibody to the nucleoprotein. Apoptosis was more prominent in lymphoid organs (bursa of Fabricius, spleen, and thymus) at 5 dpi when compared with controls using a TUNEL assay. Gross and histopathologic lesions in birds inoculated with the two APMV-1 isolates were characteristic of velogenic viscerotropic NDV (VVNDV). Extensive viral infection was evident by IHC and apoptosis in lymphoid organs was marked in the VVND-infected birds.
95: FIV MODEL OF MATERNAL-FETAL HIV-1 TRANSMISSION.
We used the feline immunodeficiency virus (FIV) model of maternal-fetal HIV-1 transmission to elucidate intrauterine virus/ host interactions which cannot be studied in humans. To determine the gestational timing of transmission, we examined 66 cesarean-derived fetuses representing 17 litters from female cats infected with FIV-B-2542. Fetal FIV infection prevalence as determined by coculture and PCR was 0%, 5%, 39%, and 60% at 3, 5, 7, and 9 weeks (term), respectively. FIV targeted fetal brain and blood mononuclear cells preferentially, followed by thymus, lymph node, marrow, spleen, and liver. At all timepoints placentas were PCR+. Maternal leukocyte counts, T cell subsets, antibody titers, and viral DNA and RNA loads were not associated with the late timing of fetal infection. Term fetal infection prevalence was similar to that seen previously in 8-week-old vaginally-delivered kittens, suggesting that normal passage through the birth canal posed little additional risk. We next evaluated virus isolates representing other FIV clades. FIV-A-Petaluma and FIV-CPgmr were transmitted to 78% and 89% of term fetuses, respectively. FIV-A-Pet produced the lowest fetal provirus burdens. Provirus was demonstrated in 100% of FIV-C but <50% of FIV-A placentas. Uniquely, FIV-A-Pet was never detected in fetal brain or liver. Taken together, our results suggest that: (a) vertical FIV transmission varies by isolate but may be more common than previously believed, (b) transmission occurs in late gestation, (c) most transmission occurs in utero rather than intrapartum, and (d) the placenta appears neither to prevent nor potentiate fetal infection risk.
96: CLONED AVIAN LEUKOSIS VIRUS-SUBGROUP J (ALV-J) INDUCES ANTIBODY NOT DETECTABLE USING EITHER OF 2 COMMERCIALLY AVAILABLE ALV-J ANTIBODY SPECIFIC IMMUNOASSAYS.
A pathogenic field isolate of avian leukosis virus subgroup J (ADOL-7501) was cloned using three terminal dilutions in cell culture. The resulting cloned virus was inoculated three and four times into 3 and 20-week-old SPF chickens, respectively. Sera (n = 27) collected one to four weeks after final exposure were examined for ALV-J antibodies using the two commercially available ELISA systems. These systems use baculovirus-expressed gp85 of ALV-J and are currently used to detect postexposure seroconversion to ALV-J, for the purposes of eradication. Experimental sera contained no ELISA-detectable antibody. However, virus neutralization testing of these same sera in vitro demonstrated neutralization titers up to 1:5,120 against homologous virus. Based on these results, an ALV-J that did not express epitopes permitting its detection by either of the presently available ELISA systems, resulted from the recombinant cloning experiments. Continued use of these ELISA systems as the mainstay of ALV-J eradication efforts is therefore suspect. Antisera produced will also be examined for in ovo use to increase resistance of hatching chicks to horizontal ALV-J exposure.
97: REAL-TIME QUANTITATIVE REVERSE TRANSCRIPTION-POLYMERASE REACTION FOR DETECTION AND QUANTITATION OF AVIAN LEUKOSIS VIRUS SUBGROUP.
A real-time system for one-tube reverse transcription-polymerase chain reaction (RT-PCR) to measure the RNA levels of avian leukosis virus subgroup J (ALV-J) was developed. The system was based on the 5’ nuclease activity of Tag DNA polymerase and fluorogenic probe which generates fluorescence when it is cleaved. The H5 and H7 primers used were known as specific for ALV-J (Smith et al. 1998). The probe, TaqMan™ probe (Perkin Elmer, Foster City, USA), is an 26bp oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with 6-FAM as reporter dye at 5’ end and TAMRA as quencher dye at 3’ end. Fluorescence was monitored in real time after each cycle by a ABI Prism- 7700 sequence detector (Perkin-Elmer). Control RNA for standard curve was prepared by in vitro transcription of a plasmid that is inserted with H5/H7 PCR product of ALV-J. The system could measure ALV-J RNA levels ranging from 25ng to 250fg and the results of cell culture material exhibited high correlation with those of quantitative competitive RT-PCR and TCID50‘s of the samples. This system was less sensitive than expected probably due to longer product size than recommended, compatibility between primers and probe, and possible other undetermined factors. However, this system allowed precise quantitation and was very specific for ALV-J. It also allowed for high sample throughout, was simpler than routine RT-PCR to perform, and can be performed with a low risk of contamination. This method will be useful for detection and quantitation in the study of ALV-J pathogenesis.
98: THE ROLE OF CD28 AND B7 IN LYMPHOPROLDPERATIVE DISEASE OF THE SCURFY MOUSE.
The scurfy (sf) mouse mutation was discovered at Oak Ridge National Lab in 1949 and has been used as a model for immunologic research. Affected animals live an average of 24 days and die of a severe generalized lymphoproliferative disease. Lesions seen in the scurfy mice include a hyperplastic dermatitis with ortho- and parakeratotic hyperkeratosis, hepato-splenomegaly, lymphadenomegaly, wasting, anemia, and thymic atrophy. Recent publications citing in vitro studies suggest that disease in the scurfy mice may be due to hyper-responsiveness of CD4+ CD8- T cell activation and/or failure of down-regulatory signals. Lymphocytes of scurfy mice have a decreased requirement for co-stimulation through CD28 and express higher levels of B7.1 and B7.2 on T cells and antigen presenting cells. To examine the effect of CD28/B7 co-stimulatory pathways of T cell activation, we created scurfy mice that lacked CD28 or B7.1/B7.2 molecules. CD28 -/- scurfy mice have a prolonged lifespan (up to 70 days) but ultimately develop scurfy disease. B7.1/B7.2 -/- scurfy mice have a prolonged lifespan (up to 98 days), and develop skin and lymphoid lesions similar to scurfy mice. However, in B7.1/B7.2 -/- scurfy mice the skin lesions are delayed and less severe and the lymphoid lesions are limited to the spleen and lymph nodes. Our findings support a role for CD28 and B7 in scurfy disease pathogenesis. However, lesions develop even in the absence of these molecules, which suggests a role for additional pathways in scurfy disease.
99: AN EXPERIMENTAL LIVER METASTASIS MODEL RETAINING EXPRESSION OF A HUMAN COLORECTAL TUMOR TARGETING GENE.
Liver metastasis is the leading cause of mortality from colorectal cancer. Guanylyl cyclase C (GCC) has been identified as a biomarker specifically expressed in human colorectal tumors and normal intestine. An experimental liver metastases model was created retaining expression of human GCC as a molecular target for development of diagnostic and therapeutic agents. Nude rats (n = 36) received intrasplenic inoculation of T-84 (2 × 107 cells), a human colorectal tumor cell line. Representative animals were terminated at weeks 3, 4, 6, 8, 10, 12, and 14, examined grossly and any neoplasms found were confirmed microscopically. Total RNA was isolated for RT-PCR from tumor tissues in the liver and spleen of several rats along with control non-neoplastic liver tissue. Primary tumors in the spleen were confirmed microscopically at weeks 3 (6/8), 4 (0/3), 6 (3/3), 8 (3/3), 10 (3/3), 12 (3/3), and 14 (5/13). Liver metastases were confirmed at weeks 3 (1/8), 4 (1/3), 6 (3/3), 8 (2/3), 10 2/3), 12 (1/3), and 14 (4/13). The overall primary tumor occurrence rate was 64%. Liver metastasis rate, 6 weeks after splenic inoculation, was 48%. Both primary and metastatic tumors retained the microscopic characteristics of the original T-84 cell line neoplasms. A single band cDNA product (∼226bp) of RT-PCR was detected in total RNA derived from tumor tissues, but not from normal rat liver. The gene expression was further verified using different GCC-specific primers.
100: OLFACTORY MUCOSAL INJURY IN EQUINE 3-METH-YLINDOLE TOXICOSIS.
Response to 3-methylindole (3MI) varies among species. Mice recover from 3MI-induced bronchiolar epithelial injury but sustain persistent olfactory mucosal injury with scarring and epithelial metaplasia. In contrast, 3MI causes persistent obliterative bronchiolitis in horses and ponies, but olfactory mucosal injury has not been reported. To evaluate the effect of 3MI on olfactory mucosa, ponies were dosed orally with 100 mg 3MI/kg (n = 9) or corn oil vehicle (n = 6). All ponies treated with 3MI developed the expected bronchiolar lesions. Mild olfactory mucosal injury was evident by light microscopy. At 3 days after 3MI, olfactory epithelium was disorganized with decreased height, uneven surface, and scalloping of the basement membrane zone. Epithelial cells of Bowman's glands were hypertrophied with enlarged nuclei. Mitotic figures and immunohistochemical labeling for proliferating cell nuclear antigen (PCNA) increased in olfactory epithelium and Bowman's glands in comparison to control ponies. Lesions were less severe at 5 days and difficult to distinguish from control olfactory mucosa at 9 days after 3MI. 11β-hydroxysteroid dehydrogenase activity, which localizes to sustentacular and Bowman's glandular epithelial cells, decreased in olfactory mucosa at 3 and 5 days after 3MI. In conclusion, 3MI damages equine olfactory mucosa without the extensive necrosis, fibrosis, and epithelial metaplasia that is seen in mice.
101: ROLE OF DNA ADENINE METHYLASE IN THE PATHOGENESIS OF DISEASE CAUSED BY NEURONINVASIVE ESCHERICHIA COLI K1.
