Commentary: the role of the toxicologic pathologist in academia.

Veterinary pathologists working as toxicologic pathologists in academic settings fill many vital roles, such as diagnosticians, educators, and/or researchers. These individuals have spent years investigating pathology problems that mainly or exclusively focus on the reactions of cells, organs, or systems to toxic materials. Thus, academic toxicologic pathologists are uniquely suited both to help trainees understand toxicity as a cause of pathology responses and also to provide expert consultation on toxicologic pathology. Most toxicologic pathologists in academia are employed at colleges of medicine or veterinary medicine, even though specific toxicologic pathology faculty appointments are uncommon in Europe and North America. Academic toxicologic pathologists typically receive lower financial compensation than do toxicologic pathologists in industry, but academic positions generally provide alternative rewards, such as higher workplace autonomy and scheduling flexibility, professional enrichment through student interactions, and enhanced opportunities for collaborative research and advanced diagnostic investigations. Regular participation by academic toxicologic pathologists in professional training opportunities (eg, as pathology and toxicology instructors in medical and veterinary medical courses, graduate programs, and residencies) offers an important means of engendering interest and inspiring veterinarians to select toxicologic pathology and toxicology as their own areas of future expertise.

Purified deoxynivalenol or feed restriction reduces mortality in rainbow trout, Oncorhynchus mykiss (Walbaum), with experimental bacterial coldwater disease but biologically relevant concentrations of deoxynivalenol do not impair the growth of Flavobacterium psychrophilum.

Diets containing deoxynivalenol (DON) were fed to rainbow trout Oncorhynchus mykiss (Walbaum) for 4 weeks followed by experimental infection (intraperitoneal) with Flavobacterium psychrophilum (4.1 × 10(6) colony-forming units [CFU] mL(-1) ). Mortality of rainbow trout fed either 6.4 mg kg(-1) DON or trout pair-fed the control diet was significantly reduced (P < 0.05) in comparison with trout fed the control diet to apparent satiation (<0.1 mg kg(-1) DON). In a second experiment, trout were fed one of three experimental diets; a control diet, a diet produced with corn naturally contaminated with DON (3.3 mg kg(-1) DON) or a diet containing purified DON (3.8 mg kg(-1) ); however, these fish were not experimentally infected. The presence of DON resulted in significant reduction (P < 0.0001) in feed intake as well as weight gain after 4 weeks. Respiratory burst of head-kidney leucocytes isolated from rainbow trout fed diets containing purified DON (3.8 mg kg(-1) ) was significantly higher (P < 0.05) at 35 day post-exposure compared with controls. The antimicrobial activity of DON was examined by subjecting F. psychrophilum in vitro to serial dilutions of the chemical. Complete inhibition occurred at a concentration of 75 mg L(-1) DON, but no effect was observed below this concentration (0-30 mg L(-1) ).

Hepatic injury correlates with apoptosis, regeneration, and nitric oxide synthase expression in canine chronic liver disease.

Assessment of the clinical severity, pathogenesis, and prognosis of canine chronic liver disease poses significant challenges to clinicians and pathologists, relating in part to a lack of standardized terminology and assessment methods and also to a lack of understanding of the pathogenesis of chronic liver disease in the dog. This study graded the severity of necroinflammatory activity in chronic liver disease in dogs using a modification of Ishak's grading scheme for human chronic liver disease and examined the association of grade score with hepatocellular apoptosis, regeneration, nitric oxide synthase isoform expression, copper and iron accumulation, and indicators of oxidative stress. Formalin-fixed, paraffin-embedded hematoxylin and eosin (HE)-stained liver biopsies from 45 dogs with chronic liver disease and 55 healthy control dogs were graded for various morphologic components of liver injury and response. The cumulative score for grade of necroinflammatory activity was strongly and significantly correlated with immunoreactive labels for hepatocellular proliferation (Ki-67); apoptosis (cleaved caspase-3); inducible nitric oxide synthase (iNOS) in lobular, portal, and septal stromal cells; endothelial nitric oxide synthase (eNOS) in hepatocytes and lobular, portal, and septal stromal cells; and total stainable hepatic iron. A weaker significant correlation was found between grade and accumulation of hepatocellular copper. No significant correlation was found between grade and immunoreactivity for malondialdehyde-protein adducts. These results document a method for grading of the severity of necroinflammatory disease in canine liver biopsies and show an association with increased iNOS and eNOS expression.

Facile production of minor metabolites for drug development using a CYP3A shuffled library.

Metabolic profiling of new drugs is limited by the difficulty in obtaining sufficient quantities of minor metabolites for definitive structural identification. Biocatalytic methods offer the potential to produce metabolites that are difficult to synthesize by traditional medicinal chemistry. We hypothesized that the regioselectivity of the drug metabolizing cytochrome P450s could be altered by directed evolution to produce minor metabolites of drugs in development. A biocatalyst library was constructed by DNA shuffling of four CYP3A forms. The library contained 11 ± 4 (mean ± SD) recombinations and 1 ± 1 spontaneous mutations per mutant. On expression in Escherichia coli, 96% of mutants showed detectable activity to at least one probe substrate. Using testosterone as a model drug-like substrate, mutants were found that preferentially formed metabolites produced in only trace amounts by parental forms. A single 1.6L batch culture of one such mutant enabled the facile isolation of 0.3mg of the minor metabolite 1ß-hydroxytestosterone and its ab initio structural determination by 1D- and 2D-NMR spectroscopy.

Finite element analysis of wall stress in the equine pulmonary artery.

