No description available.

The aim of this project was to investigate the importance of methionine depletion as a cause of cytotoxicity for paediatric CNS tumour cell lines, and also to investigate the in vitro cellular biochemical responses, as measured by changes in cellular levels of L-methionine, glutathione and O6 - alkylguanine DNA - alkyltransferase (MGMT) activity in glioma (D54) and medulloblastoma (Daoy) cell lines. A characteristic feature of many solid tumours is their requirement for both endogenous and exogenous L-methionine in order to support cellular proliferation. Normal cells are generally methionine-independent and utilise L-homocysteine to produce sufficient levels of L-methionine for cellular proliferation. The work presented in this thesis shows that Daoy and D54 cells are methionine-dependent cell lines in that they stop proliferating in methionine-depleted media supplemented with L-homocysteine. Whereas D54 cells do not exhibit detectable MGMT activity, methionine depletion markedly down-regulates the activity of this DNA repair enzyme in Daoy cells. Methionine depletion gives rise to a demonstrable decrease in methionine levels and an increase in glutathione levels for both tumour cell lines. Moreover, Daoy and D54 cells are found to be significantly more resistant to methotrexate (MTX), temozolomide (TMZ), and cisplatin (CDDP) under conditions of methionine depletion, compared to controls under normal cell culture conditions, a finding that may, at least in part, be related to the increased glutathione levels found. Further studies are required to determine the relative contribution of glutathione levels and modulation of apoptosis to explain the reasons for the reduced chemosensitivity to MTX, CDDP and TMZ for both Daoy and D54 cells. Evidence suggests that TMZ, CDDP and MTX may modulate cellular determinants of chemosensitivity through effects on methionine metabolism and MGMT levels. Therefore, the potential synergies of TMZ, CDDP and/or MTX for Daoy and D54 cells were investigated in methionine-replete conditions, and the combination of MTX and TMZ in particular was found to demonstrate synergistic effects. Further studies are needed to determine the reasons for this effect, and these may give insights into the further clinical development of these drugs in the setting of childhood CNS tumour therapy.

Adherent CD34+ cells are a stem cell enriched population with a high frequency of primitive stem cells that are genetically primed to differentiate into tissue-specific lineages. These cells make up the adherent CD34+ cells in peripheral blood which are the focus of this study. The first aim of this study was to transcriptionally characterize adherent CD34+ cells relative to nonadherent CD34+ cells using a whole human genome Affymetrix U133 plus 2 array. The subsequent analysis demonstrated transcriptional differences in genes which are involved in quiescence, cell cycle, homing and adhesion, which are likely to be relevant to the suitability of adherent CD34+ cells for clinical application. To verify the microarray analysis, a selection of representative transcripts was chosen and their relative expression was compared using real-time qPCR. To analyze further the stem cell characteristics of adherent CD34+ cells, the DNA replication timing kinetics of pluripotency-associated genes in bone marrow-derived adherent CD34+ cells, non-adherent CD34+ cells, and foetal mesenchymal stem cells were conducted and compared to those of embryonic stem (ES) cells. This showed that pluripotency-associated genes Oct4 and Nanog replicate early in the cell cycle similar to ES cells. The second aim of this study was to examine the potential use of adherent CD34+ cells in neural injury. As adherent CD34+ cells are primitive stem cells, it was reasonable to hypothesize that these cells would differentiate into neural cells in vitro, but these experiments revealed that these cells are not neurogenic and, in our hands, do not differentiate into neural cells. The experiments revealed that adherent CD34+ cells differentiate efficiently and reproducibly into microglia-like cells (CD34MG cells). CD34MG cells upregulated microglia-associated antigens and demonstrated functional characteristics of microglia cells including TNF and IL-6 release in response to lipopolysaccharide. CD34MG also stimulate responders in a mixed lymphocyte reaction, suggesting antigen presenting capabilities. To determine if these cells might be exploited for use in brain tumour therapy, it was shown that CD34MG migrate and invade in response to brain tumour glioma cells. Results also suggest that these cells are capable of presenting glioma specific antigen to immune cells in an autologous lymphocyte reaction in vitro. The combined results suggest that CD34MG cells might be exploited for therapeutic purposes in brain tumours. Stem cells are currently being explored for their therapeutic potential. This study demonstrates that adherent CD34+ cells and CD34MG cells may be useful for clinical use.