DNA adenine methylase (Dam) regulates numerous processes within bacteria, including DNA replication and repair and gene transcription. In particular, Dam modulates the expression of at least 40 virulence genes in Salmonella typhimurium, and Dam mutants are highly attenuated for virulence in the mouse model due to decreased cytotoxicity to M cells and inability to invade enterocytes. We evaluated whether Dam was required for systemic disease caused by neuroinvasive Escherichia coli K1 by construction of Dam mutants and testing in an infant rat model. Mutants were constructed by bacteriophage P1vir-mediated cotransduction of donor Dam::kan or Dam::Tn9 mutations into a clinically relevant strain of Escherichia coli K1 (RS218R). Mutant phenotypes were confirmed by sensitivity of isolated DNA to the nonmethylated adenine-specific restriction endonuclease MboI and resistance of isolated DNA to the methylated adenine-specific endonuclease DpnI. Mutant strains were inoculated intraperitoneally into infant rats and their virulence compared to positive (wild-type) and acapsular negative (neuS::Tn10) controls. Virulence of the Dam::kan strain was unaffected by loss of Dam, whereas that of the Dam::Tn9 strain was highly attenuated. These results suggest that Dam is not required for pathogenesis in our model. Attenuation of the Dam::Tn9 strain may be due to an indirect effect of the transposon insertion.
102: CHANGES IN ANIONIC PEPTIDE IMMUNOREACTIVITY IN PROGRESSION OF OVINE PNEUMONIC PASTEURELLOSIS.
Anionic peptide (AP) is a small, antimicrobial peptide composed of seven aspartic acid residues. AP is produced in the lung of sheep, cattle and humans, and is not increased in foci of acute pneumonia. The objective of this study was to assess differences in AP production in acute, subacute, and chronic lesions of bronchopneumonia. Mannheimia (Pasteurella) haemolytica or saline was inoculated intrabronchially by fiberoptic bronchoscopy in 18 mixed breed 3-month-old lambs. The lambs were euthanatized on post inoculation days (PID) 1, 15, and 45 (n = 6, each group). Lesions in lambs inoculated with Mannheimia haemolytica included fibrinopurulent bronchopneumonia in PID 1 lambs, necrosuppurative bronchiectasis with foci of bronchiolar epithelial hyperplasia in PID 15 lambs, and extensive bronchiolar hyperplasia and bronchiolitis fibrosa obliterans in PID 45 lambs. In lungs of lambs inoculated with saline or bacteria inoculation PID 1, AP immunoreactivity (AP-IR) was present in low levels in cytoplasm of multifocal bronchiolar epithelial cells. Lung tissue of saline-inoculated lambs or lobes contralateral to bacterial inoculation lacked epithelial hyperplasia and AP-IR was mild. In bacterially-inoculated sheep euthanatized PID 15 and 45, cytoplasmic AP-IR was intense in hyperplastic bronchiolar epithelial cells, suggesting that AP production is increased in hyperplastic cells in areas of chronic pneumonia. AP may protect these areas from secondary infections during lesion resolution.
103: DIHYDROCAPSAICIN TREATMENT DEPLETES PEPTIDERGIC NERVE FIBERS OF SUBSTANCE P AND ALTERS MAST CELL DENSITY IN THE RESPIRATORY TRACT OF NEONATAL SHEEP.
Dihydrocapsaicin (DHC) was administered to neonatal lambs in order to deplete C-fibers (substance P [SP] fibers) in the respiratory tract. We measured the density of SP-fibers in nasal septum to assess the effectiveness of the treatment at 3, 9, and 21 days. The numbers of mast cells in the upper and lower respiratory tract were determined at the same time points and histamine content was determined from lung tissue. DHC treatment depleted SP-fibers by 3 days and up to the 21-day time point. This depletion was estimated as 85% in comparison with controls. In both, vehicle- and DHC-treated lambs, numbers of mast cells increased progressively with time; however, the density of mast cells was augmented in the entire respiratory tract of DHC-treated animals. Apparently, DHC treatment exerts a single and initial effect in increasing mast cells whereas time maintains a continuous influence; both factors exert their influence independently. Despite large numbers of mast cells in DHC-treated animals, histamine content in the lung had similar levels as controls. Our study provides fundamental data and develops a model for a better understanding of conditions that may influence lung formation, pulmonary defense, and inflammatory processes dependent on the mast cell-nerve axis in the respiratory tract.
104: CYTOKINE EXPRESSION IN FOALS INOCULATED WITH VIRULENT RHODOCOCCUS EQUI IS ASSOCIATED WITH INCREASED PULMONARY ACTIVATION OF NUCLEAR FACTOR-κ (NF-κB).
The virulence of Rhodoccocus equi in foals is dependent on the presence of an 85- to 90-kb plasmid. Previous studies revealed foals inoculated with a plasmid-containing virulent R. equi had significantly higher pulmonary levels of IL-1β, IL-10, IL-12 p40 and TNF-α mRNA compared to foals inoculated with a plasmid-cured derivative. The purpose of this study was to determine if the increases in cytokine expression were associated with increases in pulmonary activation of NF-κB. Electrophoretic mobility shift assays (EMSA) were performed on nuclear protein extracted from the lungs of foals inoculated with either PBS, virulent or avirulent R. equi (n = 4). NF-κB activation was increased in the nuclear extracts from foals inoculated with virulent R. equi compared to those inoculated with either PBS or avirulent R. equi at the same time points increased cytokine expression was observed. UV-linked EMSA run in parallel with western analysis revealed the forms of NF-κB binding DNA contained the p65 and p50 subunits in all the foals. Western analysis for IκB found that both IκBα and IκBβ protein expression was increased in foals 14 days after they were inoculated with the virulent R. equi. These results indicate part of the cytokine response to R. equi may be mediated by NF-κB. activation.
105: IL-12 TREATMENT REDUCES SEVERITY OF SENDAI VIRUS-INDUCED AIRWAY LESIONS IN BN RATS.
We previously determined that Sendai virus-resistant F344 rats transcribe higher levels of pulmonary IL-12 p40 subunit mRNA early after viral infection than virus-susceptible BN rats. We hypothesized that administration of IL-12 to BN rats could convert their Th2-type cytokine profile to a Th1-type response and increase their resistance to virus-induced airway injury. BN rats were inoculated with Sendai virus and studied at 0, 3, 7, and 14 days following inoculation. Groups of BN rats were treated i.p. with recombinant IL-12 at 0 and 2 days after virus inoculation. IFN-gamma mRNA was detected with rat-specific primers in a competitive RT-PCR assay. In the IL-12-treated and non-treated infected rats, airway inflammation and fibrosis at 14 days following inoculation was reduced in the 0-day IL-12-treated BN rats as compared to the virus-infected controls. However, the 2-day IL-12-treated BN rats had inflammation and fibrosis that was increased compared to the virus-inoculated controls. IFN-gamma mRNA was increased in rats at 3 days postinoculation in the 0-day IL-12-treated BN rats as compared to the other groups. Results indicate that administration of IL-12 can shift the virus-susceptible BN rat to a more resistant cytokine profile and reduce severity of virus-induced airway lesions. (NTH HL56396)
106: EARLY DIFFERENTIAL GENE EXPRESSION IN SENDAI VIRUS RESISTANT F344 RATS.
BN and F344 rats differ markedly in their susceptibility to chronic airway injury and asthma-like disease induced by Sendai virus infection. Early gene responses at 2 and 3 days following virus inoculation were compared. Virus-resistant F344 and virus-susceptible BN rats were inoculated with virus at 25-days-of-age, and lung was collected for differential expression study at 2 and 3 days later. Pulmonary differential gene expression was studied by suppressive subtractive hybridization and differential screening. Comparison of gene expression patterns was performed by dot-blot hybridization. The differential screening results were confirmed by competitive quantitative RT-PCR. More than 1000 clones were picked for differential screening, respectively from F344 and BN subtracted cDNA libraries. Seven potential positive clones were further selected for sequence analysis. One of them was rat IP-10. Two clones were highly homologous to mouse interferon regulatory factor 7 (IRF-7). mRNA abundance of both IP-10 and IRF-7 was significantly up-regulated in F344 and BN rats following virus infection. IP-10 mRNA abundance in F344 was two-fold higher of that in BN rats. Since IP-10 has been identified as a factor down-regulating pulmonary fibrosis and angiogenesis, early IP-10 expression in F344 rats could contribute, at least in part, to their resistance to virus-induced airway fibrosis and asthma-like syndrome. (NIH HL61018).
107: EFFECT OF AMNIOTIC FLUID AND MECONIUM IN THE LUNGS OF NEONATAL RATS.
Meconium aspiration syndrome (MAS) is a major contributor to neonatal respiratory distress in infants and it has been recognized in foals, calves and sheep. There are few reliable animal models to evaluate the inflammatory response associated with MAS. The objectives of this investigation were: 1) to standardize a model of MAS using intratracheal inoculation (ITI) in 7-day-old Fisher-344 rats; 2) to evaluate the effect in the lungs after ITI of amniotic fluid and meconium. Groups of neonatal rats were inoculated with saline solution, amniotic fluid and meconium and their lungs were processed for histopathology and electron microscopy. Saline did not cause any change. Amniotic fluid only elicited a mild foreign body response. Meconium induced an exudative alveolitis, sequestration of PMN, aggregation of platelets, atelectasis, hyperinflation and significant (p < 0.01) thickening of alveolar septa. The early response progressed into a persistent multifocal histiocytic and granulomatous reaction. Some foci eventually calcified or became lined by cuboidal cells. There was hyperplasia of type II pneumocytes, interstitial proliferation of mesenchymal cells and intra-alveolar fibrosis. The meconium and the proliferative response remained visible up to 112 days PI. It was concluded that ITI of meconium in neonatal rats provides a reliable animal model for human MAS.
108: REDUCTION IN HELICOBACTER PYLORI COLONIZATION BY IMMUNIZED MICE IS ASSOCIATED WITH INCREASED GASTRIC mRNA FOR TH1 CYTOKINES AND INDUCIBLE NITRIC OXIDE SYNTHASE.
