Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 91-100).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 70-92).

Neurodegeneration describes the progressive loss of structure and function of neurons, leading ultimately to cell and organism death. Although the initiating factors of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and Amyotrophic Lateral Sclerosis may be different, they share common pathophysiologies. Proteinopathies, as these diseases are now termed, are characterized by atypical deposits of proteins, often due to misfolding. Associated with these deposits are dysfunctional mitochondria, oxidative stress, disrupted axonal transport, inflammation, and apoptotic cell death. If this occurs in motor neurons, as in ALS, ataxia precedes death with little or no change in cognition. On the other hand, if the deposits are found in cortical neurons, as in Alzheimer’s disease, the outcome is dementia and motor function remains largely intact. Each disease is selective for particular types of neurons and brain regions. Although research has elucidated much of the molecular biology involved in these diseases, their initiating causes remain largely unknown. Most of our current understanding originated with the identification of gene mutations that cause rare familial forms of these diseases. As a result, numerous strains of transgenic animals have been developed to study neurodegenerative disease phenomena and were central to the studies presented in this body of work. Novel routes of drug and gene delivery are described here as well as characterization of the mouse models studied. In particular, this work demonstrates that the blood brain barrier is disrupted in ALS followed by the formation of autorosettes in ALS mice. In various Alzheimer’s disease mouse models, it was demonstrated that the acute phase reactant alpha-1-antichymotrypsin (ACT) not only interacts with amyloid plaques, but also induces tau phosphorylation in vivo; tying together these disease hallmarks. It was also shown that small fragments of Aβ (1-11) could disrupt the formation of mature amyloid plaques in these mice. Lastly, it was demonstrated that mature plaques could also be decreased by intracranial delivery of granulocyte-macrophage stimulating factor (GM-CSF). My dissertation research goal was to understand and develop these treatment strategies based on protein disaggregation, neuroprotection, and inflammation, meanwhile developing novel methods for targeted delivery of molecules into the CNS of mice.

The leading cause of death among women is coronary heart disease (CHD), a multifactorial disease with polygenic heritability estimated at 50%. Polymorphisms in the family with sequence similarity 5, member C’ ( FAM5C) gene have been associated with myocardial infarction (MI), and one single-nucleotide polymorphism (SNP) has partially accounted for linkage in an acute coronary syndrome subset. The linkage peak on FAM5C corresponds directly with a quantitative trait locus for the inflammatory biomarker monocyte chemoattractant protein 1, as well as a linkage peak to metabolic syndrome (MetS). Metabolic syndrome increases the risk of developing CHD, and MI has been positively associated with elevated inflammatory biomarkers. This study was designed as a descriptive pilot gene association study. The purpose was to investigate the variability of the FAM5C SNP (rs10920501) in a cohort of women with documented CHD. It also examined the association between the variability of the FAM5C SNP (rs10920501), MetS, inflammatory markers, and the association with early onset CHD in the presence or absence of MI. A subset of 91 women was derived from an earlier study of women randomized to either a gender-tailored or traditional cardiac rehabilitation program. The results indicated the T allele of FAM5C SNP rs10920501 has a strong protective effect in women with a history of MI. Women with a history of MI and the heterozygous (AT) genotype had a mean age of onset of CHD at 62 years, compared to the homozygous wild type (AA) with a mean age of onset at 55 years, (F (3, 34) = 5.00, p < .01). No women in this study with the homozygous variant (TT) had an MI, further demonstrating the protective effective of the T allele. The genotype of FAM5C SNP rs10920501 explains approximately seven percent of the variability of age of onset of CHD in women who have had an MI, while holding body mass index (BMI) and smoking history constant. There was no significant relationship between FAM5C SNP (rs109320501) and MetS or any inflammatory biomarkers in this sample. In conclusion, FAM5C remains a gene of interest in a complex disease process.

