The influence of ageing and culture conditions on limbal epithelial cells

PhD ThesisIntroduction. The use of limbal stem cells as a cellular therapy has expanded over the last decade. Currently, the success rate of limbal stem cell transplantations is around 76% with advanced donor age being a possible reason for this figure. Medium and oxygen concentration could also have a detrimental effect on the growth of these cells for transplantation. With age the limbus undergoes a topological change which could alter the stem cells surroundings. A mouse model of corneal ageing was also used to complement the human study and avoid culturing and tissue storage artefacts. Aims. The main aim of this thesis was to assess the effect of age on corneal epithelial progenitor cells. This then led to the question; is the mouse cornea affected by age and can interventions mediate these outcomes? And finally do culture conditions affect the growth of corneal epithelial stem cell containing cultures, and how does this relate to changes to the niche with age? Methods. Limbal derived progenitor cells were extracted from human corneosclearal disks donated for research. Cells were cultured either in LEM or DKSFM and either in hypoxia (3%) or normoxia (21%). Immunofluorescence, qPCR, TRAP assay, morphological and clonal analysis where used to assess progenitor composition. The mice used were strain B6.129S-Tert, tm1Yjc/J. Old/AL and DR mice were 22 months old, young were 3 months old. Rapamycin treated mice were 16 months old and were treated for four months, starting at 12 months of age. Dietary restriction was implemented for a period of 16 months from 6 months of age, additionally in the mouse eye a DNA damage marker was also assessed, both telomeric and non-telomericly. Results and Conclusions. Age has a detrimental effect on the ability to culture a limbally derived clonal population. However age did not affect the levels of positive and negative gene expression markers or ΔNp63 protein level. Interestingly the time that corneal tissue is stored for did not affect the ability to isolate these progenitor cells. The growth of cells in hypoxia decreased negative marker KRT3 and senescence marker p21 regardless of culture medium. In the mouse model, age had a detrimental effect on the cornea with decreased TAp63, increased ɣH2A.X and TAFs. DR however, tended to have a beneficial effect on the mouse cornea. Interestingly rapamycin seems to be detrimental to the mouse cornea, with similarities with the effects of age.Biomedical Research Centre (BRC) and Newcastle upon Tyne Hospitals NHS Foundation Trust (NUTH)

Float, explode or sink: postmortem fate of lung-breathing marine vertebrates

What happens after the death of a marine tetrapod in seawater? Palaeontologists and neontologists have claimed that large lung-breathing marine tetrapods such as ichthyosaurs had a lower density than seawater, implying that their carcasses floated at the surface after death and sank subsequently after leakage of putrefaction gases (or ‘‘carcass explosions’’). Such explosions would thus account for the skeletal disarticulation observed frequently in the fossil record. We examined the taphonomy and sedimentary environment of numerous ichthyosaur skeletons and compared them to living marine tetrapods, principally cetaceans, and measured abdominal pressures in human carcasses. Our data and a review of the literature demonstrate that carcasses sink and do not explode (and spread skeletal elements). We argue that the normally slightly negatively buoyant carcasses of ichthyosaurs would have sunk to the sea floor and risen to the surface only when they remained in shallow water above a certain temperature and at a low scavenging rate. Once surfaced, prolonged floating may have occurred and a carcass have decomposed gradually. Our conclusions are of significance to the understanding of the inclusion of carcasses of lung-breathing vertebrates in marine nutrient recycling. The postmortem fate has essential implications for the interpretation of vertebrate fossil preservation (the existence of complete, disarticulated fossil skeletons is not explained by previous hypotheses), palaeobathymetry, the physiology of modern marine lung-breathing tetrapods and their conservation, and the recovery of human bodies from seawater

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used