Mucosal immunization of mice using Helicobacter pylori antigens combined with a mucosal adjuvant results in a reduction in bacterial load following challenge with H. pylori. We and others have shown that clearance of H. pylori does not require antibody production but is dependent on CD4+ T cells. To further investigate the mechanism of protection in mice, we isolated RNA from the gastric mucosa of naive, immunized/challenged (I/C), and unimmunized/challenged (U/C) mice 4 weeks following challenge with 10 million cfu of H. pylori SS1. Semi-quantitative RT-PCR was performed for INFγ, IL-12p40, TNFα, iNOS, KC, MCP-1, MIP-2, and HPRT (a housekeeping gene). At sacrifice, colonization was determined by quantitative culture of gastric mucosa from each mouse, and protection is defined as a 2 log decrease in bacterial load. Correlations of colony counts and cytokine levels from individual mice indicated that immunization was associated with increased levels of each of the 3 chemokines whether or not the mice were protected. I/C mice also had increased gastritis scores compared to naive and U/C mice. Protection was correlated with increased levels of IFNγ, TNFα, IL-12p40, and iNOS. The requirement for IFNγ, IL-12 and iNOS in reduction of bacterial load is’ currently being investigated in mice deficient in these products. In the first experiment, I/C IFNγ -/- mice had a greater than 2 log reduction in cfu compared to U/C IFNγ -/- mice, but I/C IL-12 -/- mice were not protected when compared to U/C mice. These preliminary results suggest that IL-12 may play a greater role in protection than IFNγ.
109: FETAL INOCULATION WITH FELINE IMMUNODEFICIENCY VIRUS (FIV) RESULTS IN ATTENUATED LYMPHOID TISSUE LESIONS AND ENHANCED VIRUS LOAD.
Human fetuses infected with HIV have severe thymus dysfunction and high virus load as compared to infected neonates or adults. To study this response, we inoculated fetal cats with FIV and evaluated the thymus and popliteal lymph nodes for lesion severity, lymphocyte phenotype, and virus load. These data were compared to litter-matched, sham-inoculated fetuses and neonates that received an identical FIV inoculum. All cats were evaluated at 23 or 46 days post-inoculation (pi). T-lymphocyte subsets in the peripheral blood of fetal FIV inoculates were stable despite a severe reduction in thymus weight at 23 days pi. The popliteal lymph nodes in these cats were of similar size and cell composition as controls. In comparison, cats inoculated as neonates had a gradual reduction in thymus weight, which was inversely related to changes in lymph node weight. Both tissues contained a proportionate increase in CD8+ and IgG+ lymphocytes. Numbers of productively infected cells were stable at 23 and 46 days after neonatal inoculation. In contrast, fetal inoculation resulted in a progressive increase in infected cells and a corresponding surge in plasma viremia. Together, these data suggest that local immunity within lymphoid tissues may control virus replication more effectively in neonates than in fetuses.
110: OXIDANT-INJURED AIRWAY EPITHELIAL CELLS UPREGULATE THIOREDOXIN BUT DO NOT PRODUCE IL-8.
Thioredoxin (THX), a unique chemoattractant produced by airway epithelium, is important for neutrophil transepithelial migration. IL-8, a CXC cytokine, is also produced by conducting airway epithelium and likely plays a role in neutrophil migration to sites of injury. Our goal was to characterize the phenotype of THX and IL-8 producing cells in airway epithelium following oxidant injury. We tested the hypothesis that oxidant-injured cells upregulate THX while oxidant-stressed, but not injured, cells upregulate IL-8 after injury. We exposed a monolayer of transformed human bronchial epithelial cells (BEAS-S.6) to 0, 200, 400, or 600 μM H202 for 1 hour and incubated for an additional 7 hours. Subsequently, the cells were double labeled with markers of epithelial injury (either Ethidium homodimer-1 for cellular injury or MitoTracker dye for functional mitochondria) and chemoattractants IL-8 or THX. Using stratified random sampling, we estimated the number of injured cells that were positive for IL-8 or THX in response to doses of H2O2. We also estimated the volume density of IL-8 or THX per cell in response to doses of H2O2. We found 1) significantly more cellular and mitochondrial injury with increasing H2O2 dose 2) a significant inverse relationship between IL-8 positive cells and injured cells with increasing H2O2 dose 3) a significant inverse relationship between the volume of IL-8 staining per cell and increasing H2O2 dose and 4) the volume of THX staining per cell was directly related to cell injury. We conclude that oxidant injured airway epithelial cells upregulate THX, but do not produce IL-8 which may be important in multistep navigation of neutrophils to a site of oxidant injury. Supported by NIEHS Grant ES00628 and NIH Grant ES09701.
111: DI(N-BUTYL) PHTHALATE-INDUCED TESTICULAR LESIONS FOLLOWING IN UTERO EXPOSURE: PRIMARY AND SECONDARY PHENOMENA?
Di(n-butyl) phthalate (DBP) has been previously shown to have antiandrogenic effects on male rats exposed in utero. To characterize the time course of testicular lesion development with in utero DBP exposure, time points earlier than previously examined, postnatal days (PND) 45 and 70, were chosen as end-points. Three Sprague-Dawley dams in each age group were gavaged with 500 mg/kg/day DBP in corn oil on gestation days 12–21. Histologic lesions noted in the testes at PND 45 were dilation of the rete testis and seminiferous tubules and mild degeneration of the seminiferous epithelium. At PND 70 there was dilation of the rete testis, severe degeneration of the seminiferous epithelium, and hyperplasia of Leydig cells. Epididymal lesions were similar in both age groups with malformations, including partial and complete absence, and decreased epididymal size. The lesions seen at PND 70 were consistent with those described at PND 100 and were due, at least in part, to increased intratubular pressure secondary to severe malformations of the epididymis. The mild lesions described in the testes at PND 45 may have been due to increased tubular pressure secondary to epididymal malformations but may also have been a primary effect of in utero treatment as lesions were seen in testes with histologically normal epididymides. Lesions seen following a short in utero exposure are noteworthy as they signify “imprinting” of fetal tissues for lesion development later in life.
112: INTEGRATING TRANSCRIPTOMICS INTO TOXICOLOGY: A STEEP LEARNING CURVE FOR WHICH THE PATHOLOGIST IS IDEALLY SUITED.
Gene expression patterns, in the form of mRNA transcripts, provide a wealth of information related to mechanisms of disease. Recent developments in molecular biology permit rapid assessment of the expression levels of thousands of genes simultaneously. To use these data, however, we must learn to ‘interpret the transcripts’ and exploit the information derived from them. In toxicologic pathology, through many years of investigative research, morphologic criteria have been developed to assist in the interpretation process. Such criteria are based upon a firm foundation of morphology (anatomy, cytology, histopathology) and cell physiology. Equivalent approaches are needed for transcriptomics, the study and interpretation of patterns of mRNA expression, before these rich data sets will be effectively combined with toxicologic pathology for human disease risk assessment purposes. Transcriptomics also needs to be based upon a firm scientific foundation, including knowledge of underlying transcriptional regulators, relationships of transcriptional and post-transcriptional control networks, and other related features of cell physiology and disease. Using oxidative stress as an example, such interpretative processes will be presented and discussed, with respect to the incorporation of transcriptomics into the professional lives of toxicologic pathologists.
113: MOLECULAR PROFILING OF HEPATOTOXICITY INDUCED BY AMINOGUANIDINE CARBOXYLATES (AGC)-A COMPARATIVE EXPLORATORY STUDY.
AGC's act to lower blood glucose and decrease body weight in animal models of Type 2 diabetes. Methods for the detection of differential gene and protein expression and NMR spectroscopy were applied to identify markers for hepatotoxicity seen with some AGC's. The approach included a repeated-dose study and a single dose study with histopathology and in vitro experiments with the goal to select a suitable model system for toxicity screening of new AGS-s. Sprague-Dawley rats, 5 males/group, were given AGC once daily by gavage at 0, 2.5, 10 and 50 mg/kg/day for 14 days. Rats from all treated groups showed vacuolar degeneration in cytoplasm of hepatocytes. Single cell necrosis was observed at 10 mg/kg/day and above. Liver gene expression was analyzed using the rat toxicology microarray from Affymetrix containing probes for ∼850 genes. Expression of 23 of these genes was up regulated and 15 genes were down regulated >4-fold. Furthermore, 98 genes were >2-fold up- or down-regulated. By 2D-gel electrophoresis together with image/data analysis 140 proteins were only detected in the livers of the control animals. Two unique proteins were identified in livers from rats given 50 mg/kg/day. Furthermore 26 proteins were >4 times up- or down-regulated. NMR data from urine were processed using multivariate analysis to find metabolic patterns indicative for toxicity. The high-dose animals clustered separately and markers for liver toxicity were identified. The results of the different molecular profiling techniques, which indicated hepatotoxicity, were correlated with each other and the observed histopathological findings.
114: UTILIZING TOXICOGENOMICS TO SEARCH FOR GENE EXPRESSION MARKERS OF LIVER CARCINOGENICITY.
Chronic administration of high doses of certain drugs and chemicals induces hepatocellular tumors in rodents. In many cases the exact mechanism of tumorigenesis is unknown. Previous work in our laboratories resulted in the identification of rat liver genes that were up-regulated or down-regulated following 2 or 13 weeks of treatment with the prototypical non-genotoxic carcinogen phenobarbital. To determine whether changes in gene expression patterns following short-term treatments with hepatocarcinogens could allow early prediction of tumorigenesis, we treated male Sprague Dawley rats daily for 5 days with the non-genotoxic carcinogens bemitradine, clofibrate, phenobarbital, doxylamine, or methapyrilene; the genotoxic carcinogens 2-AAF or tamoxifen; or the non-carcinogens 4-AAF or isoniazid. Fluorescently-labeled cDNAs made from individual treated animals and pooled control animals were co-hybridized to cDNA microarrays (Incyte RatGEM 1.0). Gene expression changes that correlated with toxicological endpoints such as body weight change, food consumption, and clinical chemistry endpoints were eliminated from consideration. Preliminary analysis suggests it may be possible to identify gene expression patterns associated with carcinogenicity and DNA damage.
