Investigation of the cellular and physiological effects of 5 alpha-tetrahydro reduced glucocorticoids in vitro and in vivo

Glucocorticoids have been the most widely used anti-inflammatory drugs. However, chronic excessive exposure to glucocorticoids causes numerous side effects, including obesity, hypertension and type II diabetes. Thus dissociated steroids with anti-inflammatory efficacy and reduced metabolic side effects are an attractive therapeutic goal. A previous study has shown that 5a-tetrahydro corticosterone (5aTHB), produced by steroid 5a-reductases in rodents, is an activator of glucocorticoid receptor (GR). Therefore, we hypothesized that 5aTHB may possess some of the properties of the parent steroid to transactivate and transrepress genes regulated by GR and have shown it possesses some of the characteristics of a dissociated steroid.In cultured cells, 5aTHB has weaker effects than corticosterone to induce GR translocation but was synergistic with corticosterone. 5cxTHB did not induce the expression of PEPCK or TAT or glucocorticoid responsive reporter genes. In contrast however in bone marrow derived macrophages, 5aTHB increased the secretion of anti-inflammatory cytokine IL-10 and suppressed LPS-induced production of pro-inflammatory cytokines TNFa and IL-6.In C57BL/6 mice following acute administration of 5aTHB, glucose tolerance and insulin sensitivity were not affected, and hepatic PEPCK and TAT were not increased. In mice that were chronically infused with 5aTHB, body and organ weights, glucose tolerance, insulin sensitivity, or expression of glucocorticoid responsive genes in liver and adipose tissue were not changed. Elowever circulating ACTH was suppressed and in whole blood, LPS-induced production of TNFa was suppressed. In mice suffering from peritonitis induced by LPS, 5aTHB suppressed IL-6 levels in peritoneal fluid to a similar extent as corticosterone.Therefore the contribution of 5a-reductase type 1 (5aRl) to protect metabolic tissues from adverse metabolic effects of glucocorticoids was tested in 5aRl deficient mice.Circulating corticosterone was elevated in animals receiving corticosteroid with no difference between genotypes. Infusion of corticosterone induced a greater elevation in insulin and free fatty acids during a glucose tolerance test in 5aRl-null mice, compared to wild-type controls. In the mesenteric adipose tissue, angiotensinogen expression was only increased in 5aRl-null mice receiving corticosterone. However suppression of TNFa and IL-6 by corticosterone in the whole blood was independent of genotype.In conclusion, 5aTHB is a weak GR ligand, but possesses some anti-inflammatory properties similar to those of corticosterone, potentially acting through GR-protein interactions as opposed to GR-GRE interactions. 5aTHB could be a prototype for a selective anti-inflammatory drug with limited metabolic toxicity. Thus inhibition of 5aRl may cause detrimental metabolic effects without inducing immune suppression

C-reactive protein and enhancement of tissue damage: a potential therapeutic target.

Human C-reactive protein (CRP), the classical acute phase reactant, is a member of the pentraxin family of calcium dependent ligand binding proteins. When ligand bound, particularly to phosphocholine residues found in cell membranes, bacterial cell walls and lipoprotein particles, CRP activates the complement cascade through engagement of Clq. Prior studies have suggested an important role for CRP both epidemiologically and biologically in atherothrombosis and its consequences. In the largest study to date of the epidemiological association between CRP and future cardiac events in healthy individuals, we demonstrate in contrast to previous studies, that the additive value of this inflammatory marker is less than previously thought. Further study of the biology of this ancient molecule in a number of animal models of disease has failed to confirm a protective role for CRP in endotoxaemia, and despite evidence of upregulated production in a transgenic mouse strain prone to atherosclerosis, we have demonstrated no apparent effect of human CRP on atherogenesis. Finally we have clearly and robustly shown that decamerisation of pentameric CRP by a novel low molecular weight palindromic inhibitor effectively prevents the ligand binding properties of this protein. This has established a platform for pharmacological inhibition of CRP in myocardial infarction and stroke where the complement activation initiated by CRP binding to necrotic cells has been suggested to be pro-inflammatory and deleterious

Socio-Cognitive and Affective Computing

Social cognition focuses on how people process, store, and apply information about other people and social situations. It focuses on the role that cognitive processes play in social interactions. On the other hand, the term cognitive computing is generally used to refer to new hardware and/or software that mimics the functioning of the human brain and helps to improve human decision-making. In this sense, it is a type of computing with the goal of discovering more accurate models of how the human brain/mind senses, reasons, and responds to stimuli. Socio-Cognitive Computing should be understood as a set of theoretical interdisciplinary frameworks, methodologies, methods and hardware/software tools to model how the human brain mediates social interactions. In addition, Affective Computing is the study and development of systems and devices that can recognize, interpret, process, and simulate human affects, a fundamental aspect of socio-cognitive neuroscience. It is an interdisciplinary field spanning computer science, electrical engineering, psychology, and cognitive science. Physiological Computing is a category of technology in which electrophysiological data recorded directly from human activity are used to interface with a computing device. This technology becomes even more relevant when computing can be integrated pervasively in everyday life environments. Thus, Socio-Cognitive and Affective Computing systems should be able to adapt their behavior according to the Physiological Computing paradigm. This book integrates proposals from researchers who use signals from the brain and/or body to infer people's intentions and psychological state in smart computing systems. The design of this kind of systems combines knowledge and methods of ubiquitous and pervasive computing, as well as physiological data measurement and processing, with those of socio-cognitive and affective computing

