This study tested the role of the calcium dependent cytosolic protein calpain in neutrophilic hepatitis. We hypothesized that inhibition of calpain would protect against LPS-induced neutrophilic liver damage. To test our hypothesis, a reliable LPS-mediated hepatitis model to investigate the mechanisms of hepatic neutrophil infiltration following LPS administration was developed by repeat intravenous injection of LPS at a dose of 10 mg/kg to rats. Blood was collected for hematologic and biochemical analysis and multiple organs including liver were collected for evaluation of histopathologic changes. Flow cytometry was employed to investigate L-selectin (CD 62L) and MAC-1 (CD11b/18) expression on neutrophils both in vivo and in vitro. Significant hematologic changes included neutrophilia, elevation in neutrophil to lymphocyte ratio with toxic changes and left shift. Biochemical changes were observed in several liver (AST, GGT) and kidney (BUN) parameters generally at the earliest time points. Histopathology revealed a time-dependent neutrophil and mononuclear infiltration around the periportal areas in the single dose study and mid-zonal inflammation with multifocal coagulative necrosis in the repeated dose study. CD 11b was up-regulated both in vitro and in vivo. After development of a suitable model, the first goal was to investigate the role of the intracellular enzyme calpain in the development of neutrophilic hepatitis and midzonal necrosis. A second goal was to compare the observed protective effects of calpain inhibition with a relatively selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine and an inhibitor of coagulation, heparin. When compared to rats administered LPS alone, administration of calpain 1 inhibitor prior to LPS significantly reduced hepatic iNOS expression, hepatic neutrophil infiltration and attenuated midzonal hepatic necrosis. Administration of heparin and aminoguanidine prior to LPS also decreased liver iNOS expression, hepatic neutrophil infiltration and liver pathology comparable to calpain inhibition. Blood neutrophil activation, as measured by the neutrophil adhesion molecule CD11b integrin, was upregulated in all the LPS treated groups regardless of inhibitor administration. We conclude that amelioration of liver pathology via calpain inhibition is likely dependent on the down-regulation of iNOS expression in the rat model of LPS mediated hepatitis.

This study tested the role of the calcium dependent cytosolic protein calpain in neutrophilic hepatitis. We hypothesized that inhibition of calpain would protect against LPS-induced neutrophilic liver damage. To test our hypothesis, a reliable LPS-mediated hepatitis model to investigate the mechanisms of hepatic neutrophil infiltration following LPS administration was developed by repeat intravenous injection of LPS at a dose of 10 mg/kg to rats. Blood was collected for hematologic and biochemical analysis and multiple organs including liver were collected for evaluation of histopathologic changes. Flow cytometry was employed to investigate L-selectin (CD 62L) and MAC-1 (CD11b/18) expression on neutrophils both in vivo and in vitro. Significant hematologic changes included neutrophilia, elevation in neutrophil to lymphocyte ratio with toxic changes and left shift. Biochemical changes were observed in several liver (AST, GGT) and kidney (BUN) parameters generally at the earliest time points. Histopathology revealed a time-dependent neutrophil and mononuclear infiltration around the periportal areas in the single dose study and mid-zonal inflammation with multifocal coagulative necrosis in the repeated dose study. CD 11b was up-regulated both in vitro and in vivo. After development of a suitable model, the first goal was to investigate the role of the intracellular enzyme calpain in the development of neutrophilic hepatitis and midzonal necrosis. A second goal was to compare the observed protective effects of calpain inhibition with a relatively selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine and an inhibitor of coagulation, heparin. When compared to rats administered LPS alone, administration of calpain 1 inhibitor prior to LPS significantly reduced hepatic iNOS expression, hepatic neutrophil infiltration and attenuated midzonal hepatic necrosis. Administration of heparin and aminoguanidine prior to LPS also decreased liver iNOS expression, hepatic neutrophil infiltration and liver pathology comparable to calpain inhibition. Blood neutrophil activation, as measured by the neutrophil adhesion molecule CD11b integrin, was upregulated in all the LPS treated groups regardless of inhibitor administration. We conclude that amelioration of liver pathology via calpain inhibition is likely dependent on the down-regulation of iNOS expression in the rat model of LPS mediated hepatitis.

The mortality of grouper larvae and juveniles infected by nervous necrosis virus (DGNNV) was near 100%. Vacuoles were found in photoreceptor layer, outer nuclear layer and inner nuclear layer of retina and optic tectum of mesencephalon for the dragon grouper that was infected by DGNNV, after stained with haematoxylin and eosin (H&E). Recombinantly expressed in E. coli, virus-like particles (VLPs) were used for antibody preparation. By indirect fluorescence antibody test (IFAT) with the mouse anti-VLP serum, DGNNV was detected in retina inner nuclear layer and mesencephalon optic tectum. At 96 hours post infection of DGNNV with intraocular injection, vacuoles were observed, with H&E staining, in zebrafish retina photoreceptor layer and mesencephalon optic tectum. In IFAT test, DGNNV was also detected in outer nuclear layer and optic tectum of zebrafish. This study showed antibody stimulated by the recombinant VLPs was sutible for DGNNV detection in fish nervous tissues.