115: USE OF LASER CAPTURE MICRODISSECTION TO ANALYZE SEGMENTAL RENAL GENE EXPRESSION IN NEPHROTOXICITY.
Gene expression studies aimed at understanding mechanisms of toxicity and developing screens to evaluate compounds have the potential to provide valuable data for drug safety assessment. However, the use of homogenized kidney samples for these studies may result in an ‘averaging out’ of the heterogenous elements of the sample as certain nephrotoxic compounds primarily affect specific cells/segments of the nephron. Therefore, the use of laser capture microdissection (LCM) to select populations of cells to extract RNA from may have advantages over RNA extracted from homogenized rat kidneys. Known genes identified as being differentially expressed in kidney following treatment with nephrotoxins using Suppressive Subtractive Hybridization (SSH) and microarrays were examined using the LCM approach. This revealed 1) whether experiments using homogenized tissue can identify genes whose expression is affected in a subpopulation of cells rather than in all cell types within the tissue, and 2) permitted comparisons of the applicability and limitations of both approaches individually and in combination.
116: DNA DAMAGE MAY PLAY A MAJOR ROLE IN PARAQUAT-INDUCED MITOTIC ARREST AND CELL DEATH IN HEP-G2 CELLS.
Paraquat (PQ) produces superoxide that is converted to hydroxyl radical with resulting toxicity. Patterns of gene expression which may shed light on mechanisms of chemically-induced cell damage, were investigated in HEPG2 cells exposed to 0 or 600 μM PQ (EC50 for cytolethality) for 4, 12 or 24 hrs. Confluent HEP-G2 cells were cultured on collagen-coated dishes, containing a glass cover slip with DMEM-10%FCS, 37 C, 5% CO2. Cells were lysed and poly A+mRNA was purified and reverse-transcribed with 33P-dATP. The [33P]-cDNA was hybridized to a gene expression array (Atlas Human cDNA Expression Array, Clontech, Palo Alto, CA). Levels of up- or down-regulation were estimated to yield mRNA expression patterns. HEP-G2 cells had PQ-induced loss of intracellular droplets, abnormal metaphase, absence of telophase stages of mitosis (control ∼10/high power field) at 12 and 24 hours, and scattered degenerating cells. Gene expression patterns included evidence of early response (increased c-jun and fra-1), DNA damage and mitotic arrest (increased GADD45 and p21 and decreased cyclins and top2). Genes on the array expected to respond to oxidative stress (e.g., Cu/Zn SOD, GPx1, and GSH reductase) were unaffected, as were apoptosis-associated genes (e.g., adenosine A1r, bc1-x, apo-1, caspases, p53). Observations were consistent with a significant role for DNA damage in PQ-induced cell death and mitotic arrest. Decreasing medium depth increased toxicity, suggesting the importance of oxygen and redox cycling in the mechanism(s) of toxicity.
117: TOXICOLOGIC PATHOLOGY ASSOCIATED WITH USE OF A GENE GUN (PARTICLE MEDIATED DNA DELIVERY).
A DNA vaccine for the treatment of Hepatitis B virus (HBV) infection is currently under development at Glaxo Wellcome in collaboration with PowderJect Vaccines Inc. This product utilizes the concept of ‘particle mediated DNA delivery’, in which a novel device (gene gun) is used for the cutaneous delivery of gold beads coated with plasmid DNA encoding HBV surface antigen (HBVsAg). With these vaccines the regulatory agencies require the use of a relevant animal model in the preclinical safety program to assess local tolerance of the device, systemic toxicity, biodistribution of the plasmid, and genomic integration. We validated minipig skin as a suitable human skin model by measuring several parameters at the intended inoculation sites, including epidermal thickness and Langerhans cell density. Overall, good similarity between the two species was demonstrated. The gene gun was well tolerated during dosing to the ventral abdomen of the minipig and only mild lesions were observed including erythema and inflammatory cell infiltration in the upper dermis. The fate of gold particles was monitored in the post-dosing interval and they were shown to be almost completely eliminated from the epidermis by 28 days. Expression of the plasmid DNA in transfected epidermal cells was confirmed by in situ hybridization and immunohistochemistry for HBV mRNA and surface antigen, respectively. An intranuclear gold particle was often present in a cell that exhibited a positive signal for either HBV mRNA or protein. Finally, PCR was used to assess the fate of plasmid DNA in the injection sites, drainage lymph nodes and internal organs at various time points following dosing.
118: LESIONS OF ADENOVIRALLY TRANSFERRED PULMONARY IL-13 OVEREXPRESSION IN C57BL/6 MICE.
Data from animal models and human asthmatics implicate IL-13 as a major mediator of lung lesions. To observe effects of high IL-13 levels alone in lungs, we administered adenovirus vector carrying mIL-13 cDNA that expresses IL-13 for prolonged periods thus producing continuous lung exposure. Groups of either 2 (vehicle) or 3 control adenovirus-expressing alkaline phosphatase (AdSSEAP) and 3 IL-13 expressing adenovirus (Ad5IL-13) female C57BL/6 mice were sacrificed and their lungs examined microscopically 7, 14, 23, 28, 35, and 42 days after intranasal exposure. Lungs from vehicle-treated mice were within normal limits at all timepoints. Mice administered Ad5SEAP developed minimal to mild peribronchiolar and perivascular lymphocytic infiltrates on days 14, 23, and 28; by days 35 and 42 Ad5SEAP lungs were within normal limits. Mice administered Ad5IL-13 had prominent lesions at day 7 that progressed to severe lesions by day 42. Initial leukocyte infiltrates were perivascular and peribronchial lymphocytes, macrophages, neutrophils, and eosinophils; later infiltrates included diffuse alveolar and interstitial lymphocytes, neutrophils, eosinophils, macrophages, multinucleated giant cells and plasma cells. Bronchiolar changes were epithelial degeneration, necrosis, and hyperplasia, and increased mucus production. Epithelial cells, bronchiolar lumens, and giant cells occasionally had large linear eosinophilic crytalline bodies. By day 42, vessels had prominent smooth muscle hyperplasia. In this study, C57BL/6 mice administered high concentrations of IL-13 via an adenovirus vector develop marked pulmonary lesions many of which are similar to lung lesions in chronic asthmatics. These results support the hypothesis that IL-13 may be a key mediator of asthmatic lesions.
119: HEPATIC CHANGES IN A MACAQUE MODEL OF ADENOVIRAL GENE THERAPY.
120: NEPHROTOXICITY OF CI-1029, THE HIV PROTEASE INHIBITOR, IN HUMANS AND NONHUMAN PRIMATES.
The nephrotoxic potential of antiviral protease inhibitors is not well characterized. CI-1029 is an aspartyl protease inhibitor with anti-HIV activity. Male and female Cynomolgus monkeys received CI-1029 at 200, 500 and 750 mg/kg. Monkeys at 750 mg/kg became moribund and died or were sacrificed by week 2; the remaining groups were treated for 4 weeks. To study reversibility, a subset of monkeys from the 500 and 750 mg/kg groups were withdrawn from drug and sacrificed. Renal lesions in monkeys treated with 500 and 750 mg/kg were dose-related. Distal convoluted tubules showed epithelial degeneration and necrosis with cellular casts. By day 9, serum creatinine levels were increased 2 to 4-fold in the 750 mg/kg group and correlated with tubular injury. In phase I clinical trials, 2 of 8 volunteers given 8 or 16 mg/kg of CI-1029 as a single dose developed renal dysfunction with BUN of 13 and 33 mg/dL and serum creatinine of 1.8 and 3.5 mg/dL respectively. After drug withdrawal of 4 weeks in monkeys and 3 weeks in humans, renal function was normal. Monkeys given 200 mg/kg had no renal lesions at plasma drug exposures up to 28 times higher than 2 affected volunteers. These CI-1029 data demonstrated nephrotoxic properties of anti-HIV compound, and relative species sensitivity when comparing humans with nonhuman primates.
121: LYMPHOMAS AND OTHER HEMATOPOIETIC NEOPLASMS IN GENETICALLY ENGINEERED AND CONVENTIONAL MICE.
Lymphomas are commonly naturally-occurring neoplasms of mice while other hematopoietic tumors are less common. The incidence and type of tumor depends on the strain or stock of the mouse. Some lines of genetically-engineered mice (GEM) have high incidences of these lymphomas. Application of the human hematopoietic lymphoma/leukemia classifications may be useful. The classification of lymphomas and other hematopoietic tumors in mice can be successful using appropriate necropsy protocols, specific tissue fixatives, immunohistochemistry for detection of antigen expression and the analysis of molecular changes. Formalin-fixed, paraffin-embedded tissues can be used with specific protocols to detect T and B lymphocytes and histiocytes in tumors. Follicular lymphomas of splenic or mesenteric lymph node origin are the most common spontaneous lymphomas of the majority of mouse strains or stocks while thymic lymphomas are the most common lymphoma in GEM, and can be induced by chemicals, viruses and irradiation in mice. The origin and histogenesis of these tumors can best be studied by serial sacrifice experiments. By these methods, we have found novel tumors of mice including marginal zone lymphomas and T-cell rich, B-cell lymphomas.
122: THE Tg rasH2 MOUSE MODEL FOR CARCINOGENICITY TESTING.
