Abstract not available

Bibliography: leaves 168-184. The work in this study encompasses oxygen free radical related inflammation in the peripheral lung and in lung cells. Animal and human studies have been used. Methods include cell culture with function studies, protein chemistry, animal and human physiology, and cell and lung structure through histopathology, and various forms of electron microscopy. The work resulting from this thesis has formed an important basis for understanding acute and chronic lung injury.

Measuring the level of exhaled NO (eNO) in the breath is a new method to monitor airway inflammation in asthma and may have a role in the management of asthma. The hypotheses were that eNO will reflect the degree of inflammation in chronic asthma, and will indicate how anti- inflammatory therapy should be altered to improve asthma control. Three studies were performed to test the hypotheses. A cross sectional study was performed to define the normal range of eNO and to compare this range with those who have asthma or atopy. The second study was observational, to compare the level of eNO during and after an exacerbation of asthma. The third study was an interventional study to evaluate eNO in management of paediatric asthma. In this latter study the level of eNO was measured to monitor airway inflammation in asthmatic children with the intention of adjusting antiinflammatory drugs (inhaled glucocorticosteroids) according to the level of eNO. These studies have shown that the mean level of eNO was significantly higher in asthmatic compared with normal subjects, but not significantly different when compared with atopic non-asthmatic subjects. eNO was correlated with the number of positive skin prick tests in atopic subjects whether asthmatic or nonasthmatic. The eNO level was increased during acute exacerbations of asthma and decreased after two weeks with therapy of GCS. In a pilot study eNO appeared to be superior to FEV1 in adjusting the dose of iGCS to control asthmatic children, but this needs to be confirmed with a larger sample size. Another non-invasive method to detect inflammatory markers is the technique of exhaled breath condensate (EBC). Although NO is degraded to NOx, it was found that eNO had no significant correlation with EBC NOx but had a significant correlation with pH. Hypertonic saline challenge, an artificial model of an asthmatic exacerbation was associated with an increase in EBC volume and the release of histamine, implicating mast cell activation. These novel findings suggest that non-invasive markers can be used both for clinical and mechanistic proposes.

There is evidence that the excitotoxin quinolinic acid (QUIN) synthesized through the kynurenine pathway (KP) by activated microglia may play a role in the pathogenesis of several major neuroinflammatory diseases and more particularly in Alzheimer??s disease (AD). The hypothesis of this project is QUIN affects the function and morphology of astrocytes. In this study I used human foetal astrocytes stimulated with AD associated cytokines including IFN-gamma, TNF-alpha, TGF-alpha and different concentrations of QUIN ranging from low physiological to high excitotoxic concentrations. I found that QUIN induces IL-1beta expression in human astrocytes and subsequently, contribute to the inflammatory cascade that is present in AD pathology. Glial fibrillary acid protein (GFAP) and vimentin protein expression were complementary in expression to each other after 24 hr stimulation with different QUIN doses. However, there were marked increases in GFAP levels and reduction in vimentin levels compared to controls with QUIN treatment indicating that QUIN can trigger astrogliosis in human astrocytes. Glutamine synthetase (GS) activity was used as a functional metabolic test for astrocytes and I found a dose-dependent inhibition of GS activity by QUIN. This inhibition was inversely correlated with iNOS expression whereby reduced GS activity is accompanied with an increase expression of iNOS in human astrocytes. These results suggest that reduction in GS activity can lead to accumulation of extracellular glutamate then leading to exacerbated excitotoxicity via NMDA receptor over-activation and ultimately neuronal death. PCR array results showed that at least four different pathways were activated with pathological concentration of QUIN including p38 MAPK that is associated with pro-inflammatory cytokine production, ERK/MAPK growth and differentiation that can modulate structural proteins, mitochondrial-induced apoptotic cascade and cell cycle control pathway. QUIN-induced astrogliosis and excitotoxicity could lead to glial scar formation and prevention of axonal growth thus exacerbation of neurodegeneration via synaptosomal NMDA receptor over-activation. All together, this study showed that, in the context of AD, QUIN is an important factor for astroglial activation, dysregulation and death, which can be mediated by the previously mentioned pathways.

