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In vivo experiments were conducted to determine if elevated plasma glucocorticoid concentrations would suppress intrafollicular prostaglandin F2$ alpha$ (PGF2$ alpha$) synthesis and, thereby, inhibit ovulation in the pig. Following ACTH administration, PGF2$ alpha$ concentrations in FF tended to be lower than in controls. Injections of betamethasone partially suppressed the preovulatory rise of PGF2$ alpha$ in FF at 40h, although the effect was less marked than that produced by indomethacin. While no ovulations occurred in the indomethacin-treated group at any time, betamethasone resulted in a lower number of ovulated follicles at 44h than in the control animals. Progesterone concentrations were unaffected by the treatments. / In vitro studies were conducted with a spontaneously established cell line developed through continuous culturing of primary granulosa cells collected from prepuberal gilts six hours after they had received PMSG. Characterization of these cells revealed that aromatase and steroidogenesis were functional but gonadotropin receptors were not present. When extracellular PGF2$ alpha$ levels were measured, dexamethasone was able to significantly suppress PGF$ sb{2 alpha}$ concentrations, but not as effectively as with indomethacin. (Abstract shortened by UMI.)

For over a century, documented scientific research and debate has revolved around the therapeutic properties of the medicinal plant Echinacea angustifolia. With overwhelming evidence demonstrating the biological activity of its root phytochemical constituents, the genetic improvement of E. angustifolia by selecting phytochemically rich genotypes has garnered both scientific and commercial interest. This dissertation presents results of multi-disciplinary experiments intended to help establish scientifically based guidelines for breeding efforts aimed at developing therapeutically superior varieties of E. angustifolia in British Columbia. Cultivated E. angustifolia populations from British Columbia and Washington were grown in a common greenhouse environment to identify possible genetically superior populations with respect to root concentrations of therapeutically relevant caffeic acid derivatives (CAD) and alkamides. However, none of the studied cultivated E. angustifolia populations showed significant genetic differences in terms of root phytochemical traits. In the second part, an investigation into correlations between root and shoot phytochemical concentrations in field- and greenhouse-grown plants revealed that concentrations of therapeutically relevant marker compounds in shoots were generally poor predictors of concentrations in roots. Some weak yet significant positive correlations were observed between root and shoot concentrations of CADs but were inconsistent between the two environments. Significant genotype by environment interactions were documented for the first time in phytochemical traits of E. angustifolia in a study of five genetically homogeneous populations grown in three different environments, including 1 greenhouse and 2 field sites in British Columbia. For the final objective, in vitro bioassays showed that environmentally and genotypically related differences in concentrations of CADs and alkamides in E. angustifolia ethanolic root extracts did not translate into significant differences in their anti-inflammatory potential as measured by pro-inflammatory interleukin (IL-6 and IL-8) secretion in human bronchial epithelial (BEAS-2B) cells challenged with rhinovirus. When used in isolation however, pure tetraene alkamide showed a significant inhibitory effect on secretion, thereby further supporting the use of high alkamide production as a selection criterion for therapeutic E. angustifolia cultivar development. A series of recommendations derived from these findings are presented along with ideas for important future studies in the field of Echinacea research.

Dissertation submitted in partial compliance with the requirements for a Masters Degree in Technology: Chiropractic, Durban University of Technology, 2009. / Headaches are one of the most common clinical conditions in medicine, and 80% of these are tension-type headaches (TTH). TTH has a greater socioeconomic impact than any other type of headache due to its prevalence. Within the TTH category, episodic TTH are more prevalent than chronic TTH. The mainstay in the treatment of TTH are simple analgesics and NSAIDs. Unless contraindicated, NSAIDs are often the most effective treatment for ETTH. However patients suffering with TTH tend to relate their headaches to increased muscle stiffness in the neck and shoulders and thus the non-pharmacological treatment of ETTH could be directed at the associated musculoskeletal components of ETTH. It is therefore proposed that the Kinesio Taping® Method may have an effect in the treatment of the muscular component of ETTH. Method: This study was a prospective randomised clinical trial with two intervention groups (n=16) aimed at determining the relative effectiveness of a NSAID and the Kinesio Taping® Method in the treatment of ETTHs. The patients were treated at 5 consultations over a 3 week period. Feedback was obtained using the: NRS – 101, the CMCC Neck Disability Index and a Headache Diary. Results: The Headache Diary showed a reduction in the presence and number, mean duration and pain intensity of ETTH in both groups. These treatment effects were sustained after the cessation of treatment with the exception of mean pain intensity in the Kinesio Taping® Method group. The mean NRS score decreased in both groups but at a slightly faster rate in the Kinesio Taping® Method group. The CMCC showed an improvement in the functional ability of the patients in both groups. Conclusion: There seems to be no significant difference in the relative effectiveness of the treatment modalities. We can thus state that the overall short-term reduction in symptomatology supports the use of NSAIDs or Kinesio Taping® Method in the treatment of ETTH.