The Tg rasH2 mouse, containing multiple copies of the human c-H-ras proto-oncogene and promoter, has been proposed as a model for rapid chemical carcinogenicity testing. These mice have enhanced sensitivity to development of spontaneous and chemically-induced neoplasms early in life. The most common spontaneous neoplasms are lung adenomas and carcinomas, splenic and cutaneous hemangiosarcomas, thymic lymphomas and thymomas, and epithelial neoplasms of forestomach and skin. In the ILSI protocol for testing carcinogenicity of chemical agents in Tg rasH2 mice, 15 mice per sex per treatment group are treated for six months, all major organs are examined microscopically, and tumor incidence data are analyzed statistically. The recommended positive control treatment is a single IP injection of 75 mg/kg methylnitrosourea. Of 34 mutagenic and nonmutagenic compounds tested in the Tg rasH2 mouse model, 83% of these chemicals produced concordant results with conventional rodent bioassays. For 16 nonmutagenic agents, the results from the Tg rasH2 model demonstrated 75% concordance with conventional rodent bioassays. Compounds that produced statistically significant increases in specific neoplasms were considered positive. Compounds producing increased incidences of neoplasms above historic control ranges that were not statistically significantly different from control values were considered equivocal. The U.S. FDA Carcinogenicity Assessment Committee has determined that the Tg rasH2 model has been adequately evaluated to consider for use with pharmaceutical candidates. We are requesting to use the Tg rasH2 mouse model as an alternative to a life time mouse bioassay for testing the carcinogenic effects of an oral pharmaceutical candidate.
123: 26-WEEK CARCINOGENICITY BIOASSAYS IN GENETICALLY-ALTERED Tg-rasH2, p53± AND FVB/Tg.AC MICE WITH D-MANNITOL AND DIELDRIN.
As part of an ILSI-sponsored international collaborative research program on Alternatives to Carcinogenicity Testing (ACT), our laboratory evaluated D-mannitol (classified as non-carcinogen) and dieldrin (classified as rodent carcinogen/putative human noncarcinogen based on epidemiology) in 3 genetically-altered rodent models, namely the p53± knockout mouse (heterozygous for p53 tumor suppressor gene), and the Tg-rasH2 and FVB/Tg.AC transgenic mice (Ha-ras transgene-carrying models). Bioassays consisted of 26-week dietary studies, except for the dieldrin study in FVB/Tg.AC mice in which the compound was tested topically. All studies included a vehicle control, an established positive control (dependent on model/route) and the evaluated compound at 3 dose levels. In addition, a nontransgenic (background strain) vehicle control and high-dose group were included in most studies. In all assays, there were 15 mice/sex/group. The positive control compounds consisted of p-cresidine, N-methyl-N-nitrosourea (MNU) and 12-O-tetrade-canoylphorbol-13-acetate (TPA) for the p53± Tg-rasH2 and FVB/Tg.AC assays, respectively. D-mannitol was administered in the diet at 2.5, 5.0, or 10.0%. Dieldrin was given in feed at concentrations of 1, 5, and 10 ppm or 1, 10 and 20/10 ppm. In the study involving FVB/Tg.AC mice, dieldrin was administered on the skin surface at doses of 0.03, 0.3, or 1.0 mg/kg/day. Study endpoints included toxicokinetic analyses, comprehensive histopathology and, if appropriate, tumor genotyping. Results of the studies conducted in our laboratory are discussed.
124: TOXICOLOGIC ASSESSMENT OF B LYMPHOCYTE STIMULATOR (BLYS), A NOVEL TNF FAMILY LIGAND, IN MICE.
B Lymphocyte Stimulator (BLyS), a member of the tumor necrosis factor family of ligands, is a 152-amino acid soluble protein with a molecular mass of 17 kD. A 4-week GLP study was conducted to assess the toxicity of BLyS in mice. In this study, 15 mice/gender/dose were injected subcutaneously (SC) 5 days/week for 4 weeks with BLyS doses of 0 (vehicle), 0.03, 0.3, or 3.0 mg/kg. Five of the 15 mice/gender/dose were allowed to recover for 2 weeks following treatment. BLyS-induced changes in clinical pathology parameters included increases in globulins, IgA, IgG, IgM, and IgE, and a decrease in A/G ratio. At necropsy, splenic weights were increased and microscopic changes consisted of lymphoid hyperplasia in the spleen and inflammation at the injection site at all BLyS doses. Splenic plasmacytosis and lymphoid hyperplasia in mesenteric and mandibular lymph nodes and gut-associated lymphoid tissues were detected only at the high dose. All these changes reversed after a 2-week recovery period. It was concluded from this study that the administration of BLyS to mice was well tolerated and induced lymphoid hyperplasia in B cell areas, with subsequent plasmacytosis and increased serum immunoglobulin levels. These findings were considered to result from the B cell-specific pharmacodynamic effects of BLyS, and are consistent with its in vitro activity as a B lymphocyte stimulator.
125: DIFFERING SUSCEPTIBILITY OF INBRED STRAINS OF MICE TO INHALED RICIN INTOXICATION.
Groups of six mice for each of seven inbred strains (A/J, BXSB, BALB/c, CBA/J, C57BL/6J, C3H/HeJ, C3H/HeN) and an outbred stock (NIH Swiss) of laboratory mice received whole-body graded doses of cGMP ricin of plant-origin as small-particle respirable aerosols. At a given lethal dose, BXSB, CBA/J, and C3H/HeJ mice died sooner than C57BL/6J, A/J, C3H/ HeN and Swiss mice, but BALB/c mice survived until study termination. To study subacute lesions, 31 mice that died on day five or later were examined histopathologically as were two mice killed on day 13 with head tilt due to otitis. Lesions included subacute pulmonary inflammation (31/33; most severe in BXSB and C57BL/6J), pulmonary spindle cell proliferation, (30/33; most severe in C57BL/6J), alveolar emphysema (20/33; most severe in A/J), renal perivascular edema (30/33; most severe in C3H/HeJ and BALB/c), and unilateral otitis media and interna (4/33; 3 were C3H/HeJ). Cause of death was attributed to direct and indirect effects of toxin. Renal perivascular edema was a possible manifestation of vascular leak syndrome. Pulmonary fibroproliferation was reminiscent of bleomycin-induced pulmonary fibroplasia, a toxin-induced condition with a known susceptibility in C57BL/6 mice.
126: INHIBITION OF ATHEROSCLEROSIS EN RABBITS BY DIETARY HEMATEIN.
The dried heartwood of Caesalpinia sappan has been used in oriental medicine as an analgesic and anti-inflammatory agent. Hematein, one of the compounds isolated from Caesalpinia sap-pan, has an inhibitory action on Cu2+-induced LDL oxidation. In this study, we examined the antiatherogenic potential of hematein using cholesterol-fed New Zealand White rabbits. After 8 weeks of treatment of probucol and hematein, the plasma lipoprotein profile was measured and the extent of fatty streak lesions in thoracic aorta was investigated. The severity of fatty streak lesions was histologically examined. We also observed the effect of hematein on monocyte chemoattractant protein-1 (MCP-1) expression in the aorta by northern blots. All rabbits developed the Oil red O-positive fatty streak lesions at the sub-endothelial space and the elastic lamina was destroyed as the lesion progressed. The lesions were significantly reduced in hematein- and probucol-supplemented group without changing plasma lipoprotein levels. The expression of MCP-1 in thoracic aorta was significantly reduced in the hematein-supplemented group compared to the control group. The results of the present study suggest that the anti-atherogenic effect of hematein is not related to controlling plasma lipid profile but probably related to inhibition of expression of MCP-1 resulting in amelioration of the lesion development. However, further studies will be needed to determine the mechanism of the anti-atherogenic action of hematein.
127: ERB B RECEPTOR INHIBITOR, CI-1033, INDUCES DEGENERATION AND/OR ATROPHY IN DIGESTIVE TRACT, KIDNEYS AND TESTES OF MONKEYS.
Epidermal growth factor receptor family, including erbBl-4, regulates epithelial cell function, differentiation, and proliferation. CI-1033 is a candidate anticancer agent that inhibits erbBl-4 receptor activity by irreversibly attaching to the ATP binding pocket of tyrosine kinase receptor. CI-1033 was studied in cynomolgus monkeys treated once daily for 7 days; females received 5, 10 or 20 mg/kg and males received 5, 10 or 35 mg/ kg. The severity of toxicity was proportionate to dose and corresponding plasma drug concentration. Clinical signs included diarrhea, emesis and dehydration with associated electrolyte changes. Increased BUN (4 to 10-fold) and serum creatinine (3 to 7-fold) levels correlated with renal tubular epithelial necrosis and cellular casts in dilated distal convoluted tubules. Erosions were noted in stomach and large intestine. Mucosal atrophy occurred in tongue, esophagus, and stomach; small intestine showed blunted villi with compensatory hyperplastic crypts. Hyperplastic colonic crypts were lined by abnormal epithelia. Decreased testicular weights correlated with hypocellular spermatogenic epithelia, syncytial cell formation and atrophy of prostate and seminal vesicles. Toxicity fully reversed at 5 mg/kg in males and females following 8 weeks of treatment cessation. Drug-related changes were consistent with expected pharmacological effects of erbB receptor inhibition.
128: TRANSCRIPTIONAL RESPONSES TO UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION.
Uncouplers of oxidative phosphorylation have relevance to bioenergetics and obesity. Studies were designed to identify genetic markers (mRNA) for uncouplers of mitochondrial oxidative phosphorylation using FCCP (carbonyl cyanide p-(trifluo-romethoxy)phenylhydrazone), a classical uncoupling agent, in a mouse skeletal muscle cell line (ATCC CRL-1456). Changes in mitochondrial membrane potential were used as the biological dosimeter. The cells were exposed in suspension to 100 nM–10 μM FCCP. Membrane depolarization correlated positively with increasing concentrations of FCCP (34% at 100 nM; plateau of 80% at 4 μM). Intracellular ATP concentrations exhibited a linear decrease with increasing doses of FCCP. The concentration at plateau (4 μM) was chosen to study gene expression responses using Clontech microarray platforms (Atlas Mouse 1.2’ and Mouse stress’). Changes in gene expression were observed after 6 hr exposure to FCCP. Significantly up-regulated genes included: mitogen and stress-activated protein kinase 2, several cathepsins, oxidative stress-induced protein mRNA, osp 94 osmotic stress protein, quinone oxidoreductase and quinone reductase. Significantly down-regulated genes included myogenic factor 5, phospholipase A2, ubiquitin, cyclin dependent kinase inhibitor 1, cellular tumor antigen p53 and p53-associated protein (MDM2). It was concluded that gene expression changes may provide a sensitive indicator of uncoupling in response to chemical exposure.
