Abstract Genetic factors have been shown to have an important role in intervertebral disc disease. The associations of known genetic risk factors and whole-body vibration, a proposed environmental risk factor, for intervertebral disc disease (IDD) were evaluated. Eleven variations in eight genes (COL9A2, COL9A3, COL11A2, IL1A, IL1B, IL6, MMP-3 and VDR) were genotyped in 150 male train engineers with an average of 21-year exposure to whole-body vibration and 61 male paper mill workers with no occupational exposure to vibration. The number of individuals belonging to the IDD group was significantly higher among train engineers (42% of train engineers vs. 17.5% of sedentary workers; p = 0.005). In addition, the IL1A-889T allele represented a risk factor for the IDD-phenotype. In order to clarify the role of genetic variations in the genes coding for several proinflammatory mediators, hundred fifty-five Finnish individuals with IDD were analyzed for mutations in the genes coding for inflammatory mediators IL-1α, IL-1β, IL-6 and TNF-α. In addition, sixteen single nucleotide polymorphisms (SNPs) in inflammatory mediator genes were genotyped. An association was identified between IDD and IL6 polymorphism +15T>A in exon 5 (p = 0.007). In addition, IL6 haplotype GGGA of -597G>A, -572G>C, -174G>C and +15T>A in exon 5 associated with IDD (p = 0.0033). A functional SNP in the CILP gene has been suggested to cause IDD in the Japanese population. This functional variation was analyzed in 243 Finnish IDD patients and 259 controls, and in 348 Chinese individuals with degenerative MRI findings and 343 Chinese individuals with normal MRI. No association was found in the Finnish and Chinese study populations. In order to reveal chromosomal susceptibility loci and new candidate gene(s) for IDD a genome-wide scan was performed on 14 Finnish families with 186 individuals. Genome-wide and fine mapping analysis provided maximum two-point LOD scores of 2.71, 2.36 and 2.04 for chromosomes 21, 4, and 6, respectively. Second fine mapping confirmed the susceptibility of chromosome 21. Two candidate genes, ADAMTS-1 and ADAMTS-5, were analyzed in the region suggesting linkage, leading to the identification of thirteen sequence variations. However, none of the variations were disease causing.

The pro-inflammatory cytokine interleukin 1 (IL-1) is involved in numerous physiological and pathological processes. It contributes to thermoregulation, sleep, feeding behaviour and notably to the exacerbation of non-communicable disorders such as cancer, heart disease, stroke and epilepsy, which are the greatest cause of mortality worldwide. Given this important role, IL-1 is tightly regulated, with regulation mechanisms present at the level of its synthesis, activation and receptor engagement. However, when studying IL-1 in vitro, little notice is taken of the disease microenvironment in which it acts. Acidosis is a hallmark of disease, often due to poor perfusion resulting in a shift to anaerobic respiration, a build-up of lactic acid and poor clearance of CO2. Additionally, highly active infiltrating immune cells favour anaerobic respiration and can contribute to this local acidosis. This thesis utilised primary cell cultures, cell lines and reporter cells to explore the mechanisms of IL-1 signalling under disease-relevant acidic conditions. Subsequently, a murine seizure model was developed to further explore IL-1 signalling in disease conditions in vivo. This work demonstrated that acidic pH itself did not induce IL-1β release, however, it did promote release of minimally active 20 kDa IL-1β in response to damage associated molecular patterns (DAMPs) such as ATP, monosodium urate crystals or calcium pyrophosphate dihydrate crystals. The cleavage of pro-IL-1β into 20 kDa IL-1β was mediated by cathepsin D and was also induced on addition of lactic acid to the culture media. This 20 kDa IL-1β was not further cleaved to the active mature 17 kDa IL-1β thus its production limits the spread of inflammation. The intranasal administration of kainic acid induces seizures in C57Bl/6J mice, however, IL-1β was not observed acutely in this model thus the presence of 20 kDa IL-1β in vivo was not confirmed. In recent years, the contribution of IL-1 to disease has become well established. However, despite successes in the development of novel therapeutics targeted at blocking IL-1 activity, such as anakinra, canakinumab or rilonacept to treat cryopyrin associated periodic syndromes, a number of studies have demonstrated poor efficacy and only minor improvements in patients when targeting IL-1. Thus further knowledge of the mechanisms of IL-1 signalling in disease is required to understand this system and develop improved novel therapeutics.