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to suppress bone formation when administered before, but not if administered after, an acute bout of mechanical load in rats. The NSAID ibuprofen inhibits cyclooxygenase-2 enzyme, effectively reducing loading induced prostaglandin E2. Whether this affects eventual bone mass gains after multiple sessions of a more physiological mechanical loading regimen is unclear. Therefore, the aim of this study was to test the hypothesis that gains in bone mass and size will be diminished in adult rats given ibuprofen before, but not after, each exercise bout during 20 days of simulated resistance training (SRT). Virgin female Sprague-Dawley rats (5-mo-old, n=29) completed 9 SRT sessions using stimulated muscle contractions under anesthesia at 75% peak isometric strength on alternate days; each of 20 contractions included 1 sec isometric + 1 sec eccentric contraction. Animals were blocked assigned by body weight to one of three groups: 1) ibuprofen (30mg/kg) before exercise, placebo after (I:P)(n=9), 2) placebo before, ibuprofen after (P:I)(n=10) and 3) placebo before and after (P:P)(n=10). In vivo pQCT scans measured changes in total volumetric bone mineral density (vBMD) and total bone mineral content (BMC) at the proximal tibia (cancellous), and total vBMD, total BMC and total area at midshaft tibia on days -7 and 21. Dynamic histomorphometry on both midshaft tibiae (exercised and non-exercised legs) determined mineralizing surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR) on the periosteal surface. There were no differences in body weights among groups at baseline or at day 21. There were significant gains due to SRT, but not ibuprofen treatment in total BMC (+10.50 ± S.D. +8.15%) and total vBMD (+5.29 ± 3.41%) at the proximal tibia. The midshaft tibia exhibited significant gains in total vBMD (6.68 ± 3.03%), total BMC (19.18 ± 5.51%) and total area (11.68 ± 5.49%) due solely to SRT. Furthermore, there were significant increases in periosteal BFR (pre 21.89 µm3/µm2/d ±2.63; post 717.81 µm3/µm2/d ±100.57) at the midshaft tibia in the exercised vs. non-exercised legs in all groups but no effect of ibuprofen regimen was detected on these indices of bone formation. In the context of robust increase in BFR and bone mass within this simulated resistance protocol, we were unable to detect any impact of ibuprofen administration on the response to bone loading.

For over a century, documented scientific research and debate has revolved around the therapeutic properties of the medicinal plant Echinacea angustifolia. With overwhelming evidence demonstrating the biological activity of its root phytochemical constituents, the genetic improvement of E. angustifolia by selecting phytochemically rich genotypes has garnered both scientific and commercial interest. This dissertation presents results of multi-disciplinary experiments intended to help establish scientifically based guidelines for breeding efforts aimed at developing therapeutically superior varieties of E. angustifolia in British Columbia. Cultivated E. angustifolia populations from British Columbia and Washington were grown in a common greenhouse environment to identify possible genetically superior populations with respect to root concentrations of therapeutically relevant caffeic acid derivatives (CAD) and alkamides. However, none of the studied cultivated E. angustifolia populations showed significant genetic differences in terms of root phytochemical traits. In the second part, an investigation into correlations between root and shoot phytochemical concentrations in field- and greenhouse-grown plants revealed that concentrations of therapeutically relevant marker compounds in shoots were generally poor predictors of concentrations in roots. Some weak yet significant positive correlations were observed between root and shoot concentrations of CADs but were inconsistent between the two environments. Significant genotype by environment interactions were documented for the first time in phytochemical traits of E. angustifolia in a study of five genetically homogeneous populations grown in three different environments, including 1 greenhouse and 2 field sites in British Columbia. For the final objective, in vitro bioassays showed that environmentally and genotypically related differences in concentrations of CADs and alkamides in E. angustifolia ethanolic root extracts did not translate into significant differences in their anti-inflammatory potential as measured by pro-inflammatory interleukin (IL-6 and IL-8) secretion in human bronchial epithelial (BEAS-2B) cells challenged with rhinovirus. When used in isolation however, pure tetraene alkamide showed a significant inhibitory effect on secretion, thereby further supporting the use of high alkamide production as a selection criterion for therapeutic E. angustifolia cultivar development. A series of recommendations derived from these findings are presented along with ideas for important future studies in the field of Echinacea research.