129: VASCULAR MALFORMATIONS IN THE Tg rasH2 MOUSE.
The transgenic CB6F1-TgN (RasH2) mouse, more commonly known as the Tg rasH2 mouse, has been proposed as a model for rapid carcinogenicity testing of genotoxic and non-genotoxic chemicals. The genome of these mice contains multiple copies of the human c-H-ras proto-oncogene and promoter. Anomalous vascular channels were observed in multiple organs of 3/120 male mice and 8/120 females mice in a 6-month Tg rasH2 mouse carcinogenicity study. The adipose tissue of the ventral subcutis and salivary glands, skeletal muscle, stomach, and uterus contained these vascular malformations. Most vascular malformations were not observed at necropsy. In the subcutis, salivary gland, and skeletal muscle, the irregular vascular channels dissected between adipocytes and myocytes. The vascular spaces were lined by flattened or hypertrophied endothelial cells, and in some cases were accompanied by infiltrates of lymphocytes and macrophages. Erythrocytes were rarely seen within abnormal vascular spaces. In stomach and uterus, the vascular malformations had thicker muscular walls and appeared to be irregular arterial vessels. Hemangiosarcomas were present within or adjacent to vascular malformations in the salivary gland and subcutis of two mice, suggesting that these vascular structures might predispose affected mice to vascular neoplasms. Hemangiosarcomas are relatively common neoplasms in Tg rasH2 mice, with an incidence of 14/120 in males and 4/120 in females in this study. Pathologists interpreting lesions in Tg rasH2 mice should be aware that vascular malformations in these mice occur and may be associated with hemangiosarcoma.
130: PROTEIN ADDUCT FORMATION BY NORCOCAINE NITROXIDE, AN N-OXIDATIVE METABOLITE OF COCAINE.
It was demonstrated over 20 years ago that cocaine N-oxidative metabolism leads to formation of a reactive metabolite that binds irreversibly to proteins, and this binding correlates with cocaine-induced liver injury. The identity of the reactive metabolite has not been established with certainty. Early reports suggested that the stable nitroxide metabolite of cocaine (norcocaine nitroxide; NNX) was not sufficiently reactive to be responsible for protein binding. To test this directly, 14C-labeled NNX was synthesized and incubated with mouse hepatic microsomes. Rapid, irreversible binding of the radio-label to protein was observed. The addition of NADPH to the incubation produced only a modest increase in radiolabel binding, indicating that oxidation of NNX is not required for protein-adduct formation. In subsequent experiments, incubation of unlabeled NNX with mouse whole liver homogenate was found to result in selective adduction of specific proteins as determined by western blot analysis. Many of these proteins appeared to have similar molecular masses as proteins adducted during cocaine hepatotoxicity in vivo. Two of the proteins adducted during cocaine hepatotoxicity in vivo have been identified recently as hsp60 and transferrin. Incubation of NNX with these proteins in vitro resulted in protein adduct formation. Incubation with cocaine under these conditions, in comparison produced little or no protein adduct formation, These observations suggest that NNX formed from N-oxidative metabolism of cocaine is spontaneously reactive with proteins and that it could be responsible for much, if not all, of the protein adduct formation associated with cocaine hepatotoxicity. Supported by National Institute on Drug Abuse, DA 06601.
131: CELL DEATH NOMENCLATURE: RECOMMENDATIONS OF THE SOCIETY OF TOXICOLOGIC PATHOLOGISTS.
In toxicity study reports detailed descriptions of lesions are usually included in the pathologist's narrative report, but it is the short morphologic diagnoses in incidence tables that provide the major impressions of the study outcome. It is imperative that such terms accurately indicate what the pathologist has seen. Considerable confusion regarding the terms apoptosis and necrosis in recent years prompted the STP to charter an ad hoc committee charged with making recommendations about the use of these terms. The word “necrosis” historically has been used to describe the presence of all types of dead cells in a living organism. It was concluded that the recent literature-based dichotomy between necrosis and apoptosis is inappropriate and recommend that “necrosis” be used to describe findings comprising dead cells in situ regardless of the mechanism. “Apoptotic necrosis” may be used to describe cells that shrank prior to death and “oncotic necrosis” (from the Greek onko for swelling) may be used to describe cells that swelled prior to death. “Mixed apoptotic and oncotic necrosis” may be used when appropriate. Modifiers indicating distribution and severity may also be used. “Individual cell necrosis” may be either of the apoptotic or oncotic type. In many cases more traditional terms such as “coagulation necrosis” may be used to convey a meaning similar to oncotic necrosis.
132: SPONTANEOUS DEGENERATIVE MYOPATHY IN THE Tg rasH2 MOUSE.
The transgenic CB6F1-TgN (RasH2) mouse, more commonly known as the Tg rasH2 mouse, has been proposed as a model for rapid carcinogenicity testing of genotoxic and non-genotoxic chemicals. The genome of these mice contains multiple copies of the human c-H-ras proto-oncogene and promoter. Spontaneous skeletal muscle degeneration and regeneration is a common finding in these mice. In one six-month Tg rasH2 mouse study, the incidences of minimal to moderate skeletal muscle degeneration were 96/119 (81%) in male mice and 109/119 (92%) in female mice. The incidences of degeneration of the muscles of the hind leg were similar in control mice and mice treated with a variety of chemicals, and there was no evidence that these lesions were related to treatment. Multifocal lesions included myofiber atropy, formation of strap cells with rows of central nuclei, loss of cross-striations, myofibrillar fragmentation, and infiltrates of macrophages, lymphocytes, and occasionally neutrophils. Skeletal muscle of the hind leg was the only skeletal muscle examined routinely in this study, however, similar lesions were seen occasionally in muscles of the neck, thoracic cavity, and ventral abdominal wall. Similar lesions were not observed in cardiac muscle. It is likely that skeletal muscle degeneration and regeneration could be found in nearly all Tg rasH2 mice if sufficient samples were examined.
133: SUBCUTANEOUS SARCOMAS ASSOCIATED WITH BIO-MEDIC TRANSPONDERS IN P53 ± MICE.
In a six month p53 ± mouse carcinogenicity study, the incidence of poorly differentiated subcutaneous sarcomas over the dorsal thorax and abdomen was 5/75 (6.7%) in male C57BL/ 6TacBR-[KO]p53 mice and 9/15 (12%) in females, for an overall incidence of 14/150 (9.3%). Six of the sarcomas were present in control animals (3 males and 3 females), and 3 or fewer sarcomas were present in any other treatment group. Mice treated with the positive control agent, p-cresidine, had no subcutaneous sarcomas. Twelve sarcomas were associated with glass and plastic-coated transponders implanted subcutaneously prior to the beginning of the study. Most sarcomas were well demarcated and expansile, but a few infiltrated deeply into subcutaneous fat and muscle. Sarcomas were composed of pleomorphic fusiform to polyhedral cells with poorly demarcated cell margins. Very large, irregular nuclei were seen within very large cells in most neoplasms. Very large multinucleated cells containing up to 30 nuclei were observed in some neoplasms. Mitotic figures were common in some neoplasms and unusual in others. Transponder-induced sarcomas have been reported previously in C57BL/ 6TacBR-[KO]p53 heterozygous p53 knockout mice (Blanchard et al., Toxicol. Pathol. 27:519–527, 1999). The incidence of subcutaneous sarcomas in this report were 7/90 (8%) in males and 8/90 (9%) in females. Our results support the findings of Blanchard et al. that a significant proportion of p53 ± mice develop sarcomas at the site of subcutaneously implanted Biomedic transponders within 6–7 months of implantation.
134: CHARACTERISTICS OF EOSINOPHILIC GLOBULES IN VINYL CARBAMATE-INDUCED MOUSE LIVER TUMORS.
Eosinophilic globules (EGs) often appear in neoplastic hepatocytes in mice. Using immunohistochemistry, erythropoietin (EP) and α1-antitripsin (AAT) have been previously reported as constituents of EGs in spontaneously occurring hepatocellular carcinomas and diethylnitrosamine-induced hepatomas, respectively. Hepatocellular proliferative lesions including altered foci, adenomas, and carcinomas induced by vinyl carbamate (VC) in 5 strains of male mice were examined histologically to confirm the presence of EGs. The incidence of EG was highest in adenomas in all strains; the incidences in adenomas were C57BL/6 (97.4% of 73 mice) > B6CF1 (75.0% of 106 mice) > B6C3F1 (73.9% of 66 mice) > B6D2F1 (69.8% of 73 mice) > C3H (1.2% of 146 mice). Several formalin-fixed samples were examined immunohistochemically and ultrastructurally to clarify the constituents and ontogenesis of the EGs. Most of the EGs were immunopositive for α-fetoprotein (AFP), but consistently negative for EP or AAT. Ultrastructurally, EGs developsed as aggregates of electron-dense granules within the rough endoplasmic reticulum. Our present study showed that the EGs seen in VC-induced hepatocellular tumors contained AFP. Thus, the major constituent protein of EGs in mouse liver tumors may differ among hepatic carcinogens.
135: PATHOLOGICAL CHARACTERIZATIONS OF ESTROGEN RECEPTOR AGONIST AND ANTAGONIST IN UTEROTROPHIC ASSAY USING IMMATURE RATS.
In this study we performed uterotrophic assay to investigate the dose-response effects of ER agonist in uterine response and the alteration of weights. A positive control substance, 17-ethinyl estradiol (EE), was daily administered by subcutaneous injection for three consecutive days to immature rats at various doses (0.01–10.0 μg/kg). Additionally, anti-estrogenic material, ZM189,154 (0.1–1.0 mg/kg) was administered to EE-treated rats immediately after EE administration to determine its inhibitory effects. In EE-treated rats, the uterine weights increased dose-dependently at doses higher than 1.0 μg/kg of EE (P < 0.01). A similar dose-response relationship was observed at vaginal weights. Moreover, the uterine responses increased (uterine or vagina weights) by EE (0.3 μg/kg) were blocked by ZM189.154 at the dose of 1.0 μg/kg (P < 0.05). Histopathologically, distinctive cytoplasmic vacuolation with vaginal epithelium was observed at doses from 0.1 μg/kg to 1 μg/kg. Excessive vaginal keratinization was observed at doses higher than 3.0 μg/kg. Uterine epithelial cell proliferation was observed at doses higher than 3.0 μg/kg and uterine stromal cell proliferation was even seen at 0.03 μg/kg. In conclusion, these findings indicate that the effective dose of EE for positive control of the assay should be over 0.3 μg/kg and we suggest that the 3-day uterotrophic assay using immature rats is an effective in vivo short-term system for screening potential EDC.