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily. The result of RAGE-ligand interactions augments the proinflammatory mechanisms acting in chronic inflammatory diseases. RAGE recognises a wide range of ligands that have no apparent structural similarities. It is unclear what controls this promiscuity of RAGE. The extracellular domain of RAGE has two potential glycosylation sites. It is speculated that N-linked glycosylation may have significant impact on ligand recognition, especially of S100 calcium binding protein ligands. Two objectives of this thesis were to establish whether S100A9 acts as a ligand for RAGE and to investigate whether glycosylation of RAGE has any influence on ligand recognition. These were achieved by generating two forms of RAGE. HEK 293 cells were transfected to express full-length, membrane-bound RAGE or a secreted form comprising the extracellular domain of RAGE. Site-directed mutagenesis of RAGE showed that asparagine at position 25 is the pre-dominant N-linked glycosylation site. The carbohydrate added to asparagine 25 was further modified to a non-sialylated carboxylated N-linked glycan, specifically recognised by monoclonal antibody GB 3.1. Binding studies showed that different RAGE ligands have individual requirements for glycosylation of the receptor. Binding of AGE-modified AGE-BSA or of S100B to RAGE occured independent of N-linked glycosylation of the receptor. RAGE also binds the S100 protein, MRP-14 (S100A9). In contrast to AGE-BSA or S100B, the non-sialylated carboxylated N-glycan expressed on RAGE is crucial for binding to MRP-14. However, RAGE produced in tunicamycin containing medium and thus lacking N-linked glycosylation, shows strong binding to MRP-14. It was concluded that two forms of binding are involved: the first mechanism relies on the non-sialylated carboxylated N-glycan attached to RAGE and acts in a "tethering" fashion. The second mechanism involves a conformational change of RAGE, which results in exposure of a binding site(s) and a more conventional receptor-ligand interaction. Another objective for this thesis is to study the expression of RAGE and its alternatively spliced variants. PCR analysis has revealed several variants of RAGE that result from alternative splicing mechanisms. The variant proteins are soluble due to a lack of membrane localising sequence. PCR results confirmed the presence of transcripts encoding for spliced variants of RAGE in several tumour cell lines. Among these were transcripts that should encode a soluble form of sRAGE 2. Furthermore, it was shown that sRAGE 2 transcript can be present in forms that contain the ligand-binding V-domain of RAGE or that are N-truncated and lack the V-domain. This is the first report of a soluble, N-truncated sRAGE 2 variant. The results in this thesis add to our knowledge of RAGE biology. MRP-14 (S100A9) is identified as a new ligand. The control of MRP-14/RAGE interaction relies on N-linked glycosylation of the receptor and further modification of the carbohydrate. "Tethering" or stronger receptor-ligand interactions are suggested as mechanisms for controlling RAGE recognition of multiple ligands. Soluble RAGE variants that lack or contain V-domain binding regions, and hence sites for glycosylation were produced. These have the capacity to compete with membrane-bound receptor for available ligand. The control of the expression of soluble RAGE variants, in concert with the control of various modification to carbohydrate expressed on the receptor, adds a level of complexity to ligand specificity. This may ultimately result in different paradigms of the inflammatory process.