Doctor of Philosophy / Department of Human Nutrition / Sara Rosenkranz / The aims of this dissertation were to provide innovative, applicable insights regarding the impact of single-meal consumption on metabolic and inflammatory responses in the acute post-meal (“postprandial”) period. In Chapter 2, the connection between large postprandial glucose and triglyceride (TG) fluxes and cardiovascular disease (CVD) risk were reviewed. A new marker of metabolic status, Metabolic Load Index (MLI), calculated by adding glucose and TG, was proposed based on several considerations: 1) independent associations between postprandial glucose and TG with CVD risk, although the substrates are considered to increase risk through similar mechanisms; 2) postprandial glucose and TG responses are interrelated; and 3) meals consumed in daily life typically contain both carbohydrate and fat. MLI may be useful in characterizing metabolic status/risk in both clinical and research settings. Chapter 3 was a systematic review with the purpose of objectively describing postprandial responses (i.e. magnitude and timing) to a high-fat meal (HFM) in five commonly assessed inflammatory markers: interleukin (IL)-6, C-reactive protein (CRP), tumor necrosis factor (TNF)-α, IL-1β, and IL-8. IL-6 increased in >70% of studies, starting at ~1.4 pg/mL pre-meal and peaking at ~2.9 pg/mL ~6 hours post-HFM. Other markers (CRP, TNF-α, IL-1β, and IL-8) did not change after the HFM in the majority of studies. These findings suggest that IL-6 is an inflammatory marker that routinely increases following HFM consumption. Future postprandial studies should further investigate IL-6, as well as explore novel markers of inflammation. In Chapter 4, we compared the metabolic and inflammatory responses to a HFM (17 kcal/kg, 60% fat), representative of meals used in previous postprandial studies, to two meal trials that were more reflective of typical eating patterns: a moderate-fat meal (MFM; 8.5 kcal/kg, 30% fat), and a biphasic meal (BPM), in which the MFM was consumed twice, three hours apart. The HFM elicited a greater total area-under-the-curve (tAUC) TG response (1348.8 ± 783.7 mg/dL x 6 hrs) compared to the MFM (765.8 ± 486.8 mg/dL x 6 hrs; p = 0.0005) and the BPM (951.8 ± 787.7 mg/dL x 6 hrs; p = 0.03), but the MFM and BPM were not different (p = 0.72). It appears that the large postprandial TG response observed in previous studies may not be representative of the daily metabolic challenge for many individuals. Chapter 5 assessed the impact of both aging and chronic physical activity level on postprandial metabolic responses by comparing three groups: younger active (YA), older active (OA), and older inactive (OI) adults. The TG tAUC response was lower in YA (407.9 ± 115.1 mg/dL x 6 hr) compared to OA (625.6 ± 169.0 mg/dL x 6 hr; p = 0.02) and OI (961.2 ± 363.6 mg/dL x 6 hr; p = 0.0002), while the OA group TG tAUC was lower than OI (p = 0.02). Thus, it is likely that both aging and chronic physical activity level impact the postprandial metabolic response. This series of projects provides needed clarification regarding the postprandial metabolic and inflammatory responses to single-meal intake, particularly in the context of real-life application.

M.Tech. / The plants Salacia leptoclada and Warburgia salutaris possess antioxidant properties and are commonly used in Southern Africa for the treatment of inflammatory and other diseases. In order to determine their therapeutic use in crystalline silica-induced injury, the extracts of S. leptoclada and W. salutaris were investigated on silica-induced increased levels of (i) TNF-a, IL-113, INF-y, (ii) the activation of the transcription factor NE-KB, and (iii) the induction of DNA damage and lipid peroxidation. Through its antioxidant property, W. salutaris exhibited a protective effect against crystalline silica-induced inflammatory cytokine expression, NF-KB activation and DNA strand breakage. The extracts of W. salutaris also inhibited cellular membrane peroxidation induced by crystalline silica. Similarly, the extracts of S. leptoclada showed protection of cells against crystalline silica-induced membrane peroxidation. However, S. leptoclada proved ineffective in protecting against silica-induced DNA damage, proinflammatory cytokine expression and NF-KB activation. Since crystalline silica-induced inflammation, NE-KB activation, DNA damage and lipid peroxidation are involved in the process of crystalline silica-induced fibrogenecity and carcinogenicity, W. salutaris may be a potential therapeutic agent against crystalline silica-induced cellular injury.