136: APPLICATION OF MICROARRAYS IN TOXICOLOGY.
Toxicogenomics allows for the application of molecular techniques to elucidate the mechanisms of toxicity, allowing for efficient toxicity testing and prioritization of compounds early in the drug discovery process. Microarrays present a valuable tool in this regard. This study was conducted to examine the application of microarrays to toxicology to interpret mechanisms of toxicity. As a part of the HESI initiative to investigate alternative approaches for testing the carcinogenic potential of compounds, reserpine is being tested in transgenic animal models. Reserpine (RES) is a rodent carcinogen, but not a human carcinogen that is currently being evaluated in transgenic animal models. In order to understand how changes in gene expression correlate with cytotoxicity, initial studies determined the toxicity of RES in HepG2 cells by measuring changes in MTT activity and ATP levels following a 24-hr exposure. The TC50 for MTT and ATP was estimated at 50 and 70 μM respectively. These studies indicated alterations in mitochondrial functions and cell viability. To evaluate RES-induced changes in gene expression, HepG2 cells were seeded at a density of 300,000/ml in a final volume of 6 ml. Following the 96 hr growth period, media was removed and the cells were dosed by adding media with 50 μM RES and 0.5% DMSO. After a 24-hr exposure the media was removed, cells were processed to recover mRNA and the target RNA was hybridized to human oligonucleotide microarrays. Preliminary results revealed alterations in expression of approximately 340 genes; based on changes of ±3 SD as a level of significance for analysis. Up-regulation of about 170 genes and down-regulation of approximately the same number of genes was observed. These and additional studies examining alteration in gene expression patterns may lead to a better understanding of the mechanisms of toxicity and allow for better prediction of toxicity.
137: CORRELATION OF HISTOPATHOLOGY, OPACITY AND PERMEABILITY OF BOVINE CORNEAS EXPOSED IN VITRO TO KNOWN OCULAR IRRITANTS.
The Bovine Corneal Opacity and Permeability (BCOP) assay is an ex vivo model for the detection of potential ocular irritants. Viable corneas excised from normally discarded bovine eyes are directly exposed to test materials in vitro, and resulting damage is assessed by measuring changes in corneal opacity and permeability. Recently, a major international validation study did not show an acceptable performance for the BCOP assay. We investigated whether histopathologic evaluation of the bovine corneas would reveal damage that was not suggested by the conventional in vitro endpoints of opacity and permeability. In fact, three materials (parafluoroaniline, quinacrine, and sodium oxalate) which were severely underpredicted by the conventional endpoints showed diverse microscopic cellular damage (including cell necrosis, loss of tissue and/or extensive vacuolation) throughout the epithelium and extending into the stroma. Three moderately underpredicted materials showed multifocal separation of the basal epithelial cells from the basal lamina, as well as other changes that were more reflective of the in vivo scores than were the in vitro opacity and permeability measurements. We conclude that the excised bovine cornea does respond appropriately in vitro to the above test materials, and that it will be necessary to develop a histopathologic scoring system to extend the utility of the BCOP assay.
138: OLFACTORY DEFICITS AND LESIONS IN MURINE 3-METHYLINDOLE TOXICOSIS.
3-Methylindole (3MI) injures olfactory mucosa in mice. We measured olfaction by the ability of mice injected i.p. with 150 mg (low-dose) or 300 mg (high-dose) 3MI/kg or vehicle (control) to avoid drinking 0.1% isoamyl acetate (odorant), 0.5% quinine monohydrochloride (aversive tastant) solution. Water-deprived control mice rarely (0/24, 1/18, 0/12) tried the solution, whereas 3/24, 3/18, and 2/12 low-dose; and 10/24, 11/18, and 6/12 high-dose mice tasted it 7, 14, or 21 days after 3MI, respectively. High-dose mice performed worse than controls (P < 0.01) by the Fisher exact test. Olfactory mucosa from 2 test and 1 control mouse was examined by transmission electron microscopy at ½, 1, 2, 4, 6, 24, 48, and 72 hr after i.p. 400 mg 3MI/ kg, and at 7, 14, and 21 days after 300 mg 3MI/kg or vehicle. Degeneration was evident in epithelial cells of Bowman's glands and olfactory sustentacular cells by ½ hr, but not in neurons until 24 hr, by which time necrosis was fully developed in sustentacular cells and Bowman's glands. Epithelial cells detached from the basal lamina by 48 hr. Fibroblasts were hypertrophied by 72 hr and, with collagen fibrils, formed the bulk of the mucosa at 7 days. By 21 days, fibroblasts were less numerous, Bowman's glands were few with epithelial cells that lacked secretory granules, and mucosal epithelium was reconstituted, but disorderly and lacked neurons. Though olfactory neurons are initially spared, their absence in the regenerated epithelium explains olfactory deficits in murine 3MI toxicosis.
139: EVALUATION OF THE EKER RAT FOR SHORT-TERM CARCINOGENESIS BIOASSAYS.
This study examined the response of the Eker rat to nephrotoxic compounds and to genotoxic non-renal carcinogens. Groups of male Eker rats received: no treatment; a vehicle control; a noncarcinogenic nephrotoxin [aluminum nitrilotriacetate (2 mg/kg/day of aluminum, intraperitoneally, 3 days/week) or cyclosporine A (30 mg/kg/day, orally by gavage, 7 days/week)]; a genotoxic non-renal carcinogen [furan (8 mg/kg/day, orally by gavage, 5 days/week) or 2, 4-diaminotoluene (6.5 mg/kg/day, orally by gavage, 7 days/week) or 2-nitropropane (89 mg/kg/ day, orally by gavage, 3 days/week)]. Duration of treatment was four and/or six months. A complete set of tissues was evaluated microscopically and numbers of proliferative renal lesions were counted. Administration of nephrotoxic compounds (A1-NTA and cyclosporine) significantly increased the numbers and incidence of preneoplastic and neoplastic renal lesions in the Eker rat compared to concurrent vehicle controls. The genotoxic nonrenal carcinogens had no consistent effect on numbers or incidence of preneoplastic or neoplastic renal lesions and did not produce neoplasms in the expected target organs (liver). A four or six month study in Eker rats does not appear to be a reliable predictor of the results of a 2-year rodent carcinogenesis bioassay. However, this conclusion may not be definitive due to the short treatment durations studied.
140: EVALUATION OF CONTACT HYPERSENSITIVITY RESPONSE IN MICE EXPOSED TO PERMETHRIN AND CIS-UROCANIC ACID.
Contact hypersensitivity (CH) is a cell-mediated or delayed-type hypersensitivity reaction that is characterized by a local epidermal reaction at the site of topical allergen exposure. CH is a sensitive indicator of local immunocompetence and is a commonly used technique in immunotoxicity studies. Two agents were evaluated for effect on the CH response 48 hours after exposure: permethrin and cis-urocanic acid (cUCA). Permethrin, a topical pyrethroid insecticide used in the treatment and prophylaxis of ectoparasites, was applied to the shaved dorsal interscapular region of female C57B1/6 mice at doses ranging from 0.5–25.0 μ1 (5.6–28 mg). This agent produced as much as 71% inhibition of the CH response in mice. Further, this inhibition was persistent, lasting 30 days post-exposure. C-UCA is a chromophore that is isomerized in sunlight-exposed stratum corneum, and is thought to be involved in sunlight-induced local immunosuppression. Intradermal exposure to cUCA at doses ranging from 5.0–100.0 ug resulted in up to 76% inhibition of CH 48 hours after exposure. Permethrin and cUCA were also given together to determine if additive, more than additive, or less than additive effects on CH resulted.
141: EXPRESSION OF CYCLOOXYGENASE-2 IN EMBRYONIC AND FETAL TISSUES DURING ORGANOGENESIS AND LATE PREGNANCY.
Cyclooxygenase (COX) catalyzes the committed step in prostaglandin (PG) biosynthesis and exists as two related but unique iso-forms, COX-1 and COX-2. COX-1 is constitutively expressed in many tissues including the kidney, GI tract, and platelets, where it functions to produce prostaglandins necessary for homeostatic physiologic processes. In contrast, COX-2 expression is induced in response to a variety of mitogens and cytokines where the resulting prostaglandins mediate inflammation and cell growth. In light of the known role of prostaglandins in pregnancy and cell growth (placentation and decidualization), we evaluated COX-2 expression throughout embryonic and fetal development in the rat. COX-2 protein and mRNA expression were evaluated in the developing rat on gestation days 7, 11, 13, 15, 17 and 20 (5–6/age group) by immunohistochemistry and in situ hybridization, respectively. As anticipated, COX-2 expression was marked in decidualized uterine tissue on gestation days 7–13 and in fetal membranes on gestation days 17–20. In contrast, COX-2 expression was not detectable in the developing embryo during gestation days 7–13. COX-2 expression was, however, observed in the fetal growth period (gestation days 15–20); expression was evident in epidermal keratinocytes, hair follicles, ganglionic neurons, vascular endothelial cells, stromal cells of the atrioventricular valve, chondrocytes and the renal cortex. The apparent absence of COX-2 expression during gestation days 7–13 suggests that this enzyme does not play a major role in embryonic organogenesis. COX-2 expression in rat fetal tissues in mid to late pregnancy suggests a role for COX-2 derived prostaglandins in organ growth.
142: CYCLOOXYGENASE-2 DISTRIBUTION IN THE MALE REPRODUCTIVE TRACT.