Purpose: The aim of this study was to investigate the usefulness of formulation approaches to minimise injection site reactions for poorly soluble drugs. The specific objectives were to modify the injection site reactions by identification of irritant components in the formulation and control of their release kinetics; and to gain understanding of formulation approaches to create a favourable microenvironment in the tissues allowing better tissue tolerance and drug absorption. Methods: Physicochemical properties of the model drug, ricobendazole (RBZ) were characterised using conventional methods. Three formulation approaches to minimise irritancy of the low pH RBZ solution were assessed. An in vitro method using 96-well microplates and a microtiter plate reader was used for detection of drug precipitation on dilution for formulation characterisation. Cellular damage by the formulations was investigated in L929 fibroblasts using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase (LDH) assays. Tissue tolerance and pharmacokinetics were simultaneously investigated after subcutaneous injection in sheep. A low pH RBZ solution was used as a reference formulation. Results: Preformulation studies showed that RBZ was practically insoluble in water and oils, and was slightly soluble in commonly used co-solvents. Solubility was slightly improved by complexation with hydroxypropyl-β-cyclodextrin (HP-β-CD, K₁:₁ = 311 M⁻�) or a combination of low pH (> 2) with surfactants or co-solvents. A U-shaped pH-solubility profile in aqueous solutions indicated that RBZ is an ampholyte. pKa values measured by absorbance spectroscopy and pH solubility methods were 3.45 and 3.76 (basic) and 9.82 and 9.53 (acidic) respectively. The partition coefficient was 14.3 - 15.2 at pH 6 - 9 and less at higher or lower pH. In aqueous solutions, RBZ showed a V-shaped pH-degradation rate profile and was most stable at pH 4.8. Degradation pathways were identified as hydrolysis and oxidation. Three RBZ injectables (5%) were obtained by modification of the low pH RBZ solution; addition of 20% HP-β-CD, incorporation into a w/o emulsion, and a microemulsion (ME). On dilution with SPB, the onset time of drug precipitation was prolonged and the rate was reduced in the presence of HP-β-CD. The w/o emulsion had a low viscosity (< 60 mPa.s) and exhibited Newtonian flow. Drug release versus the square root of time was linear and the release rate could be adjusted by phase ratio and droplet size. Drug release was found to be by diffusion. A coarse emulsion layer appeared at the interface between the ME and buffer. Drug release from the ME was faster than from the emulsion and was linear with the square root of time. On titration into SPB, the three formulations showed controlling effects on the release of H₃O⁺ compared to the reference formulation. RBZ (0.1 mg/ml) was more toxic to L929 cells than the co-solvent propylene glycol (50 mg/ml). The formulations showed greater cytotoxicity than their vehicles in the order: ME > RBZ solution = emulsion > HP-β-CD. HP-β-CD and emulsion excipients showed little or no cytotoxicity. The MEs exhibited more toxicity in the LDH assay than in the MTS assay. A reversed phase HPLC assay for simultaneous determination of RBZ and its metabolite in sheep plasma using an isocratic system with UV detection was developed and used in the pharmacokinetic studies. Plasma samples were prepared by solid phase extraction. A suitable internal standard was selected by quantitative structure-retention relationships analysis. The composition of a ternary mobile phase was optimised with the assistance of multiple linear regression. The assays were linear over the concentration range 10 - 1000 ng/ml for both analytes (r > 0.999) with satisfactory inter-day and intra-day precision and accuracy (CV < 10%). The recoveries for all analytes were > 96%. A pilot study in sheep suggests that injection of the vehicles (the CD, emulsion and ME) caused virtually no pain on injection or site reactions. Both the reference formulation and its vehicle induced pain on injection and resulted in swollen tissues. Histology after two weeks showed granulation for the formulation, but not the vehicle. In contrast, animals showed virtually no injection site reactions with the ME and emulsion. The HP-β-CD formulation gave transient pain on injection but a two-fold increase in bioavailability compared with the reference. The emulsion produced sustained drug release and increased drug absorption. In the main study, the HP-β-CD vehicle showed good tissue compatibility. Irritation by the HP-β-CD formulation was attributed to the low pH. Cmax, tmax and AUC0-[infinity] for the reference formulation were 1.3 � 0.3 [mu]g/ml, 9.6 � 2.9 h and 36.7 � 9.2 [mu]g�h/ml respectively, while the corresponding data for the HP-β-CD formulation were 2.9 � 0.8 [mu]g/ml, 5.0 � 0.6 h and 54.5 � 15.3 [mu]g�h/ml respectively. The half-life following the injection of the HP-β-CD formulation (5.5 � 2.8 h) was shorter than that of the reference formulation (8.5 � 3.4 h). Conclusions: Injection site reactions may be minimised by identification of irritant components in a formulation and by controlling their release. Controlling the burst release of the poorly water soluble drug RBZ in a low pH solution could improve tissue tolerance and minimise post-injection precipitation, and hence increase drug bioavailability. In addition, HP-β-CD was a useful local injectable carrier which significantly enhanced the absorption of RBZ after subcutaneous injection in sheep.

