The presence of inflammation in the progression of fatty liver disease induced by fasting was determined in mink. Tumour necrosis factor alpha (TNF-?), and monocyte chemoattractant protein 1 (MCP-1) liver mRNA levels were quantified by real-time PCR. Mink fasted for 5 and 7 days had significantly higher levels of TNF-? and MCP-1 liver mRNA, compared to mink fasted for 0, 1, and 3 days. Mink fasted for 7 days, but re-fed for 28 days had the lowest mRNA levels of both TNF-?, and MCP-1 demonstrating the liver’s ability to restore homeostasis post-fasting. TNF-? mRNA levels were correlated with MCP-1 liver mRNA and liver fat percent. To confirm the physical presence of inflammation, slides stained with haematoxylin and eosin were analyzed for bile ducts resulting in no significant differences. Results indicate that elevated MCP-1 and TNF-? expression are associated with fasting induced fatty liver in mink.

The development of cellular senescence is not consistent in cultured bovine fibroblasts. Numerous factors may be contributing to the variable onset of cellular senescence, including oxidative stress, telomere shortening and DNA damage. Recent results indicate that cellular senescence is also associated with markers of DNA replication. This suggests that cells may be more likely to enter a senescent state depending on their cell cycle progression. The purpose of this study was to determine the effect of cell cycle phase on the development of stress-induced premature cellular senescence (SIPS). Bovine fibroblasts were synchronized at various cell cycle phases, followed by treatment with increasing doses of H2O2. Senescent cells were detected using SA-β-galactosidase staining assay. As H2O2 dosage increased, the amount of cell death by necrosis increased in both unsynchronized and cell cycle synchronized groups, while the amount of senescence varied depending on cell cycle phase. Our results suggest that the S phase of the cell cycle is the most resistant to oxidative damage, the G2/M phase is the most susceptible, and the G1/S phase is the most likely to enter senescence as a protective measure following low doses of H2O2. Increased senescence also resulted from an increased recovery time post-H2O2 treatment, and gene expression studies suggest SIPS bovine fibroblasts senesce via the p53-independent pathway. An improved understanding of SIPS has important implications for improving cloning efficiency and understanding age-related diseases.

This thesis is an investigation of two common skin conditions of pigs: exudative epidermitis (EE) and ear necrosis (EN). The cause of exudative epidermitis and risk factors are well understood, however the study was prompted because of reports of treatment failure. A survey of veterinary practitioners (n=15) and pork producers (n=58) was conducted to determine which treatments are commonly used. Amongst farmer respondents topical treatments were often used and in serious cases injectable penicillin G was administered. Thirty farms with a history of EE were visited and skin samples taken from affected pigs. The antimicrobial resistance pattern for isolates of Staphylococcus hyicus and Staphylococcus aureus revealed that almost all isolates were resistant to penicillin G and ampicillin. In addition, certain isolates of S. hyicus as well as S. aureus were shown to possess the mecA gene which is associated with resistance to methicillin. The presence of widespread resistance to penicillin G among staphylococci isolates suggests a reason for poor treatment response. The presence of the mecA gene in staphylococci other than S. aureus recovered from pigs has not been reported before and is of interest from a public health standpoint. A second study investigated EN. The causative agent(s) and the associated risk factors are not well understood. Eleven case farms were visited and skin biopsies and oral swabs taken from pigs in early, mid and late stages of the disease. Bacteriological culturing was performed for staphylococci and spirochetes as well as histological examination of the biopsy samples. Farm-level risk factors were assessed on 14 case farms and 9 control farms. Staphylococci were generally recovered in abundance from the majority of samples but spirochetes were not cultured and only identified microscopically in a small number of tissue samples. Histology revealed that the disease appeared to occur first as a lesion on the epidermal surface that caused tissue damage and led to subsequent invasion of the dermis. This pathogenesis was consistent with the hypothesis that staphylococci colonize the skin surface and produce exfoliating toxins. Ear biting was noted to be commonly present and may be an important contributing factor. / Ontario Pork Animal Health Strategic Initiative Fund Ontario Ministry of Agriculture, Food and Rural Affairs(OMAFRA) Ontario Veterinary College, University of Guelph

