INTRODUCTION: Platelets are involved in the inflammatory and thrombotic complications associated with atherosclerosis. Human neutrophil peptides (HNP), released from activated neutrophils, demonstrate inflammatory effects related to lesion development. HNP bind the low-density lipoprotein receptor (LR) family member LRP1 and LRP8 is the only member on platelets. HYPOTHESIS: HNP enhance platelet activation and aggregation through interactions with LRP8. METHODS: Platelet activation and aggregation in response to HNP were determined using flow cytometry and aggregometry. Activation was also examined in the presence of recombinant LRP8 and in LRP8 knockout platelets. RESULTS: HNP activate platelets as determined by P-selectin expression and the formation of microparticles. HNP sensitize platelets enhancing their aggregatory response to ADP. Lastly, LRP8 plays a role in HNP-induced platelet activation. CONCLUSIONS: With an improved understanding of the mechanism by which HNP induce platelet activation, we may be able to devise therapeutic strategies to treat patients with cardiovascular diseases.

Drug induced idiosyncratic agranulocytosis has been attributed to oxidation by hypochlorite formed by bone marrow myeloperoxidase (MPO). Idiosyncratic liver toxicity could also involve drug oxidative activation by cytochrome P450 (in hepatocytes) or MPO (in Kupffer cells or infiltrating neutrophil/macrophages). Such drug reactive metabolites could cause cytotoxicity or release “danger signals” that attract immune cells which release H2O2 resulting from nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activation. In vivo animal studies have shown that low level tissue inflammation markedly increased drug-induced tissue toxicity which was prevented by immune cell inhibitors and increased by cell activators. It is suggested that idiosyncratic drugs are much more toxic, taken during symptomless inflammation periods. Furthermore, it is hypothesized that hepatocytes are much more susceptible to some idiosyncratic drugs if they are exposed to hydrogen peroxide (H2O2)/myeloperoxidase or cytokines released by inflammatory cells. A hepatocyte inflammation model, in which hepatocytes were exposed to a non-toxic H2O2 generating system and peroxidase, was found to be much more vulnerable to four idiosyncratic drugs e.g., troglitazone, isoniazid, hydralazine and amodiaquine. The molecular cytotoxic mechanisms for this marked increase in cytotoxicity were investigated as follows: 1) A P450/H2O2-catalyzed pathway not involving oxidative stress e.g., hydralazine and isoniazid; 2) A P450/H2O2-catalyzed oxidative stress-mediated cytotoxic pathway e.g., hydrazine (an isoniazid metabolite) and hydralazine; and 3) A peroxidase/H2O2-catalyzed oxidative stress-mediated cytotoxic pathway e.g,, hydralazine, amodiaquine and troglitazone. Before cytotoxicity ensued, GSH oxidation, protein carbonyl formation and often lipid peroxidation occurred followed by a decrease in mitochondrial membrane potential indicating that oxidative stress was the molecular mechanism of cytotoxicity. In summary, a H2O2-enhanced hepatocyte system in the presence and absence of peroxidase may prove useful for a more robust screening of drugs for assessing the enhanced drug toxicity risk associated with taking drugs during periods of inflammation.

A growing demand for information on the human health and environmental effects of materials produced using nanotechnology has led to a new area of investigation known as nanotoxicology. Research in this field has widespread implications in facilitating the medical applications of nanomaterials but also in addressing occupational and environmental toxicity concerns. Improving our understanding of these issues also has broad appeal in the stewardship of nanotechnology and its acceptance by the public. This work represents some of the early research in burgeoning field of nanotoxicology. Using a variety of in vivo and in vitro models, as well as cellular and molecular techniques I first studied a possible role for the novel cytokine endothelial monocyte activating polypeptide-II (EMAP-II) in acute lung inflammation in rats (Chapter 2). This work demonstrated a significant increase in total EMAP-II concentration in lipopolysaccharide inflamed lungs as early as 1h post-treatment (P<0.05). Increased numbers of monocytes and granulocytes were also observed in lungs treated with mature EMAP-II relative to control rats (P<0.05), and the recruitment of cells did not occur via upregulation of either Interleukin-1β or Macrophage inflammatory protein-2. I further studied whether mature EMAP-II can be induced in pulmonary nanotoxicity studies by exposure to rosette nanotubes (RNT) (Chapters 3-5). In the first in vivo experiments in mice on the RNT(1)-G0 (Chapter 3) I showed an acute inflammatory response at the 50 µg dose by 24h, but this response was resolving by 7d post-exposure as evidenced by a decreased number of cells in the bronchoalveolar lavage fluid (P<0.05) and from histological examination. The results of this study indicated that water soluble and metal-free rosette nanotubes can demonstrate a favorable acute pulmonary toxicity profile in mice. Subsequently, I studied the responses of the pulmonary epithelium using the human Calu-3 cell line (Chapter 4). This experiment indicated that RNT(2)-K1 neither reduces cell viability at 1 or 5 µg/ml doses nor does it induce a dose-dependent inflammatory cytokine response in pulmonary epithelial cells in vitro. My final experiment (Chapter 5) studied the human U937 pulmonary macrophage cell line since the macrophage is one of the key defense mechanisms to encounter RNT in the lung environment. The data indicate that this cell line lacks a robust inflammatory response upon exposure to RNT and that when RNT length is changed by altering the conditions of nanotube self-assembly, cytokine release into the supernatant is not affected profoundly. Although, EMAP-II is upregulated in a lipopolysaccharide model of lung inflammation, it does not serve as a good marker of RNT exposure. The data indicate that RNT have a favourable toxicity profile and these experiments provide a framework upon which rosette nanotubes can be investigated for a range of biomedical applications. Furthermore, in light of media and scientific reports of nanomaterials showing signs of toxicity, this work demonstrates that a biologically inspired nanostructure such as the RNT can be introduced to physiological environments without acute toxicity.