REASONS FOR PERFORMING STUDY: Arterial calcification is found frequently in the pulmonary artery of racehorses, but the aetiology is unknown. Calcification might be associated with increased wall stress due to arterial geometry (shape) and exercise-induced hypertension. HYPOTHESIS: High wall stress levels are found in the regions associated with calcified lesion formation, exacerbated as transluminal pressure increases to levels associated with exercise. METHODS: The pulmonary arteries of 5 horses, unaffected by calcification, were dissected and pressurised to resting and exercising physiological transluminal pressures and scanned with MRI. Arterial geometries were reconstructed to form 3D computer models and finite element analyses performed. Wall stress levels were measured in 4 regions of interest: the arterial trunk and bifurcation, the wall ipsilateral and contralateral to the bifurcation. Measurements were made for arterial transluminal pressures of 25, 50 and 100 mmHg. RESULTS: High wall stress levels were consistently found at the pulmonary artery bifurcation and wall ipsilateral to the bifurcation, where calcified lesions typically form. Lower wall stress levels were found along the trunk and the wall contralateral to the bifurcation where lesions are less frequently found. Wall stress levels increased 5-fold over a 4-fold increase in pressure. The wall stress levels ranged 10 kPa in the wall of the branch contralateral to the bifurcation at 25 mmHg to 400 kPa in the bifurcation at 100 mmHg. CONCLUSIONS: Wall stress from arterial geometry and increased pulmonary artery transluminal pressure are factors that may be associated with calcification of the equine pulmonary artery. POTENTIAL RELEVANCE: Arterial calcification may increase the risk of arterial wall failure in racing horses.

Pathologic fracture healing after femoral limb salvage in a dog.

BACKGROUND: Bone sarcomas are a significant cause of pain, disability, and mortality in dogs. A variety of surgical limb salvage options are available to preserve limb function with comparable prognosis to amputation. CASE REPORT: This report describes successful healing after plate fixation of an undifferentiated sarcoma pathologic femoral fracture in a dog. The fracture was treated surgically with curettage of the tumour site, placement of autogenous bone graft, and then stabilized using a locking plate rod construct. The patient regained excellent mobility after surgery and was managed with monthly pamidronate therapy. Serial radiographs demonstrate progressive healing of the pathologic fracture. Ultimately, the patient developed a maxillary fibrosarcoma and was euthanased 1 year after treatment of the femoral fracture. Postmortem histopathological evaluation of the pathologic fracture site demonstrated complete bone healing. CONCLUSION: This case highlights the possibilities of limb salvage by fracture stabilization and bone healing as a viable option in select patients.

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss).

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.

Proteins associated with the early intrauterine equine conceptus.

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF(2 alpha). Two forms of uteroglobin were distinguished by partial amino acid sequences of approximately 6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF(2 alpha) one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.

An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice.

Radioiodinated transforming growth factor-beta 1 (TGF-beta 1) bound to the plasma proteinase inhibitor, alpha 2-macroglobulin (alpha 2M), as determined by chromatography on Superose-6 and native polyacrylamide gel electrophoresis. When alpha 2M conformational change was induced with methylamine, 125I-TGF-beta 1 binding significantly increased. Intravenously injected 125I-TGF-beta 1 cleared from the circulation of mice rapidly at first; however, intravascular radioactivity stabilized near 20% of the initial level. At necropsy, radioactivity was recovered predominantly in the liver (65%); however, the density of radioactivity (disintegrations per minute/g organ wt) was highest in the lungs. Markedly different results were obtained with purified 125I-TGF-beta 1-alpha 2M-methylamine complex. Clearance of the complex occurred as a first-order process with a t1/2 of 4 min. Greater than 90% of the radioactivity was recovered in the liver. The clearance and distribution of 125I-TGF-beta 1-alpha 2M-methylamine were equivalent to those observed with 125I-alpha 2M-methylamine and 125I-alpha 2M-trypsin. The latter two radioligands clear via specific alpha 2M receptors in the liver. Large molar excesses of alpha 2M-trypsin or alpha 2M-methylamine competed with 125I-TGF-beta 1-alpha 2M-methylamine for plasma clearance. Native alpha 2M, which does not bind to the alpha 2M receptor, did not compete. The receptor binding domain of alpha 2M-methylamine was blocked by chemical modification or enzyme treatment. The resulting alpha 2M preparations still bound 125I-TGF-beta 1; however, the complexes did not clear when injected intravenously in mice. The studies presented here demonstrate that alpha 2M can mediate the plasma clearance of a growth factor via the alpha 2M receptor system. We propose that alpha 2M, the alpha 2M receptor, and proteinases may function as a concerted system to regulate TGF-beta 1 activity and the activity of related factors in vivo.

Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control.

We have developed transgenic mouse models to determine whether endogenous expression of phytase transgenes in the digestive tract of monogastric animals can increase the bioavailability of dietary phytate, a major but indigestible form of dietary phosphorus. We constructed phytase transgenes composed of the appA phytase gene from Escherichia coli regulated for expression in salivary glands by the rat R15 proline-rich protein promoter or by the mouse parotid secretory protein promoter. Transgenic phytase is highly expressed in the parotid salivary glands and secreted in saliva as an enzymatically active 55 kDa glycosylated protein. Expression of salivary phytase reduces fecal phosphorus by 11%. These results suggest that the introduction of salivary phytase transgenes into monogastric farm animals offers a promising biological approach to relieving the requirement for dietary phosphate supplements and to reducing phosphorus pollution from animal agriculture.