Obesity is a known risk factor for postmenopausal breast cancer, and is associated with worse disease prognosis in pre- and postmenopausal women. Adjuvant hormonal therapies improve disease prognosis in obese women, but many still recur. Given that obesity induces inflammation and increases levels of cyclooxygenase-2 (COX-2) enzyme, resulting in tumor proliferation, this retrospective study investigated if women on anti-inflammatory drugs would have improved disease outcomes by reduced production of prostaglandins by COX-2 pathway. Four hundred and forty women treated for invasive breast cancer in San Antonio clinics were included. Cases were classified as NSAID users if notes included daily use of aspirin, ibuprofen, celecoxib or another COX-2 inhibitor; patients were categorized as NSAID nonusers if they were not taking any NSAIDs, or if they used COX-2 drugs for pain as needed rather than daily. Patients on NSAIDs were more likely to be older, be slightly more obese and postmenopausal. NSAID and NSAID nonusers did not statistically significantly differ in regards to BMI categories, tumor stage, hormone receptor status, type of invasive tumor, ethnicity/race and type of surgery. NSAID users had significantly less recurrence rates compared to nonusers (p=0.05). Further, time to disease progression was delayed by almost 28 months in patients who were NSAIDs users. Although this trend was non-significant statistically due to low number of total recurrences, it is promising in the clinical setting. In a logistic regression model using NSAID use, BMI categories and hormonal therapy drug as independent variables to predict recurrence, use of NSAID was only statistically significant in the univariate model. Overweight women were more likely to develop recurrence than normal weight when holding NSAID use and endocrine therapy constant. Obese women had increase recurrence risk, but the trend was not statistically significant. Females using aromatase inhibitors were less likely to recur than those on tamoxifen. The results of this exploratory study had limited power to determine multiple modulating factors, but because they suggest a major clinical benefit, further analyses in a larger sample size are needed to confirm these findings. / text

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Risks of metabolic syndrome including cardiovascular disease and diabetes are significantly affected by maternal nutrition. This concept of developmental programming had been investigated in our laboratory and in an earlier study, it was established that maternal high-fat diet predisposed rat offspring to insulin resistance and higher triglyceride in serum, liver, skeletal muscle and adipose tissue. These abnormalities, however, were ameliorated by supplementing green tea extract to dam’s diet throughout gestation and lactation. The overall objective of this thesis was to examine lipid metabolism, oxidative stress and inflammatory responses in heart of offspring born to dams receiving high-fat diet with or without green tea supplementation during pregnancy and lactation. Female Sprague-Dawley rats were fed an obesogenic diet which was a high-fat diet (HF,30%), low-fat diet (LF,7%) or HF diet containing 0.75% green tea extract prior to conception and throughout gestation. During lactation, half of the dams had their diet switched from HF to GT and vice versa. Pups were weaned to the HF or LF diet, forming 10 offspring groups (gestation/lactation/postweaning): LF/LF/LF, LF/LF/HF, HF/HF/LF, HF/HF/HF, HF/GT/LF, HF/GT/HF, GT/GT/LF, GT/GT/HF, GT/HF/LF and GT/HF/HF. Except a larger fibrotic area, maternal HF diet did not affect lipid accumulation, oxidative status and inflammatory response in the heart of offspring. Analysis of variance revealed different, and even opposite, effects of GT supplementation during gestation and lactation. In offspring born to dams receiving GT supplementation during gestation, they had suppressed fatty acid oxidation (FAO) and higher triglyceride (TG) level in the heart. In contrast, when GT was supplemented to dams during lactation, offspring had elevated heart TG, cholesterol and free fatty acid levels but up-regulated FAO. Since FAO is associated with reactive oxygen species (ROS) production, modulation