Dissertation (MSc)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, August 2001. / Includes bibliographical references. Also available on the Internet.

published_or_final_version / Dental Surgery / Master / Master of Dental Surgery

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Tumor necrosis factor's (TNF) cytotoxic mechanism of action was examined using cultured cancer cell lines. TNF demonstrated cytolytic and cytostatic effects on L929 fibrosarcoma and MCF-7 adenocarcinoma cells. TNF failed to show any specific effects on RNA, DNA or protein synthesis or ATP content in tumor cells in vitro. It did not cause DNA single strand breaks. Decreased cellular levels of reduced thiols did not predict sensitivity to the cytotoxic effects of TNF. Depletion of cellular glutathione failed to increase the sensitivity of TNF-sensitive or resistant cells. However, various non-specific and specific lysosomotropic agents lead to an inhibition of TNF's cytotoxic action. Differences in enzyme activity, primarily lysosomal, were noted between TNF-sensitive and resistant cells. These changes involved a general halving of lysosomal proteins and enzymes in the TNF-resistant cells. The antitumor activity of TNF does not involve specific inhibition of macromolecular synthesis but may involve alterations in lysosomes.

published_or_final_version / Medicine / Master / Doctor of Medicine

Lameness continues to cause significant problems in profitability, productivity, and animal welfare in the feedlot industry. Toe tip necrosis syndrome (TTNS) is a new name for a previously reported condition. By definition, TTNS is separation of the apical white line with tissue necrosis and clinical lameness. This definition includes complications such as pedal (P3) osteitis, middle (P2) and proximal (P1) phalangeal osteomyelitis, tendonitis, tenosynovitis, cellulitis, and embolic pneumonia. Anecdotal experiences from practitioners report this lameness in feedlot cattle will develop within weeks after feedlot entry. Often the hindlimbs, specifically the lateral claw, are affected where a separation of the dorsal wall and sole will be noticed. Secondary infections will progress deeper into the foot and become systemic. Unfortunately, despite treatment, these animals can become very lame and will need to be euthanized. The overall objective of this project was to describe the epidemiology of TTNS in western Canadian feedlot cattle. The specific objectives were 1) to use clinical examinations, imaging modalities, and necropsy findings to aid in description, classification, and characterization of TTNS lesions, 2) to describe the epidemiology of TTNS in feedlot cattle, and 3) to evaluate risk factors for TTNS. Upon further investigation into this arrival related condition it became apparent that there were many different descriptors: P3 necrosis, toe abscess, apicus necrotica, apical pedal bone necrosis or toe necrosis. These names and descriptors of toe tip lesions were based on anecdotal experiences and previous case reports. As a result, traditional epidemiological approaches that included field investigations, clinical and necropsy examinations were implemented to identify, characterize and describe this condition. Based on clinical findings, imaging modalities, and necropsy specimens examined during September to December 2012, inclusive, a more specific name and descriptive case definition were introduced. TTNS descriptive epidemiology was described by use of a retrospective database analysis from Feedlot Health Management Services (FHMS) with 702 veterinarian confirmed TTNS cases by necropsy examination. From this database, there were 30% (210/702) of necropsy cases treated for TTNS and 70% of cases (492/702) that were not treated. Of those animals treated, the mean and standard deviation (median) interval from feedlot arrival to first treatment was 18.9 ±1.7 d (12 d). The mean (standard deviation) days on feed until death from TTNS was the earliest in grass-fed calves (32.4 ± 22.1 d), followed by auction-derived (40.6 ± 40.6 d), ranch direct (44.1 ± 53.1 d), and back-grounded calves (69.0 ± 75.6 d) (P < 0.001). Yearlings were on feed for a mean (standard deviation) days of 37.1 ± 32.0 d when compared to calves at 49.5 ± 57.0 d before death (P < 0.001). The greatest proportion of deaths occurred from September to November. There were 96.2% (1,832/1,904) of lots without one case of TTNS and 3.8% (72/1,904) of lots had one or more TTNS cases. A prospective case-control study to identify TTNS risk factors consisted of 148 total necropsy submissions (82 cases, 66 controls) from three feedlot veterinary practices and 16 feedlots during October 2012 to January 2013, inclusive. Confirmation of feet samples by the principal investigator at the Western College of Veterinary Medicine reduced the total to 135 animals: 67 cases and 68 controls. The measure of agreement (kappa) on classification of TTNS cases and controls between the veterinary practice and WCVM was 0.778 (P < 0.001). Bacterial culture results revealed that 75% of pure isolates in TTNS cases were attributed to Escherichia coli, Streptococcus spp., Trueperella pyogenes, Fusobacterium necrophorum. TTNS cases were 3.8 times more likely than control animals to have BVDV isolated (95% CI 1.7-8.5; P < 0.001). TTNS animals were 2.2 times more likely than control animals to have histopathological evidence of vasculitis (95% CI 1.0-4.6; P = 0.04). BVDV samples were 11.2 times more likely to show histopathological evidence of vasculitis than non-BVDV samples (95% CI 4.7-27.0; P < 0.001). A decreased difference was found in sole thickness at the toe tip (P < 0.001). There was no evidence of pedal bone rotation between case and control animals (P = 0.15). In summary, TTNS is a specific term for apical white line separation with tissue necrosis and clinical lameness. A practitioner's field diagnosis of TTNS based on apical white line separation and tissue necrosis is accurate on clinical signs alone. TTNS is a transport or arrival related condition in feedlot cattle that has a propensity for cases to cluster together. Pure bacterial isolates provide an understanding of the pathogens responsible for TTNS and that environmental pathogens contribute to an ascending infection. BVDV, vasculitis and apical sole thickness were risk factors associated with TTNS; however, their exact role requires further investigation.