Inflammatory bowel disease (IBD) alters neuronal regulation of the gastrointestinal (GI) tract. The superior mesenteric ganglia (SMG) contain sympathetic neurons that modulate GI functions such, as motility and blood flow. IBD reduces the release of noradrenaline, a sympathetic neurotransmitter. We hypothesized that the reduction in NA release is due to inhibition of voltage-gated calcium current (ICa), as calcium influx is a regulator of neurotransmitter release. We also hypothesized that tumor necrosis factor α (TNFα), a proinflammatory cytokine elevated during IBD, can also inhibit the ICa of SMG neurons. Therefore, we compared ICa amplitude in neurons from normal mice and mice with dextran sulphate sodium (DSS; 5% w/v)-induced colitis. Neurons dissociated from the SMG were cultured overnight and changes to ICa were investigated using electrophysiological, Ca2+ imaging, PCR and neurotransmitter release techniques. Colitis significantly reduced ICa of SMG neurons by selectively inhibiting N-type Ca2+ channels. This was accompanied by a reduction in mRNA encoding the N-type channel alpha subunit (CaV 2.2) and a rightward shift in the voltage dependence of activation of ICa. Colitis reduced the NA release from the colon and jejunum. Depolarization-induced release of tritiated-NA was inhibited by ω-Conotoxin GVIA (300 nM). These results suggest that the changes in VGCC observed at the cell bodies of SMG neurons were also occurring at the nerve terminals during colitis. Similar experimental techniques were performed using SMG neurons incubated overnight in TNFα (1nM). TNFα decreased ICa and depolarization-induced Ca2+ influx in SMG neurons. Similar to DSS-induced colitis, the reduction in ICa was limited to N-type Ca2+ channels. Preincubation of neurons with SC 514 (20μM) and Bay 11 7082 (1µM), inhibitors of nuclear factor kappa B signaling, prevented the reduction in ICa. Preincubation with the p38 MAPK inhibitor, PD 169316 (30µM), recovered a smaller portion of the reduction in Ca2+ influx. These data suggest that DSS colitis and TNFα inhibit N-type VGCC ICa in sympathetic neurons and identify a novel role for NF-κB and p38 MAPK in the regulation of neurotransmitter release. These findings also suggest that DSS colitis inhibits NA release by altering sympathetic N-type VGCC in the colon and jejunum. / Thesis (Master, Physiology) -- Queen's University, 2008-09-02 12:06:20.438

No description available.