Research examining psychological outcomes of people diagnosed with primary brain tumours indicates that distress is common. However, research demonstrates mixed findings with regards to those factors associated with psychological distress. This thesis explores factors that relate to psychological outcomes in this group, using the self regulation model as a specific framework for understanding the individuals' experience. In section one, a narrative review examines research pertaining to the experience of living with a primary brain tumour and explores the relevance of the self regulation model to this population. The review suggests a number of illness perception dimensions are linked to psychological distress, specifically, illness coherence, consequences and causal attributions. However, the review highlights that the field of psychosocial brain tumour research has yet to fully exploit the potential of the self regulation model. Section two reports findings from a quantitative study carried out using a sample of 74 adult participants from the UK who have been diagnosed with a low grade primary brain tumour. The relationship between the domains of the self regulation model and psychological outcomes is investigated. Other, more established variables, which have been shown to predict psychological distress, are also investigated. Findings indicate a biopsychosocial causal attribution predicts both anxiety and depression in this group. In addition, the illness perceptions variables of illness coherence and illness identity contributed to the prediction of anxiety and depression, respectively. Furthermore, cognitive difficulties were found to predict scores on all three psychological outcomes. Personal and methodological reflections are discussed in section three along with considerations of areas for future research.

No description available.

In this thesis, a novel case-based reasoning (CBR) approach to radiotherapy treatment planning for brain cancer patients is presented. In radiotherapy, tumour cells are destroyed using ionizing radiation. For each patient, a treatment plan is generated that describes how the radiation should be applied in order to deliver a tumouricidal radiation dose while avoiding irradiation of healthy tissue and organs at risk in the vicinity of the tumour. The traditional, manual trial and error approach is a time-consuming process that depends on the experience and intuitive knowledge of medical physicists. CBR is an artificial intelligence methodology, which attempts to solve new problems based on the solutions of previously solved similar problems. In this research work, CBR is used to generate the parameters of a treatment plan by capturing the subjective and intuitive knowledge of expert medical physicists stored intrinsically in the treatment plans of similar patients treated in the past. This work focusses on the retrieval stage of the CBR system, in which given a new patient case, the most similar case in the archived case base is retrieved along with its treatment plan. A number of research issues that arise from using CBR for radiotherapy treatment planning for brain cancer are addressed. Different approaches to similarity calculation between cases are investigated and compared, in particular, the weighted nearest neighbour similarity measure and a novel non-linear, fuzzy similarity measure designed for our CBR system. A local case attribute weighting scheme has been developed that uses rules to assign attribute weights based on the values of the attributes in the new case and is compared to global attribute weighting, where the attribute weights remain constant for all target cases. A multi-phase case retrieval approach is introduced in which each phase considers one part of the solution. In addition, a framework developed for the imputation of missing values in the case base is described. The research was carried out in collaboration with medical physicists at the Nottingham University Hospitals NHS Trust, City Hospital Campus, UK. The performance of the developed methodologies was tested using brain cancer patient cases obtained from the City Hospital. The results obtained show that the success rate of the retrieval mechanism provides a good starting point for adaptation, the next phase in development for the CBR system. The developed automated CBR system will assist medical physicists in quickly generating treatment plans and can also serve as a teaching and training aid for junior, inexperienced medical physicists. In addition, the developed methods are generic in nature and can be adapted to be used in other CBR or intelligent decision support systems for other complex, real world, problem domains that highly depend on subjective and intuitive knowledge.