Systemic injections of the neurosteroid progesterone (P) have been shown to improve cognitive, sensory and motor recovery after traumatic brain injury (TBI). Progesterone withdrawal (PW), however, increases the risk of ischemia, anxiety, seizure, and excitotoxicity. Given these side effects, it is possible that acute PW during recovery from TBI may retard the healing process. In this project, we investigated the effect of acute PW for short and long-term intervals, and optimized post-TBI P treatment through tapered P injections and slow-release implanted capsules. Male Sprague-Dawley rats received either frontal-bilateral cortical contusion injury or sham surgery. P-treated animals displayed increased anxiety in the elevated plus maze at the peak of acute withdrawal compared to tapered P doses or vehicle. Inflammation and apoptosis, as measured by TNF and #61537;, NF and #61547;B, and active caspase-3, among others, were decreased for all P-treated animals; these effects were further reduced with tapered treatment. Three weeks after injury, animals that received tapered P administration displayed fewer sensory deficiencies and increased motor activity. In addition, reducing the effects of acute PW increased the activity of HSP70 and BDNF while decreasing necrotic lesion size and reactive astrocyte staining, indicating increased neuroprotection. Finally, the beneficial effects of P administration after TBI were further enhanced through a steady-state release of P from a subcutaneously implanted silastic capsule. Compared to animals receiving a daily bolus through subcutaneous injections, capsule animals demonstrated decreased anxiety and edema. All P-treated animals, regardless of delivery method, had reduced inflammation and apoptosis compared to vehicle-treated animals. This system also serves as a model of steady-state intravenous P administration used in human clinical trials. In conclusion, all P treatment enhances both short and long term recovery after TBI. Acute PW, however, has a negative effect on both behavior and tissue recovery. At the peak of withdrawal, animals undergoing acute PW exhibit an increase in anxiety, sensory deficits, inflammation and apoptosis, and a decrease in locomotor activity, all of which are further exacerbated by injury. Tapered withdrawal enhances neuroprotection and plasticity, while a steady-steady application of P further decreases edema and the anxiogenic effects of withdrawal.

Thèse de doctorat : Sciences pharmaceutiques. Pharmacologie cellulaire et moléculaire : Strasbourg 1 : 2006. / Titre provenant de l'écran-titre. Bibliogr. 24 p.

Thèse de doctorat : Pharmacologie : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.

The link between obesity and the risk of prostate cancer (PCa) is inconclusive. However, studies demonstrate a correlation between obesity, advanced PCa and mortality. Investigating the underlying biological mechanisms by which obesity promotes advanced PCa is necessary to develop potential therapeutic targets that may aid in the efficacy of treating obese men. Obesity-associated changes in tumor biology may modulate key aspects of the hallmarks of cancer; acquisition of characteristics essential for the development and progression of cancer. We hypothesized obesity-induced inflammation promotes PCa progression. Our studies incorporated cell culture and murine models to investigate the role of obesity-related systemic factors on AR signaling, inflammation-stimulated invasive PCa, and the paracrine interaction of the tumor-microenvironment (TME). We sought to recapitulate the systemic effects of obesity to investigate characteristics of the metastatic cascade. Briefly, sera from mice fed 60% or 10% kcal from fat diet for 12 weeks were used for in vitro studies. PCa cells exposed to sera from obese mice increased AR transcriptional activity, proliferation, invasion, migration, MMP-9 activity and EMT: e-cadherin, vimentin and β-catenin. PCa cells exposed to sera from 1 hour maintained the invasive phenotypes similar to PCa cells directly exposed to sera from obese mice. IL-6 is associated with advanced PCa cancer. Depleting sera of IL-6 or IL-6 shRNA suppressed obesity-induced proliferation, invasion, migration and MMP-9 activity in LNCaP cells. Furthermore, in a PTEN spontaneous model of PCa, IL-6 protein and mRNA levels corresponded with progression of PCa in mice fed a high-fat diet. These results suggest IL-6 mediates obesity-induced PCa progression. Stromal cells that comprise the TME vary in their contribution to the growth of tumors. Our studies show macrophage-like and myofibroblasts increased NF-kB activity in PCa cells exposed to sera from obese mice. An increase in NF-kB activity corresponded with proliferation, prostaglandin E2, and invasion and recruitment of stromal cells by PCa cells. In summary, obesity-related systemic factors promote an invasive PCa phenotype, which may be mediated by Akt, AR, IL-6 and the TME. Obesity-induced changes in tumor biology and the microenvironment provide a niche suitable for invasive prostate cancer. / text

abstract / Anatomy / Doctoral / Doctor of Philosophy

Insulin resistance, the cause of type 2 diabetes mellitus, is intimately linked to the dysregulation of adipose tissue. Recent decades have witnessed the discovery and characterization of numerous hormones produced by adipocytes, including leptin, adiponectin and resistin, underscoring the endocrine functions of adipose tissue. To better understand the role of the adipocyte in the mediation of obesity-related insulin resistance and inflammation, this study has optimized the primary adipocyte isolation technique to minimize inflammation inherent to the isolation procedure and has analyzed adiponectin levels and insulin sensitivities of various adipocyte size populations both in vitro and ex vivo.The data described herein suggest that cell size plays an important, but not solitary, role in the regulation of insulin action and adiponectin production. It is possible that obesity-related insulin resistance is associated with the failure of a population of small adipocytes to expand and produce the insulin sensitizing protein hormone, adiponectin.