Humin substances have been used as folk remedies for the last 3000 years. Recent studies have shown that humates possess anti-inflammatory properties, but the mechanism of how it affects inflammation is still unclear. In this study the anti-inflammatory properties of potassium humate, a water soluble humic acid salt, was investigated on different inflammatory pathways in vitro and in vivo. The effect of potassium humate on human mononuclear lymphocyte proliferation showed that potassium humate stimulated lymphocyte proliferation of resting-, PHA- and PWM-stimulated lymphocytes in vitro from concentrations of 20 to 80 µg/ml, in a dose dependant manner, where a maximum proliferation was observed at 80 µg/ml whereas lymphocyte proliferation decreased at 100 µg/ml. On the contrary potassium humate, at 40 µg/ml, significantly inhibited the supernatant concentrations of the following cytokines; TNF-α, IL-1ß, IL-6 and IL-10 by PHA stimulated lymphocytes. The effect of potassium humate on the alternative as well as the classical complement pathway was investigated in vitro using the haemolytic complement assay. Results indicated that potassium humate inhibits both the alternative and classical complement pathways without affecting the red blood cell membrane stability. Different inflammatory mechanisms were investigated in vivo, using the carrageenan-induced paw oedema model and the delayed type hypersensitivity reaction model. The carrageenan-induced paw oedema model was used to determine the effect of potassium humate on acute inflammation in the hind paw. Carrageenan was injected into the right hind footpad of a rat which caused an increase in paw volume due to oedema, which was measured with a plethysmometer. Potassium humate significantly inhibited the oedema at a dose of 60 mg/kg bodyweight and compared favourably with indomethacin at 10 mg/kg bodyweight. The effect of potassium humate on the delayed type hypersensitivity reaction model was also investigated whereby rats were sensitised with sheep erythrocytes. Potassium humate was administered daily by oral gavage at a dose of 60 mg/kg bodyweight. After 7 days, rats were challenged by injecting sheep erythrocytes into the right hind footpad. The degree of inflammation was determined by measuring the increase of paw volume with a plethysmometer. It was found that potassium humate did not have an anti-inflammatory effect on the delayed type hypersensitivity reaction as opposed to the inhibition caused by dexamethasone at a dose of 30 mg/kg bodyweight. This study showed that potassium humate selectively inhibited the inflammatory pathway of the carrageenan-induced paw oedema as opposed to the delayed type hypersensitivity. The mechanism of the anti-inflammatory property of potassium humate might possibly be due to the inhibition of the complement cascade. This study clearly shows that potassium humate possesses anti-inflammatory properties that can be utilised in the future as a potential treatment for inflammatory disorders associated with the activation of complement. However further investigation in the mechanism by which potassium humate inhibits complement activation needs to be done. / Dissertation (MSc (Pharmacology))--University of Pretoria, 2008. / Pharmacology / unrestricted

Many researchers in the academic and clinical communities theorise that inflammation may underpin the placental dysfunction to which the majority of fetal growth restriction (FGR) and stillbirth cases are attributed. Villitis of unknown etiology (VUE) is an inflammatory condition of the placenta characterised by lesions of macrophages and T cells in the villous stroma. This study addressed the hypothesis that VUE is a maternal-mediated immune reaction that contributes to FGR and stillbirth by detrimentally affecting placental function. The hypothesis was tested by: 1) completing a systematic review of the literature to confirm implied links of VUE to poor pregnancy outcome, 2) performing a detailed characterisation of the cellular phenotype of VUE in stillbirth, 3) developing an in vitro model of VUE and 