Ex vivo culture of patient tissue and examination of gene delivery

Gene therapy has emerged as a realistic prospect for the treatment of cancer due to its potential for selective tumour cell targeting. The greatest challenge gene delivery vectors face is the ability to safely and efficiently deliver genes into target cells. The overall objectives of this thesis are to evaluate the efficacy of various gene delivery methods in a clinically relevant tumour model and to also investigate potential strategies for tumour selective delivery. We began with the development of a tumour slice model system using patient waste tissue. This model involves the use of fresh human tumour tissue, cut into thin slices and maintained ex vivo and is universally applicable to gene delivery methods, using a real-time luminescence detection method to assess gene delivery. The nature of the ex vivo culture system permitted examination of specific physiological variables, the influence of intratumoural factors and tissue specific effects on vector expression. Adenoviral vectors under the control of the human CXCR4 promoter demonstrated a 'tumour on' and 'normal off' expression profile when compared with the ubiquitously active CMV promoter when tested in patient tumour tissue. In addition, we developed an ex vivo system of changing oxygenation using the hypoxia inducer, cobalt, to mimic the transient hypoxic conditions found in solid tumours. We found that Adenoviral transgene expression was robust in the cycling hypoxic conditions relevant to solid tumours and re-oxygenation of chronically hypoxic tissue enhanced transgene expression. Finally, we demonstrated an AAV-based tumour targeting strategy using a tumour-selective promoter allowing for the efficient targeting of AAV vectors to cancer cells and the sparing of normal tissue in both murine metastatic liver tumours models and patient tissue. The thesis highlights the importance of indepth preclinical assessment of novel therapeutics and may serve as a platform for further testing of novel gene delivery approaches.

Managing periodontal disease: the clinician's role and the patient's responsibility

This manuscript highlights the roles and responsibilities of the clinician and patient in the successful management of periodontal disease. Clinical relevance: This article highlights the variety of factors that need to be addressed for periodontal diseases to be successfully managed. Learning objective: The reader should understand the broad range of issues that require consideration for patients to be successfully managed for their periodontal problems.

Comparison between local anaesthetic agents, lidocaine and bupivacaine, in patients undergoing third molar extraction in terms of patient satisfaction.

AIMS: (1) To determine if anaesthetic agent bupivacaine, has a prolonged effect on the period of acute postoperative pain when compared to lidocaine, a shorter acting agent. (2) To determine patient’s post-operative satisfaction and preference with regard to anaesthetic choice. METHODS: This double blind, randomised, interventional clinical trial included 85 patients. All patients had bilateral impacted lower third molars of removed under general anaesthetic. All patients received 0.5% plain bupivacaine on one randomly allocated side, with 2% lidocaine (with adrenaline) administered on the opposite side. Pain was measured using visual analogue scales at 0, 30, 60 minutes and 3, 4, 6 and 8 hours post-surgery. Pain was analysed for 1 week following surgery. Psychological evaluations and patient reported outcomes, including patient satisfaction were evaluated. RESULTS: A significant difference in pain (P=0.001) was seen during the 3-8 hour post-operative period. The upper limit of the 95% confidence interval was 10.0 or above at 3hours and 4 hours post-surgery. Two-thirds of patients preferred bupivacaine. CONCLUSION: Longer lasting anaesthetics such as bupivacaine offer a longer period of analgesia, and improve overall patient satisfaction.

Paracetamol metabolism in postoperative patients

Introduction: Despite being available for more than 50 years, there is still much to learn about paracetamol. Postoperative analgesic regimens that maintain good pain control while minimising exposure to opiates are beneficial and paracetamol has had a resurgence in this role since an IV formulation came to market. However there is evidence to suggest currently licensed doses are sub-therapeutic, especially when administered orally or rectally. Higher, unlicensed doses are now being advocated but, prior to this study, there was little evidence of their safety in surgical patients. When assessing drug safety in surgical patients a number of surgery and patient related factors influence results, and these must be considered. Methods: Major and intermediate surgical patients were recruited from two hospitals in Ireland. They were administered IV paracetamol at either 9g or 4g daily doses. In addition they received daily sub therapeutic doses of four other medicines to indicate the activity of their CYP450 enzymes that are involved in paracetamol metabolism. Urine and blood samples were collected to determine paracetamol pharmacokinetics, CYP450 activity, inflammatory cytokine concentration and for evidence of hepatotoxicity. Results: There were 33 patients that participated in the study. There was no evidence of clinically significant hepatotoxicity occurring in any patient during the study period, but there could have been changes following this time. Paracetamol disposition was shown to change, however half-life remained relatively constant. There were a number of changes to the way paracetamol was metabolised following surgery that maintained this rate of elimination. Conclusion: Doses of up to 9g per day given to major surgical patients for up to five days postoperatively produced no evidence of hepatotoxicity. Further research is warranted to determine the clinical utility of these higher doses