Protein-energy wasting (PEW) is commonly seen in patients with chronic kidney disease (CKD). The condition is characterised by chronic, systemic low-grade inflammation which affects nutritional status by a variety of mechanisms including reducing appetite and food intake and increasing muscle catabolism. PEW is linked with co-morbidities such as cardiovascular disease, and is associated with lower quality of life, increased hospitalisations and a 6-fold increase in risk of death1. Significant gender differences have been found in the severity and effects of several markers of PEW. There have been limited studies testing the ability of anti-inflammatory agents or nutritional interventions to reduce the effects of PEW in dialysis patients. This thesis makes a significant contribution to the understanding of PEW in dialysis patients. It advances understanding of measurement techniques for two of the key components, appetite and inflammation, and explores the effect of fish oil, an anti-inflammatory agent, on markers of PEW in dialysis patients. The first part of the thesis consists of two methodological studies conducted using baseline data. The first study aims to validate retrospective ratings of hunger, desire to eat and fullness on visual analog scales (VAS) (paper and pen and electronic) as a new method of measuring appetite in dialysis patients. The second methodological study aims to assess the ability of a variety of methods available in routine practice to detect the presence of inflammation. The second part of the thesis aims to explore the effect of 12 weeks supplementation with 2g per day of Eicosapentaenoic Acid (EPA), a longchain fatty acid found in fish oil, on markers of PEW. A combination of biomarkers and psychomarkers of appetite and inflammation are the main outcomes being explored, with nutritional status, dietary intake and quality of life included as secondary outcomes. A lead in phase of 3 months prior to baseline was used so that each person acts as their own historical control. The study also examines whether there are gender differences in response to the treatment. Being an exploratory study, an important part of the work is to test the feasibility of the intervention, thus the level of adherence and factors associated with adherence are also presented. The studies were conducted at the hemodialysis unit of the Wesley Hospital. Participants met the following criteria: adult, stage 5 CKD on hemodialysis for at least 3 months, not expected to receive a transplant or switch to another dialysis modality during the study, absence of intellectual impairment or mental illness impairing ability to follow instructions or complete the intervention. A range of intermediate, clinical and patient-centred outcome measures were collected at baseline and 12 weeks. Inflammation was measured using five biomarkers: c-reactive protein (CRP), interleukin-6 (IL6), intercellular adhesion molecule (sICAM-1), vascular cell adhesion molecule (sVCAM-1) and white cell count (WCC). Subjective appetite was measured using the first question from the Appetite and Dietary Assessment (ADAT) tool and VAS for measurements of hunger, desire to eat and fullness. A novel feature of the study was the assessment of the appetite peptides leptin, ghrelin and peptide YY as biomarkers of appetite. Nutritional status/inflammation was assessed using the Malnutrition-Inflammation Score (MIS) and the Patient-Generated Subjective Global Assessment (PG-SGA). Dietary intake was measured using 3-day records. Quality of life was measured using the Kidney Disease Quality of Life Short Form version 1.3 (KDQOL-SF™ v1.3 © RAND University), which combines the Short-Form 36 (SF36) with a kidney-disease specific module2. A smaller range of these variables was available for analysis during the control phase (CRP, ADAT, dietary intake and nutritional status). Statistical analysis was carried out using SPSS version 14 (SPSS Inc, Chicago IL, USA). Analysis of the first part of the thesis involved descriptive and bivariate statistics, as well as Bland-Altman plots to assess agreement between methods, and sensitivity analysis/ROC curves to test the ability of methods to predict the presence of inflammation. The unadjusted (paired ttests) and adjusted (linear mixed model) change over time is presented for the main outcome variables of inflammation and appetite. Results are shown for the whole group followed by analyses according to gender and adherence to treatment. Due to the exploratory nature of the study, trends and clinical significance were considered as important as statistical significance. Twenty-eight patients (mean age 61±17y, 50% male, dialysis vintage 19.5 (4- 101) months) underwent baseline assessment. Seven out of 28 patients (25%) reported sub-optimal appetite (self-reported as fair, poor or very poor) despite all being well nourished (100% SGA A). Using the VAS, ratings of hunger, but not desire to eat or fullness, were significantly (p<0.05) associated with a range of relevant clinical variables including age (r=-0.376), comorbidities (r=-0.380) nutritional status (PG-SGA score, r=-0.451), inflammatory markers (CRP r=-0.383; sICAM-1 r=-0.387) and seven domains of quality of life. Patients expressed a preference for the paper and pen method of administering VAS. None of the tools (appetite, MIS, PG-SGA, albumin or iron) showed an acceptable ability to detect patients who are inflamed. It is recommended that CRP should be tested more frequently as a matter of course rather than seeking alternative methods of measuring inflammation. 27 patients completed the 12 week intervention. 