4) examining the functional effects of VUE using this model. A systematic review of the literature revealed that VUE occurred in 28.6% of placentas from FGR pregnancies compared to 15.6% of placentas from appropriately grown infants (p < 0.0001), confirming the implied association. There were insufficient published studies to be able to corroborate a link with stillbirth. Elevated numbers of macrophages, CD4 and CD8 T cells were quantified in VUE lesions. There were significant increases in pan-placental CD4 and CD8 T cell presence in placentas from stillborn infants with VUE (p < 0.0001). A greater staining area of pro-inflammatory cytokines interleukin (IL)-2 (p < 0.05) and IL-12 (p < 0.0001) was recorded in VUE lesions and a reduction in the anti-inflammatory cytokine IL-4 in the stillbirth with VUE cohort. Dual immunofluorescence of cell markers and cytokines implies that the immune response in VUE is directed towards Th1-type cell-mediated immunity. An in vitro model of VUE was developed that enabled co-culture of explants with fluorescently labelled T cells isolated from matched maternal whole blood samples. Placental tissue and T cells could be maintained in culture for the required duration of the experiment and placental function was not affected by preparation and culture conditions. In vitro co-culture with maternal T cells resulted in a significant reduction in placental function as measured by hCG secretion (p=0.015). There were significant increases in culture supernatant concentrations of IL-1β (p=0.008), IL-10 (p=0.02), interferon-γ (p=0.02) and IL-1Ra (p=0.05) and tissue lysate concentrations of IL-6 (p=0.008) and IL-1β (p=0.02). Culture of explants with a combination of IL-2, IL-12 and anti-IL-4 significantly reduced hCG secretion compared to control (p=0.03).These studies indicate that VUE involves a Th1-type immune response that may affect placental function, the impact of which might be impaired fetal growth that could contribute to stillbirth. The novel in vitro model facilitates future investigations into the pathophysiology of VUE.

Neuroinflammation is an important contributor to the pathogenesis of many neurological diseases. A key component of the innate immune response in the central nervous system is the migration of neutrophils into the brain parenchyma, where they exacerbate neuronal injury and worsen clinical outcome. A greater understanding of the mechanisms underlying neutrophil influx into the brain may aid the development of novel therapeutic interventions for the variety of diseases to which neutrophils contribute, notably including stroke and epilepsy. In vitro evidence implicates the pro- inflammatory cytokine, interleukin-1α (IL-1α), derived from platelets as a key mediator of cerebrovascular inflammation and neutrophil migration across brain endothelial cells.The aim of the work in this thesis was to test if this mechanism is important in vivo.We investigated the contribution of platelets and IL-1 in a murine model of neutrophil migration into the peritoneal cavity in response to injection of lipopolysaccharide (LPS). Depletion of platelets abrogated the migration of neutrophils in response to LPS- induced peritonitis, indicating an important role for platelets in the process. Genetic knockout of IL-1 had no effect on neutrophil influx, demonstrating that migration in the peritoneum occurs independently of IL-1.The discovery that neutrophil migration in LPS-induced peritonitis was independent of IL-1 contrasted with the finding that platelet-derived IL-1 was a mediator of neutrophil influx across mouse brain endothelial cells in vitro. The question arose as to whether IL-1 was required as a mediator of neutrophil migration in extra-cerebral tissues. Hence, we tested the contribution of platelets and IL-1 in two further in vivo models of neutrophil migration: LPS injection into a subcutaneous air pouch, and acute lung injury induced by LPS inhalation. Platelet depletion significantly reduced neutrophil migration into the air pouch in response to LPS, yet had no effect in acute lung injury. This