The development of the science of micropropagation from the very initial concept of totipotency to the modern day advancement and sophistication has been affected by a wide range of problems such as hyperhydricity, shoot-tip necrosis and somaclonal variation. These problems are largely the result of the obvious fact of trying to grow plants in an environment that is different from the one plants are used to naturally. The extent of these problems ranges from minor technical inconvenience to significant economic loss. Characterization and control of micropropagation problems has been one of the priorities of plant tissue culture research due to the enormous contribution of this discipline for plant production, improvement and conservation. The prevalence and severity of these tissue culture problems varies widely among plant species. The rationale of this research project was therefore, to identify plant species most affected by the problems studied, characterize the problem and find mechanism(s) to control or minimize the damage caused by the problem. The literatures reviewed provide sufficient background information for the experimental chapters. Due to the different nature of the problems and variation in the plant species they affect, the model plant, the methodologies used and parameters analysed were also different. The findings of these investigations, in their own different way, addressed certain problems that individually and collectively pose difficulties to the micropropagation industry. The difference in the content of the experimental chapters is therefore the result of the broader objective of the research project to tackle such difficulties. The success and failure of tissue culture system greatly depends on the choice of PGR’s. This choice can be made based on comparative study of their biological activity. Some promising reports on the role of topolins in micropropagation led to the idea of testing these cytokinins for their potential in tissue culture. As a prerequisite to subsequent investigations, the biological activity of some selected topolins and BA derivatives was tested using the soybean callus bioassay. The activity of the cytokinins tested varied significantly. The results demonstrated that the structure of a cytokinin dictates its activity. Modifications of side-chain improved the activity of oT but had no effect on pT. The presence of the methyl group had an enhancing effect on cytokinin activity of topolins or at least it did not reduce it. BA derivatives BA9THP (conjugated at N9 position), 3FBA and 2Cl6(3OHBA)R (halogenated derivatives) also showed good cytokinin activity and hold good promise for future research. In an attempt to alleviate hyperhydricity in Aloe polyphylla and optimize the micropropagation protocol, meta-topolin and its derivatives were tested at various concentrations together with BA and zeatin. Of all the cytokinins tested mT produced the best results in terms of shoot and root growth. Five μM was found to be the optimum concentration at which complete control of hyperhydricity was achieved without compromising shoot and root growth. Plantlets rooted in a multiplication media. BA generally had a negative effect on growth and development both in vitro and ex vitro. Acclimatization of plantlets was achieved easily by initially transferring plantlets to a mist house (for three weeks) followed by transfer to the greenhouse. The type of cytokinin also had an effect on ex vitro growth with BA-treated plants producing the lowest shoot and root biomass. Various experiments were conducted to characterize and control factors affecting STN in Harpagophytum procumbens. Media type and strength, PGR, carbon sources, sub-culturing, calcium and boron were tested. Results indicated that all of the tissue culture components tested affected STN. From the different media types tested, half strength was MS found to be the preferred medium. Increasing cytokinin concentration increased the incidence of STN and the problem was aggravated by the addition of auxin to the multiplication medium. Optimum shoot multiplication was achieved by omitting auxin and using the cytokinin mTR. Plantlets produced basal callus which interfered with rooting. The quantity of this basal callus was minimum when mTR was used. Sub-culturing plantlets onto fresh medium every two weeks helped minimize STN. Off all the sugars tested 3% sucrose was optimum. Other sugars either aggravated STN or inhibited growth when compared at equi-molar concentration. Increasing the concentration of either Ca or B prevented the development of necrotic shoots. When the concentration of both elements is increased simultaneously negative effects on both growth and STN were observed. Using 6 mM Ca in half strength MS medium was optimum. B was toxic at higher concentrations. Plantlets rooted readily in half strength cytokinin-free MS media supplemented with 2.5 μM IAA. Rooted plantlets produced using the optimized protocol were acclimatized successfully by transferring directly to a greenhouse in a 1:1 ratio of sand and soil mixture. The effect of meta-toplins on micropropagation and somaclonal variation of banana was investigated. Tissue cultured explants of cultivars ‘Williams’ and ‘Grand Naine’ were cultured in MS media containing the cytokinins BA, mT, MemT, MemTR and mTR at various concentrations. Results of the investigation revealed that superior multiplication and lower abnormality index was recorded from the mTR and mT treatments at 22.2 μM concentration. These treatments, however, had an inhibitory effect on rooting. The effect of these treatments (22.2 μM mT and mTR) in comparison with equi-molar concentration of BA on somaclonal variation of ‘Williams’ banana was tested using RAPD-PCR at the 7th multiplication cycle. No significant difference was found between the treatments. It should however be highlighted that cultures were initially maintained for three multiplication cycles in media containing BA. The inherent stability and initial effect of BA could have influenced the results. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

No description available.