of FAO is believed to affect cellular stress responses in heart. Consistent with FAO, cardiac stress, apoptotic and inflammatory biomarkers including B-type natriuretic peptide (BNP), bcl 2 associated-x (bax) and interleukin-1β (IL1b) were down-regulated in offspring born to dams given GT during gestation, whereas GT supplementation during lactation increased the expression of pro-apoptotic markers: bax and caspase-3 (Cas3) concurrent with activation of antioxidant defense system: catalase, glutathione peroxidase (GPx) and glutathione S-transferase (GST) as adaptive mechanism against increased ROS. Uncoupling protein 2 (UCP 2) and subsequent higher bcl 2 /bax ratio has been reported to stimulate apoptosis. In agreement with this, mRNA expression of BNP, bax and Cas3 were found to correlate with that of UCP 2. This suggests UCP 2may play an important role in apoptosis under the impact of maternal GT supplementation. The present data suggest that the effect of maternal high-fat diet is organ specific causing apparently lesser damage to the heart. When GT is given in conjunction with a high-fat diet to dams during gestation, there is no clear cut advantage to the offspring. However, potential adverse effects could not be ruled out when GT is supplemented to dams during lactation possibly due to higher catechin exposure via milk. Future study should focus on establishing the benefits and safety use of GT during gestation. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

abstract / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Hyperbaric oxygen (HBO) therapy is the intermittent inhalation of 100% oxygen at a pressure greater than one atmosphere absolute. It is an effective treatment for various inflammatory conditions, including chronic wounds which are characterized by an excessive influx of neutrophils and their prolonged persistence at the wound site. Neutrophil apoptosis and clearance have been shown to be required for resolution of inflammation. The mechanisms by which HBO aids wound healing are well documented, but its effects on cellular inflammatory response are not well understood particularly with respect to neutrophils. The hypothesis presented in this thesis is that increased oxygenation via HBO assists chronic wound healing by enhancing non-inflammatory neutrophil defences and cell death through apoptosis. An investigation was carried out into the effects of HBO on neutrophil antimicrobial function and apoptosis using differentiated HL-60 cells as an in vitro neutrophil model. The data clearly showed that a single HBO treatment for 90 min caused an increase in the oxidative burst activity of neutrophil-like cells as shown by increased NBT staining, superoxide (cytochrome c reduction) and H2O2 production (Kruskal-Wallis, P < 0.05), and phagocytosis of Staphylococcus aureus. HBO treatment displayed a pro-apoptotic effect, enhancing caspase 3/7 activity both in the presence and absence of a TNF-α stimulus (Kruskal-Wallis, P < 0.05) and causing morphological changes (observed using Giemsa and SYBR® Safe staining) associated with apoptosis. Although no consistent pattern was observed, both hyperoxia and pressure alone seemed to contribute to both the increase in antimicrobial activity and the increase in apoptosis induced by HBO in these neutrophil-like cells (Chapters 4 and 5). HBO-enhanced neutrophil clearance by macrophages was investigated using bovine neutrophils and monocyte-derived macrophages (MDMФ). A single 90 min HBO exposure significantly increased the clearance of fresh and 22 h-aged neutrophils by MDMФ (two-way ANOVA, P < 0.05), suggesting an increase in phosphatidylserine (PS) exposure in apoptotic neutrophils after HBO treatment (Chapter 6). Importantly, a long-term repetitive exposure to HBO in patients with chronic wounds caused a significant decrease in the antioxidant enzyme defence system (one-way repeated measures ANOVA, P < 0.05), plasma TNF-α and IL-1β after 30 HBO sessions, with down regulation of expression of the anti-apoptotic factors, NF-B and Bcl-2 (Chapter 7). These findings may go some way towards explaining the effectiveness of HBO treatment not only for chronic wounds but also for other inflammatory conditions that may be affected by this treatment.