indicated that neutrophil migration into the air pouch was dependent on platelets, and that migration into the lungs was platelet-independent. LPS induced the same degree of neutrophil migration in wild-type and IL-1 knockout mice, demonstrating that IL-1 was not required for neutrophil migration in either model.To determine the contribution of platelets and IL-1 to neutrophil migration in response to cerebrovascular inflammation, we injected LPS into the mouse striatum. In this model, neutrophil influx to the brain parenchyma in response to LPS was reduced by depletion of circulating platelets, and inhibition of the platelet adhesion molecule, GpIb. Genetic knockout of IL-1α significantly reduced the number of invading neutrophils induced by LPS. These data confirmed that both platelets and IL-1α were important contributors to cerebral neutrophil migration in vivo. To determine whether platelets in systemic circulation may be the source of IL-1α, we treated mice with IL-1 receptor antagonist or anti-IL-1 antibodies to block systemic IL-1 action. Neither intervention affected cerebral neutrophil migration in response to LPS, suggesting that the IL-1α that mediates neutrophil migration may originate in the brain.Overall, these data demonstrate that IL-1α and platelets make an important contribution to neutrophil migration to the brain, yet independently of each other. Our data also suggest there may be specific mechanisms driving innate immune responses in vivo even in response to the same inflammatory stimulus.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic, inflammatory condition, characterised by airflow limitation. The use of glucocorticoids (GC) as an anti-inflammatory treatment in COPD has limited clinical benefits, and as such, new treatments are needed. Identifying key pathways involved in the inflammatory response in COPD may enable the development of novel treatments. The aims of this thesis were to examine the steroid sensitivity of an in vitro mixed sputum culture cell model, comparing COPD cells to smoking and non-smoking controls, examine expression of the intracellular signalling molecule p38 Mitogen Activated Protein Kinase (MAPK) in COPD lungs compared with controls, examine the GC and p38 MAPK inhibitor and dual therapy sensitivity of a bronchial epithelial cell line and finally, to understand the mechanisms by which a p38 MAPK inhibitor in combination with a GC synergistically inhibit pro-inflammatory mediator production in a bronchial epithelial cell line. Dexamethasone inhibits mixed sputum cell pro-inflammatory mediator release, with no differences in sensitivity observed between COPD and control cells. Isolated sputum neutrophils demonstrate modest sensitivity to dexamethasone, which is in contrast to blood neutrophils. There are increased numbers of cells positive for activated p38 MAPK in COPD lungs compared with controls, specifically localised to follicular B and CD8+ T cells, bronchial epithelial cells and alveolar and sputum macrophages. Lung and sputum neutrophils are devoid of activated p38 MAPK, and a pharmacological p38 MAPK inhibitor has no effect on pro-inflammatory mediator production from these cells. This is in contrast to blood neutrophils, whereby p38 MAPK activation can be induced following LPS stimulation and in vitro cell culture, and pro-inflammatory mediator release is inhibited by a p38 MAPK inhibitor. Dexamethasone and birb 796 inhibit stimulated pro-inflammatory mediator release from a bronchial epithelial cell line in a dose-dependent manner. Sensitivity to either drug is dependent on stimuli and the pro-inflammatory mediator analysed. There is additive and synergistic inhibition of pro-inflammatory mediator production when combination therapy comprising dexamethasone and birb 796 is used compared with either drug alone. This may be due to Birb 796 enhancing dexamethasone-mediated nuclear translocation of the glucocorticoid receptor, which may enhance the GC-mediated anti-inflammatory effects. Combination therapy may therefore be a useful therapeutic in the treatment of COPD.