Rationale: CMF (cyclophosphamide: CP; methotrexate: MTX; 5-fluorouracil: 5-FU) is a chemotherapy combination associated with the cognitive impairments which many cancer patients experience after treatment. A reduction in hippocampal neurogenesis is a known means by which cytotoxic drugs alter cognition and is the mechanism investigated in the present study. There is currently no way of treating or preventing the cognitive deficits produced by chemotherapy and a simple pharmacological approach to achieving this could potentially have significant benefits for patients. Objectives: The studies in the present thesis use an animal model to investigate the effects of the individual agents in the CMF combination on spatial working memory and the proliferation and survival of neural precursors involved in hippocampal neurogenesis. It was also investigated whether the cognitive impairment produced by chemotherapy could be reversed or prevented by the antidepressant fluoxetine. Methods: In 4 separate experiments, adult male Lister-hooded rats were chronically administered with CP (30mg/kg, 4 or 7 i.v. doses), MTX (75mg/kg, 2 i.v. doses) or 5-FU (25mg/kg, 5 i.p. doses). Some rats were co-administered with fluoxetine (10mg/kg/day, in drinking water) for different time periods. Spatial memory was tested using the novel location recognition (NLR) task and the spontaneous alternation in the T-maze memory tasks. Proliferation and survival of hippocampal cells was quantified using immunohistochemistry and the levels of doublecortin (DCX) and brain-derived neurotrophic factor (BDNF) were quantified in the hippocampus and frontal cortex using Western blotting. Neural stem cells (NSC) were also isolated from the adult mouse hippocampus, to examine the direct effects of 5-FU, fluoxetine and its active metabolite, norfluoxetine (0.01-100µM) in vitro. Results: Rats treated with 5-FU and MTX showed impairment in the NLR task but not the spontaneous alternation in the T-maze task. They also exhibited a reduction in cell proliferation (Ki67-positive cells) and survival (BrdU-positive cells) in the dentate gyrus, compared to saline treated controls, but no difference was seen in the levels of DCX of BDNF. The induced cognitive and cellular impairments were not seen when fluoxetine was co-administered with the chemotherapy. The impairments caused by 5-FU were counteracted when fluoxetine was co-administered before and during 5-FU treatment but not when it was only administered after treatment. CP did not impair performance in the NLR task or hippocampal cell proliferation; however it significantly reduced cell survival. 5-FU, fluoxetine and norfluoxetine all decreased cell viability in vitro. Conclusions: These results demonstrate that MTX and 5-FU have more pronounced effects on spatial memory and hippocampal cell proliferation than CP in the CMF combination. Furthermore these impairments can be reversed by fluoxetine in a mechanism of prevention but not recovery. Although further work is required, it would be beneficial to establish an in vitro model of chemotherapy-induced cognitive impairment to justify this conclusion and to investigate the potential benefits of fluoxetine in cancer patients.

Elucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the b-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type.