20 patients were considered adherent based on changes in % plasma EPA, which rose from 1.3 (0.94)% to 5.2 (1.1)%, p<0.001, in this group. The major barriers to adherence were forgetting to take the tablets as well as their size. At 12 weeks, inflammatory markers remained steady apart from the white cell count which decreased (7.6(2.5) vs 7.0(2.2) x109/L, p=0.058) and sVCAM-1 which increased (1685(654) vs 2249(925) ng/mL, p=0.001). Subjective appetite using VAS increased (51mm to 57mm, +12%) and there was a trend towards reduction in peptide YY (660(31) vs 600(30) pg/mL, p=0.078). There were some gender differences apparent, with the following adjusted change between baseline and week 12: CRP (males -3% vs females +17%, p=0.19), IL6 (males +17% vs females +48%, p=0.77), sICAM-1 (males -5% vs females +11%, p=0.07), sVCAM-1 (males +54% vs females +19%, p=0.08) and hunger ratings (males 20% vs females -5%, p=0.18). On balance, males experienced a maintainence or reduction in three inflammatory markers and an improvement in hunger ratings, and therefore appeared to have responded better to the intervention. Compared to those who didn’t adhere, adherent patients maintained weight (mean(SE) change: +0.5(1.6) vs - 0.8(1.2) kg, p=0.052) and fat-free mass (-0.1 (1.6) vs -1.8 (1.8) kg, p=0.045). There was no difference in change between the intervention and control phase for CRP, appetite, nutritional status or dietary intake. The thesis makes a significant contribution to the evidence base for understanding of PEW in dialysis patients. It has advanced knowledge of methods of assessing inflammation and appetite. Retrospective ratings of hunger on a VAS appear to be a valid method of assessing appetite although samples which include patients with very poor appetite are required to confirm this. Supplementation with fish oil appeared to improve subjective appetite and dampen the inflammatory response. The effectiveness of the intervention is influenced by gender and adherence. Males appear to be more responsive to the primary outcome variables than females, and the quality of response is improved with better adherence. These results provide evidence to support future interventions aimed at reducing the effects of PEW in dialysis patients.

Chaperonin 10 (Cpn10) is a mitochondrial protein with protein folding function. There is substantial evidence that extracellular Cpn10 regulates the immune response. Prior research has shown that Cpn10 binds to T cells, inhibits LPS-induced RAW264.7 macrophage cell- and healthy donor peripheral blood mononuclear cell (PBMC)-activation, and downregulates lipopolysaccharide (LPS)-induced membrane distribution of the MHC II molecule on dendritic cells (DC). Recent Phase IIa rheumatoid arthritis (RA), psoriasis and multiple sclerosis (MS) clinical trials demonstrate improved disease amelioration with Cpn10. Despite compelling evidence of the anti-inflammatory properties of Cpn10, the precise mechanisms of action are unknown. The principal aim was to characterise the modulation of inflammation by Cpn10 and in the process create a bioassay that would allow for the reliable assessment of batch-to-batch variability of Cpn10 preparations. For this purpose, a Cpn10 bioassay was performed in the RAW264.7 cell line and expanded to DC and T cell lines. Furthermore, the analysis of gene expression in healthy donor PBMC was performed, as a mixed cell population experiment, to reflect possible involvement of cell-to-cell communication pathways. Initial data showed that Cpn10 reduced LPS-induced tumour necrosis factor ?? (TNF??) expression in RAW264.7 cells. However, the Cpn10 preparation was shown to contain trace lipid contaminants, which induced cellular tolerance, resulting in the observed reduction in TNF??. Experiments with a second batch of Cpn10 showed no reduction of LPS-induced TNF?? in the RAW264.7 cells, seen with the primary batch of Cpn10 and previously reported characterisation of Cpn10. The Cpn10 bioassay conducted in DC and T cell lines was shown to have the potential to decrease toll-like receptor 9 (TLR9) expression, suggesting that Cpn10 may attenuate immune responses by downregulating receptor recognition of bacterial components. The Cpn10 bioassay conducted in LPS-stimulated PBMCs revealed that Cpn10 downregulates gene expression of Th1 related genes including the polarising cytokines IL-7, IL-12B and IL-23A and Th2 related genes including the transcriptions factors GATA3, GFI1 and CEBPB. The downregulation of these genes may play an immuno-modulatory role, having improved efficacy of Cpn10 in T cell mediated autoimmune diseases, with possible therapeutic implications in Th2 mediated diseases such as asthma. The research carried out in this thesis provides insight into the success of Cpn10 in the RA, MS and psoriasis clinical trials. These results have also supported previously published data and provide additional insight into the mechanism of action of Cpn10. In addition, a Cpn10 bioassay has been established using healthy donor PBMCs stimulated with LPS and results show a reduced expression of Th1 and Th2 associated genes. The findings that in mixed cell populations, Cpn10 downregulates not only genes involved in Th1 polarisation mainly at the signal 3 level, but is also capable of downregulating Th2 polarising genes at the signal 1 level of TCR mediated transcription factors, are of particular interest. Ultimately, research from this project has confirmed the anti-inflammatory action of Cpn10 and given useful insight into how Cpn10 acts to modulate the inflammatory response.

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