The autonomic nervous system serves to maintain a constant inner environment, a process termed homeostasis. Thus, in response to the homeostatic challenge posed by infectious agents, the autonomic nervous system answers to signals from the immune system and elicits adaptive physiological and behavioral reactions. These so called sickness responses include fever, anorexia, hyperalgesia, social avoidance, and the release of stress hormones. Neuropeptides, used in the communication between neurons, are because of their release properties and sustained actions likely mediators of homeostatic responses. The enkephalinergic system constitutes one of the largest neuropeptidergic systems in the brain, but its involvement in inflammatory conditions has been little studied. We first examined the immune-induced activation of the parabrachial nucleus (paper I), an enkephalinergic autonomic relay center in the brain stem. We found that intravenous injection of bacterial endotoxin, lipopolysaccharide (LPS), activated the external lateral parabrachial subnucleus, as measured in terms of Fos expression, but that the enkephalinergic cell population in this subnucleus was largely separated from the LPS-activated neurons. Because Fos may not always be a reliable activity marker, we next examined by in situ hybridization the immune-induced expression of newly transcribed preproenkephalin (ppENK) heteronuclear RNA (hnRNA), which gives a direct indication of the utilization of enkephalin in a particular neuron (paper II). We detected induced expression of ppENK hnRNA in several autonomic structures in the brain, including the paraventricular hypothalamic nucleus (PVH) but not the parabrachial nucleus, indicating increased enkephalinergic signaling activity in the positively labeled structures during inflammatory condition. We then examined the projections of the immune-induced ppENK transcribing PVH neurons by injecting rats intraperitoneally with the retrograde tracer substance Fluoro-Gold, hence labeling neurons with axonal projections outside the blood-brain barrier, followed by systemic injection of LPS (paper III). Dual-labeling histochemical and hybridization techniques showed that the vast majority of the ppENK hnRNA expressing cells were hypophysiotropic cells, hence being involved in neuroendocrine regulation. These findings suggest that centrally produced enkephalin is involved in the coordination of the sickness responses during systemic immune challenge, including the modulation of the release of stress hormones or other hypothalamic hormones during inflammatory conditions. We next turned to the role of prostaglandins in the hypothalamic-pituitary-adrenal (HPA) axis response to inflammation. We injected mice deficient for the terminal prostaglandin (PG) E2 synthesizing enzyme mPGES-1 with LPS and studied their stress hormone release (paper IV). The genetically modified mice displayed attenuated plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone during the later phases of the HPA-axis response compared with wild type mice, and this impairment did not depend on a changed activation pattern in the brain, but instead correlated to an early decrease in corticotropin-releasing hormone mRNA expression in the PVH, hence being the likely cause of the blunted ACTH and corticosterone responses at later time-points. Based on these findings we suggest that a neural, mPGES-1-independent pathway, and a humoral, mPGES-1-dependent pathway act in concert but in distinct temporal patterns to initiate and maintain the HPA-axis response during immune challenge. In addition to activating the central limb of the HPA-axis, inflammatory mediators have been suggested to act directly on the adrenal gland to induce the release of corticosterone, but little is known about the underlying mechanisms. We examined adrenal tissue isolated from rats injected with LPS or interleukin-1β (IL-1β) (paper V), and found that immune stimulation resulted in dynamic changes in the adrenal immune cell population, implying a rapid depletion of dendritic cells in the inner cortical layer and the recruitment of immature cells to the outer layers. These changes were accompanied by an induced production of IL-1β and IL-1 receptor type 1, as well as of cyclo-oxygenase-2 and mPGES-1 in these cells, implying local cytokine-mediated PGE2 production in the adrenals, which also displayed EP1 and EP3 receptors in the cortex and medulla. Additional mechanistic studies using an IL-1 receptor antagonist showed that IL-1β acts locally to affect its own synthesis, as well as that of cyclooxygenase-2. Taken together these data demonstrate a mechanism by which systemic inflammatory agents activate an intrinsically regulated local signaling circuit that may influence the adrenals’ response to immune stress and may help explain the dissociation between plasma levels of ACTH and corticosteroids during chronic immune perturbations.