Gliomas are the most common primary tumours of the central nervous system with a lethality rate approaching 80% in the first year of glioblastoma diagnosis. Their highly invasive nature renders local therapies such as surgery and radiation ineffective; whereas novel approaches such as immunotherapy are being actively studied as possible adjuncts in the treatment of patients with malignant gliomas. A number of factors have been proposed to play a role in glioma immune escape, including their poor immunogenicity since they do not express specific glioma antigens; their location within the CNS, which is considered to be an immune-privileged organ and some indications that glioma cells are capable of releasing various immunosuppressive cytokines, which inhibit the cytotoxic function of T cells. Cytokines are multifunctional pleiotropic proteins, which are involved in intercellular communication and cellular activation, and they may exert their effects in the CNS both directly and indirectly. Since they may originate either from peripheral immune organs, and cross the blood-brain barrier or they may be produced by the neuronal cells within the CNS, their properties are both immunoregulatory and neuromodulatory. This thesis investigated gliomas for the expression of cytokines and implicated their expression in the malignancy and the angiogenesis of the tumours. Cytokine secretion was evaluated from isolated peripheral blood mononuclear cells (PBMCs) of 33 patients with malignant gliomas (grade IV) immediately before surgery, in parallel with a control group of 23 age-matched individuals, and in 5 primary glioma cell cultures using the sensitive ELISPOT methodology. Thi cytokines tumour necrosis factor (TNF-a) and interferon (IFN7) were markedly reduced compared to control levels (P=0.01 and P < 0.001, respectively). In contrast, Th2 interleukins IL-(4) and IL-(10) were strongly expressed in both peripheral lymphocytes and glioma cell cultures (P < 0.05 and P < 0.001 respectively). Immunohistochemical localisation of IL-6, IL-8, COX, IL-lO, VEGF (vascular endothelial growth factor) and CD34 expression levels was performed in formalin-fixed paraffin-embedded tissue sections taken from 23 patients. Microvessels highlighted by means of anti-CD34 irnmunohistochemistry were enumerated with computer assisted image analysis. IL-6 was identified immunohistochemically in all specimens and showed an elevation in expression as necrosis grade increased (p=O.00S). IL-6 was also increased as the histological grade of the tumour increased (p0.020). IL-S expression was detected in all cases of gliomas with a mean of 19.5% of the cells. Expression was not related to either necrosis grade or increase in tumour grade. COX immunoreactivity was detected in all cases with an average of 29.1% positive cells. However, COX immunoreactivity was not significantly correlated with either necrosis or tumour grade. VEGF immunoreactivity occurred in 58.3% of cells and demonstrated a positive correlation with tumour grade (pzr0.035). The expression level of IL-10 was low both in terms of staining intensity and percentage of positive cells, and did not correlate with either necrosis or histological grade. CD34 immunoreactivity indicated that vascular volumetric parameters were not affected by either tumour or by necrosis grade. The expression of soluble CD95 as measured using a specific ELISA was not significantly (p > 0.05) reduced in the serum of 43 glioma patients compared to normal levels. In conclusion, these results indicate that patients harbouring malignant gliomas exhibit a broad suppression of cell-mediated immunity possibly due to the marked dysregulation in their cytokinetic profile. Glial tumours seem to induce a Thi (stimulatory) to Th2-type (inhibitory) cytokine shift which further supports humoral immunity at the expense of cell-mediated immune responses, contributing to the inefficient anti-tumour responses generated in these patients.

Despite improved knowledge and advanced treatments of high-grade gliomas, the overall survival rate of glioma patients remains low due to the recurrences and locations of the tumour. Evidence shows that the existence of a subpopulation of cells - cancer stem cells (CSCs) may be the major obstacle in treating gliomas. CD133 and nestin have been suggested as the markers of CSCs and natural stem cells. The primary focus of this study was to identify CD133+/nestin+ stem-like cells and discover their association with multidrug resistance (MDR) related genes, i.e. multiple drug resistance I (mdrl) gene and anti-apoptotic gene (bcl-2) in human glioma compared to normal brain tissues and cell lines. Glioma and normal astrocyte cell lines have been employed for CD133 isolating purposes to characterise the association with MDR related genotype and phenotype. The chemosensitivity of the isolated CD 133 population was investigated using chemosensitivity assay. Meanwhile, a serum deprivation method was established in this study to enrich and select CD 133+ CSCs in a glioma (GOS-3) cell line. As a secondary focus of this project, the possibility of immortalisation enzyme hTERT being a discriminative masker between normal and cancer brain stem cells and the transcriptional correlation between cd133 and bmi-lIc-myc oncogenes were investigated. For the first time, findings of the current study demonstrated that 1) there was an evident increase of CD133 gene expression in glioma compared to normal brain tissues where the latter expressed low levels of CD133, P-gp and Bcl-2 than glioma tissues, with an exception of nestin expression, 2) serum deprivation enriched CD133 expression and demonstrated a direct coexpression between CD133 and drug resistance in GOS-3 cells, 3) hTERT may not be a discriminative marker for normal and cancer brain stem cells, 4) although there was a strong transcriptional association between bmil and cmyc, there was an inverse transcriptional association between these genes and cd133 in serum deprived glioma cells, suggesting that bmil may not be essential for the maintenance of glioma stem cells, and 5) CD133+ glioma and normal brain cells showed a significantly high resistance towards chemotherapeutic drugs compared to the autologous CD133- cells. In conclusion, an improved understanding of molecules contributing to the maintenance of CSCs may lead to a combined treatment, targeting both CSCs and their protective MDR phenotypes leading eventually to attractive strategies for the treatment of gliomas.