The male reproductive tract is a rich source for prostaglandins where they are known to affect sperm metabolism, contractility of the testicular capsule, and smooth muscle layers surrounding the seminiferous and epididymal tubules. Prostaglandins are produced by two distinct cyclooxygenase enzymes COX 1 and 2. Recently, COX-1 has been implicated in the regulation of anion secretion in cultured epididymal epithelial cells from immature rats. Although COX-2 expression was detected in principal cells of the immature rat epididymis, its role and temporal expression was not elucidated. To define the expression of COX-2 in the maturing male reproductive tract, immunohistochemical staining was performed at 14, 21, 28, 35 and 62-days-of-age. COX-2 expression was apparent in the initial segment, caput epididymis and vas deferens at 14-days-of-age. This pattern of staining increased in intensity with age and achieved adult levels by 28-days-of-age. Staining was also evident in smooth muscle cells of blood vessels in the seminal vesicle. There was no COX-2 staining in the efferent ducts, rete testis or ventral prostate. These data suggest that COX-2 derived PGs may be involved in the pubertal development of the epididymis, but not the more apical regions of the excurrent duct system including the rete testis and efferent ductules.
143: TUMOR SPECTRUM IN p53 DEFICIENT MICE ON A MIXED 129/SV X FVB/N BACKGROUND.
p53-deficient mice have provided valuable information on the role of this tumor suppressor gene in development, differentiation, cell cycle control, and tumorigenesis. Recently, these mice have been utilized by the toxicological/pharmaceutical industry in carcinogenicity studies. Their widespread use mandates knowledge of their spontaneous pathology spectrum before experimental results can be properly interpreted. The purpose of this study was to report the spectrum of spontaneous tumors in 129/SV homozygous (-/-) and hemizygous (±) p53-deficient mice. These mice were crossed with ret/PTC1 transgenic mice on an FVB/N background to investigate enhanced metastatic potential in a thyroid cancer model. For that study, 72 p53 -/-mice (49–120 days of age), 77 p53 ± mice (67–194 days of age), and 69 p53 +/+ mice (49–204 days of age) were evaluated. The spontaneous tumor spectrum in these mice was similar to that reported in the literature, and did not differ according to ret/PTC1 transgenic status. The tumor spectrum in p53 —I— mice included hemangiosarcomas (35/72; 49%), lymphosarcomas (33/72; 46%), gliomas (8/72; 11%), testicular teratomas (2/ 37; 5%), and miscellaneous tumors (6/72; 8%; pulmonary adenoma (1) and adenocarcinoma (1), 2 rhabdomyosarcomas, 1 fibrosarcoma, 1 liposarcoma, and 1 histiocytic sarcoma). In contrast, tumors were infrequent in p53 ± mice (1 glioma, 1 testicular teratoma, 1 pulmonary adenoma, and 1 anaplastic soft tissue sarcoma) and absent in p53 +/+ mice. It is important to recognize the influence of the genetic background in the resulting phenotype for any studies in which p53-deficient mice are used.
144: CANINE NECROPSY TRAINING ON CD-ROM.
Simple but subtle techniques of canine necropsy are demonstrated using the official protocol for military working dogs. Each step is documented using still frame (JPG) images annotated by an expandable outline. Field trials suggest that veterinary students, veterinarians, and technicians will learn from this software. Though some may choose to view alone, maximum utility will be realized when viewed in groups of 2–3, or with an LCD projector in the necropsy suite. Images AND instructions can be viewed with small images (300 × 200 pixels), or large (600 × 400 pixel) images viewed alone. Images are displayed by clicking hot words (Sm, Lg) following each instruction. Clicking either hot words or the currently displayed image dismisses images. Stepwise navigation can be done using navigational buttons. Random access is achieved by clicking major headings of the expandable necropsy outline. Creation of such a product poses many challenges. Judgements were made to photograph each step on a non-reflective turquoise aluminum background without the normal blood contamination usually present. Long delays between image capture and review forced the shift from film based to digital camera imaging. This enabled immediate review and reshooting of unsatisfactory images. Five different digital cameras were tested for image sharpness, macro capability, lighting requirements, and download speed and method. The Sony Mavica FD88 was chosen for superior zoom and macro capability, autofocusing, and easily transferred files using 3.5 inch floppy disks. The challenge of electronic image sorting and file naming was handled using InMedia Slides and Sound Plus software (PhotoChannel Networks Inc., Vancouver, BC, Canada). This product was developed using the Asymetrix Toolbook Instructor 6.5 (Asymetrix Learning Systems, Inc., Bellevue, WA), with QuickTime drivers (Apple, Inc., Cupertiono, CA) for image display. It runs on any Windows operating system from 3.1 to NT.
145: AFIP VETERINARY SYSTEMIC PATHOLOGY STUDY SETS ON WWW.
The Armed Forces Institute of Pathology provides consultation, education and research resources to military ana civilian medical communities. The Department of Veterinary Pathology conducts a three-year residency program for military veterinarians. The systemic veterinary pathology study sets, with more than 700 disease entities organized into eleven body systems, are the core component of the training and are used extensively by visiting students from the USA and abroad. Each system includes neoplastic, viral, bacterial, fungal, parasitic, toxic and miscellaneous conditions. Each entity includes history, microscopic description, histologic images, morphologic diagnosis, general information, pathogenesis, clinical signs, gross and histologic findings, differential diagnosis, comparative pathology and relevant references. Each case is updated every 3 years by residents under the supervision of ACVP board certified staff pathologists. We propose that this educational resource will be made available to other interested training programs via the World Wide Web (WWW). We have constructed sample web pages called “systemic pathology training” within the departmental web site (www.afip.org/vetpath) using eight cases from the Urinary System. This educational project will require funding and a team consisting of veterinary pathologists and computer specialists. These study sets can supplement as a valuable educational resource via the WWW for residency training program.
146: ON-LINE CONFERENCING: REMOVING THE BARRIERS OF TIME, COST AND DISTANCE TO CREATE A NEW TYPE OF HISTOPATHOLOGY CONFERENCE.
Wouldn't it be nice to attend and participate in international scientific meetings between regular work week responsibilities? Now you can park the jet, cancel the hotel, stay on the job, and still meet with colleagues from around the globe to be educated and to discuss innovative topics, all at a time and place of your own convenience. An entire “traditional” conference can now be offered and conducted completely on the Internet; except for the jet lag and the pile-up of work awaiting your return. In January 2000, the Registry of Toxicologic Pathology for Animals, a part of the Department of Veterinary Pathology, AFIP, broke new ground with their “Toxicologic Histopathology Web Slide Conference”. Cases are submitted by the 25 participating pharmaceutical companies, as well as government and educational institutions. In each 3 week session, 4 discussion-worthy cases are analyzed. The high quality images for each case are displayed on the Web, and accessible to the 285 registered conference participants. With the assistance of a moderator, registrants are encouraged to diagnose and discuss each case. A summary is provided, and after the discussion ends, the cases are archived. The cases are contributed anonymously and this provides a neutral forum for the exchange of knowledge and ideas, while safeguarding proprietary information. This conference has been very well received, and will be expanded to 9 sessions in 2001. A demonstration of the web site will be presented. As the pace of life continues to increase, budgets will continue to decrease. This new media has many applications, and is a powerful, flexible and economical continuing medical education option for not only the future, but also the present.
147: THE EUROPEAN CENTER OF TOXICOLOGIC PATHOLOGY AN INITIATIVE IN TRAINING VETERINARY PATHOLOGISTS.
Worldwide there is a lack of professionally trained veterinary pathologists, placing severe constraints on academia and industry. Attracting more of our most able veterinary graduates into pathology training programs would address this problem. Undergraduates and graduates should be made aware of the exciting, central role the professional pathologist plays in many scientific arenas. The European Centre for Toxicologic Pathology will foster and train veterinary pathologists for industry and academia. Creating a multinational academic center in a strong partnership with the pharmaceutical industry, the Centre provides support and expertise in veterinary and toxicologic pathology. The Centre coordinates teaching, learning and research interactions between trainees, graduate students, and industrial pathologists and scientists. A creative step is the first intercalated degree in veterinary pathology, designed specifically to attract veterinary undergraduates into the discipline. The second step is a combined residency/PhD program that leads to diplomacy in the European or American College of Veterinary Pathologists and to a Doctor of Philosophy (PhD) in Veterinary Medicine. The training will be shared between the Royal Veterinary College, the University at Bern, and industry. This will enhance training opportunities and give exposure to multicultural, multilingual concepts and provide a broad yet focussed experience in the essential skills of veterinary and toxicologic pathology. The vision is to create and maintain a pool of career biomedical researchers, in both industry and academia, well trained in veterinary pathology.
148: INEXPENSIVE DIGITAL PHOTOMICROGRAPHS FOR WEB-BASED INSTRUCTION.
Digitized images of hematologic, cytologic, and histopathologic specimens are becoming an increasingly popular method of presenting this material in veterinary and medical colleges. Such images can be made available to students outside of normal course meeting times, and can also be viewed by students at home via internet (or intranet) pages, or incorporated onto instructional media such as CD-ROM's. The quality and resolution of digital images may be somewhat less than 35mm photographic slides, but the above advantages, plus the ease with which digital images can be annotated and updated, makes them appealing for many teaching purposes. Digitization of existing 35mm slides is often used to produce computer-based lessons, but this may be costly and requires a 2-step process for images of new specimens. Expensive ($2000+) digital cameras capable of producing quality images directly through the microscope are, in general, too costly to gain widespread “end-user” availability. A relatively inexpensive 1.3 megapixel digital camera with excellent zoom, macro-focusing, and image capturing control has been adapted for simple recording of a wide range of microscopic specimens. A unique, yet widely available, adapter which allows direct attachment of the camera to a microscope was utilized. Optimal camera settings have been established and shown to generate high quality images that are easily uploaded to a PC or Mac via a USB connection. Image labeling and editing was performed with a “freeware” photo enhancement software program. The total cost of the “package” is under $800, making it far more suitable for generalized adoption in veterinary pathology departments and teaching programs. This poster will present full details on the system and a variety of sample images that have been produced with it.