Traumatic brain injury (TBI) today remains a major health and social problem for both developed and developing countries. It is the leading cause of death and disability under the age of 40, and despite the significance of this public health problem, no effective therapy exists. While a number of factors have been shown to be associated with the development of irreversible tissue injury after TBI, the formation of oedema and opening of the blood brain barrier (BBB) have been shown to be of major significance to outcome. Oedema formation in the brain, when left uncontrolled, results in increased intracranial pressure that may lead to a decrease in brain tissue perfusion, localised hypoxia and ischemia, and ultimately tissue herniation and death. The mechanisms associated with the development of oedema are largely unclear, and accordingly, current therapies are generally ineffective, often resulting in dehydration, hypotension and renal problems. This thesis describes the characterisation of neurogenic inflammation in the development of post-traumatic brain oedema and functional deficits, and particularly the role of substance P (SP) in mediating their development, using rodent models of both focal and diffuse TBI. Results from this thesis demonstrate that increased SP immunoreactivity, particularly at the level of the vasculature, is a ubiquitous feature of TBI implying a potential role in the breakdown of the blood brain barrier (BBB) following TBI. This was confirmed through the use of the NK[subscript 1] receptor antagonists, which attenuated BBB and oedema formation as well as deleterious morphological events such as dark cell change and axonal injury. The NK[subscript 1] receptor antagonists also resulted in an associated improvement in both motor and cognitive deficits, as assessed using a battery of functional outcome tests. Possible mechanisms of action of the NK[subscript 1] receptor antagonist included effects on the BBB, SP release, intracellular free magnesium concentration and aquaporin-4 channels. Neuroprotection could be facilitated with administration of a non-lipid permeable NK[subscript 1] receptor antagonist in the immediate postinjury period, or up to 12 h after TBI with a lipid permeable NK[subscript 1] receptor antagonist, suggesting that this class of drugs have a clinically viable therapeutic window. We conclude that SP has a significant role in the secondary injury process following TBI and may offer a novel target for the development of interventional pharmacological strategies. / Thesis (Ph.D.)--School of Medical Sciences, 2006.

Bibliography: leaves 234-314. Examines the role of vascular endothelial cells in inflammation with particular reference to their participation in the immune response directed against a vascularised allograft (kidney)

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 127-155).

Abstract: Introduction: Partial bladder outlet obstruction (pBOO) is a ubiquitous problem that results in renal damage. We hypothesize that change in the bladder progresses over time. Methods: Fischer rats underwent surgical pBOO for 2, 4, 8, or 13 weeks and were compared to shams. Urodynamic measurements were taken, bladders weight and thickness recorded, and tissue analyzed with microscopy. RT-PCR was performed for inflammatory mediators and spectrometry used to quantify collagen. Results: Urodynamics demonstrated an increased capacity and deterioration into high-pressure. H+E demonstrated an initial inflammatory response, and increased mRNA levels of TGF-, CTGF, HIF-1, and PDGF. Muscle hypertrophy was evident on H+E and increased bladder mass and thickness. Massons Trichrome and mass spectrometry showed an increase in collagen. Conclusion: We believe that this represents distinct phases of bladder decompensation: inflammation, hypertrophy, and fibrosis. This could lead to improved preventative strategies, with respect to biochemical pathways and the time course of their initiation. / Experimental Surgery