In our previous work we demonstrated that the integrin antagonist, HYD1, induced necrotic cell death in myeloma cell lines in vitro and in vivo as a single agent. In order to further delineate biomarkers of response to HYD1 we developed an isogenic drug resistant variant named H929-60. We show that the acquisition of resistance towards HYD1 correlates with reduced expression of the cleaved α4 integrin subunit and beta 1 integrin. Moreover, we demonstrate that HYD1 interacts with α4 integrin in myeloma cells. Consistent with reduced VLA-4 expression, the resistant variant showed ablated functional binding to fibronectin, VCAM-1 and the bone marrow stroma cell line, HS-5. The reduction in binding to extracellular matrices of the resistant variant translated to sensitivity to melphalan and bortezomib induced cell death in the bone marrow stroma co-culture model of drug resistance. Moreover, CD138 positive myeloma cells were more sensitive to HYD1 induced cell death compared to the CD138 negative fraction, and potency of HYD1 induced cell death significantly correlated with α4 integrin expression. We were also able to show that reducing α4 or β1 integrin using shRNA strategies was sufficient to cause resistance in myeloma cell lines. In addition we investigated the effects of cyclized variants of HYD1 to improve potency of the agent. One such compound, named HM-27, was determined to be 30 fold more active in H929 cells when compared to HYD1. HM-27 and HYD1 were determined to have similar mechanisms of action as H929-60 cells were shown to be resistant to both compounds when compared to H929 cells. We further characterized HM-27's mechanism of action by investigating what effects HM-27 induced Ca2+ oscillations had on HM-27 induced cell death. The increases in intracellular Ca2+ seen after treatment with HM-27 were determined to occur via release from ER stores and not through influx through plasma membrane channels. Inhibiting Ca2+ release from the ER also potentiated the effects of HM-27 in MM cells. Furthermore, inhibiting Ca2+ release from the ER was also shown to block the onset of autophagy after ER treatment. Treating cells with the lysosomotropic agent, chloroquine, was shown to potentiate the activity of HM-27 in vitro and ex vivo. HM-27 was also shown to have activity in an in vivo model with combination treatment containing bortezomib and HM-27 increasing mouse survival. Collectively our data indicate that VLA-4 expression is a critical determinant of response to HYD1 induced cell death. We also showed that increases in intracellular Ca2+ seen after treatment with HM-27 had a cytoprotective effect in MM cells. Moreover, neutralizing autophagy potentiates HM-27 induced cell death in vitro and ex vivo while combining bortezomib and HM-27 increased survival in vivo. These data continue to provide rationale for further pre-clinical development of HYD1 as a novel anticancer agent.

The kinetics of production and role of tumor necrosis factor-alpha (TNF-$ alpha$) and nitric oxide (NO) during the early phase of blood-stage infection with Plasmodium chabaudi AS were investigated using two inbred strains of mice which differ in the level of resistance to this parasite. Analysis of the in vivo expression of TNF-$ alpha$ and inducible nitric oxide synthase (iNOS) revealed that, early during infection, resistant C57BL/6 mice, which clear the infection by 4 weeks, have higher levels of TNF-$ alpha$ and iNOS mRNA in the spleen and TNF-$ alpha$ mRNA in the liver than susceptible A/J mice which succumb to the disease 10 days after initiation of infection. Moreover, resistant mice expressed high levels of IFN-$ gamma$ (a Th1 marker) and low levels of IL-4 (a Th2 marker) mRNA in the spleen, whereas susceptible A/J mice had low levels of IFN-$ gamma$ but high levels of IL-4 mRNA in the spleen early during infection. Increased levels of NO$ sb3 sp-$ were detected in serum of resistant C57BL/6 mice only at the time of peak parasitemia. Furthermore, treatment of resistant C57BL/6 mice with anti-IFN-$ gamma$ and anti-TNF-$ alpha$ monoclonal antibody demonstrated that TNF-$ alpha$, either alone or in synergy with IFN-$ gamma$, plays a major role in the up-regulation of NO production during P. chabaudi AS malaria. Moreover, treatment with the iNOS inhibitor aminoguanidine, eliminated resistance of these mice to infection with P. chabaudi AS without affecting parasitemia, suggesting that NO may not be involved in parasite killing in vivo. Taken together, these results demonstrate that a Th1-associated increase in TNF-$ alpha$ early during infection, as occurs in resistant mice, leads to the up-regulation of NO production which is crucial for survival of the host. On the other hand, our results also suggest that a Th2 response, as occurs in susceptible mice, does not result in protective levels of TNF-$ alpha$ and NO. However, susceptible A/J mice were found to