COPD is a condition characterised by chronic inflammation in which macrophages arethought to play a central role. Corticosteroids are the most widely used antiinflammatoryagents to treat COPD. There is a subset of inflammatory mediatorswhich are corticosteroid insensitive and so there is a need for novel anti-inflammatorytherapies to treat COPD. One such target is the liver X receptor (LXR), a cholesterolsensing nuclear hormone receptor with anti-inflammatory properties. Before investigating the anti-inflammatory potential of LXR, I aimed to validate thealveolar macrophage in vitro culture model. I investigated the effect of differentculture times on unstimulated and stimulated cytokine release, dexamethasoneinhibition of cytokine release, and transcription factor phosphorylation in alveolarmacrophages from COPD patients and controls. I found that freshly isolatedmacrophages release higher levels of cytokines, are less responsive to dexamethasoneand have increased levels of phosphorylated p38 MAPK.I next investigated LXR gene and protein expression levels in alveolar macrophages andwhole lung tissue from COPD patients and controls, the effect of LXR activation on thesuppression of inflammatory mediators from LPS stimulated COPD alveolarmacrophages, and the effect of LXR activation on the induction of genes associatedwith alternative macrophage polarisation. The levels of LXR mRNA were significantlyincreased in whole lung tissue extracts in COPD patients and smokers compared tonever smokers. The expression of LXR protein was significantly increased in smallairway epithelium and alveolar epithelium in COPD patients compared to controls. Nodifferences in LXR mRNA and protein levels were observed in alveolar macrophagesbetween patient groups. The LXR agonist GW3965 significantly induced the expressionof the LXR dependent genes ABCA1 and ABCG1 in alveolar macrophage cultures. InLPS stimulated alveolar macrophages, GW3965 suppressed the production of CXCL10and CCL5, whilst stimulating IL-10 production. GW3965 did not significantly suppressthe production of TNFα, IL-1β, or CXCL8. The major finding is that LXR activation hasanti-inflammatory effects on CXC10, CCL5 and IL-10 production from alveolarmacrophages. Finally, I investigated whether dysfunctional lipid homeostasis is a feature of COPDalveolar macrophages. I found that alveolar macrophages from current smokers withand without COPD had increased levels of neutral lipids compared to never smokersand ex-smokers with and without COPD. Dysfunctional lipid homeostasis may play arole in COPD.

Introduction: Appetite is often impaired in patients with gastrointestinal inflammation. Up to 75% of hospitalised Crohn's disease (CD) patients are malnourished. Recent animal research has suggested that immune mediated upregulation of enteroendocrine cell (EEC) activity plays a mechanistic role in the appetite and feeding disturbance observed during gut inflammation. The role of EEC in producing factors regulating satiety and intestinal growth is well recognised but work on their use as therapeutic targets or agents in inflammatory bowel disease (IBD) is still in its infancy. EEC peptide dynamics are further controlled through dipeptidyl peptidase (DP4) protease metabolism but no data are yet available on its expression in IBD. My aim is to understand the roles of EEC in appetite control and the maintenance of gut mucosal integrity in intestinal inflammation. Methodology: Patients with CD and healthy controls were studied. Symptoms were assessed using visual analogue scores (VAS). Gut hormone responses to a test meal were studied using a multiplex-ELISA technique, and correlated to symptoms. At the tissue level, EEC markers and transcription factors were quantified using immunohistochemistry, quantitative polymerase chain reaction (qPCR) and western blotting techniques. The same techniques were used to study DP4 expression. The effects of glucagon-like peptide-2 (GLP-2) on a gut model of the epithelial barrier were studied by measuring the transepithelial electrical resistance (TEER) across GLP-2 exposed Caco-2 cell monolayers after cytokine exposure. Tight junction protein expression in naïve and GLP-2 exposed cells was quantified by western blotting. Main Results: CD patients with active inflammation displayed a significant reduction in appetite. At the tissue level, GLP-1 and chromogranin A (CgA) were significantly upregulated. At the mRNA level significant increased expression was noted for CgA, glucagon-like peptide-1 (GLP-1), ubiquitination factor 4a and neurogenin 3. At the plasma level, total polypeptide YY (PYY) was increased. A significant correlation was seen between postprandial PYY responses and symptoms of nausea and bloating. Ghrelin, was 3-fold higher in the CD group compared to controls, and showed a reversed postprandial response with a significant correlation with the CD activity index (CDAI). Protein DP4 expression was significantly decreased at the tissue and plasma level in CD. GLP-2 increased tight junction protein expression in Caco-2 cells and maintained stable TEER and tight junction protein expression after cytokine exposure. Conclusions: The data presented are compatible with a potential role of EEC in appetite dysregulation in intestinal inflammation. An enhanced EEC response to food intake may directly affect appetite in such patients through increased gut-brain signalling. These may present tractable therapeutic targets. The decrease in mucosal DP4 expression in CD may make bioactive GLP-2 more available in the affected gut, hence improving gut mucosal integrity in intestinal inflammation. This pilot work has shown that GLP-2 has a role in maintaining gut mucosal integrity in intestinal inflammation through a positive effect on tight junction protein expression.