Smoking and Second Hand Smoking in Adolescents with Chronic Kidney Disease: A Report from the Chronic Kidney Disease in Children (CKiD) Cohort Study

The goal of this study was to determine the prevalence of smoking and second hand smoking [SHS] in adolescents with CKD and their relationship to baseline parameters at enrollment in the CKiD, observational cohort study of 600 children (aged 1-16 yrs) with Schwartz estimated GFR of 30-90 ml/min/1.73m2. 239 adolescents had self-report survey data on smoking and SHS exposure: 21 [9%] subjects had “ever” smoked a cigarette. Among them, 4 were current and 17 were former smokers. Hypertension was more prevalent in those that had “ever” smoked a cigarette (42%) compared to non-smokers (9%), p\u3c0.01. Among 218 non-smokers, 130 (59%) were male, 142 (65%) were Caucasian; 60 (28%) reported SHS exposure compared to 158 (72%) with no exposure. Non-smoker adolescents with SHS exposure were compared to those without SHS exposure. There was no racial, age, or gender differences between both groups. Baseline creatinine, diastolic hypertension, C reactive protein, lipid profile, GFR and hemoglobin were not statistically different. Significantly higher protein to creatinine ratio (0.90 vs. 0.53, p\u3c0.01) was observed in those exposed to SHS compared to those not exposed. Exposed adolescents were heavier than non-exposed adolescents (85th percentile vs. 55th percentile for BMI, p\u3c 0.01). Uncontrolled casual systolic hypertension was twice as prevalent among those exposed to SHS (16%) compared to those not exposed to SHS (7%), though the difference was not statistically significant (p= 0.07). Adjusted multivariate regression analysis [OR (95% CI)] showed that increased protein to creatinine ratio [1.34 (1.03, 1.75)] and higher BMI [1.14 (1.02, 1.29)] were independently associated with exposure to SHS among non-smoker adolescents. These results reveal that among adolescents with CKD, cigarette use is low and SHS is highly prevalent. The association of smoking with hypertension and SHS with increased proteinuria suggests a possible role of these factors in CKD progression and cardiovascular outcomes

The role of Annexin-A1 in the pathophysiology of diabetes.

PhDDiabetes is a complex disease characterised by hyperglycaemia, which often leads to microvascular complications including diabetic nephropathy and cardiomyopathy. In this thesis, I have investigated the role of Annexin-A1 (ANXA1), an endogenous anti-inflammatory peptide, in two experimental murine models of diabetes caused by streptozotocin (STZ) or high-fat high-sugar diet (HFD), which mimic type-1 (T1DM) and type-2 diabetes (T2DM) respectively. I have also investigated the levels of ANXA1 in patients with either T1DM or T2DM. Patients with T1DM have increased plasma ANXA1 levels. In a murine models of type 1 diabetes loss of endogenous ANXA1 aggravates both cardiac and renal dysfunction in mice. Specifically, I have shown that key mediators of the MAPK pathway (p38, JNK and ERK1/2) are constitutively activated in ANXA1-/- mice, and activation of these pathways is exacerbated in diabetic ANXA1-/- mice. Administration of human recombinant (hr) ANXA1 did not alter the diabetic phenotype in diabetic WT mice, but attenuated the cardiac and renal dysfunction caused by STZ. Interestingly, late administration of ANXA1 (after significant cardiac and renal dysfunction had already developed) halted the progression of both cardiac and renal dysfunction. Patients with T2DM have increased plasma ANXA1 levels. HFD-fed ANXA1-/- mice have a more severe diabetic phenotype compared to HFD-fed WT mice. Therapeutic administration of hrANXA1 prevented the development of a diabetic phenotype. Specifically, I have shown that the insulin signalling pathway is further perturbed in diabetic mice resulting in severe insulin resistance, and that these signalling abnormalities were prevented by therapeutic administration of hrANXA1. In addition, loss of endogenous ANXA1 aggravates both cardiac and renal dysfunction in mice with experimental T2DM. The GTPase RhoA is constituently activated in ANXA1-/- mice leading downstream activation of MYPT1. Feeding a HFD also activated the small GTPase RhoA, leading to increased MYPT1 activity, which could be attenuated with treatment with hrANXA1. Mice subjected to HFD for 12 weeks had a more ‘leaky’ blood brain barrier (BBB), which is further exacerbated in ANXA1-/- mice fed a HFD. Compared to mice fed a chow diet, mice fed a HFD had an augmented CD4+ T-cell profile; with a clear decline in CD4+FoxP3+ (anti-inflammatory) and increase in CD4+RORgt+ (pro-inflammatory) cells. Administration of hrANXA1 to mice fed on HFD restored BBB integrity and CD4+ T-cells profile similar to mice fed on normal chow diet. Mice fed a HFD also had more activated CD4+ T-cells, which adhered more readily and transmigrated through a brain endothelial mono-layer ex vivo. In contrast, administration of hrANXA1 to mice fed on HFD reduced re-activity of CD4+ T-cells, reducing the number of adherent CD4+ T-cells to the brain endothelial mono-layer.British Heart Foundatio

The Molecular Biology of Sickle Cell Anaemia

Sickle cell anaemia (SCA) is a haemolytic anaemia that reduces life expectancy and places a great burden on healthcare systems worldwide. Despite being a monogenic disorder, the phenotypic severity varies greatly between patients, ranging from patients that experience multiple strokes and organ failure during childhood, to those that live largely unaffected lives. Some genetic variants that affect globin gene expression are known to influence phenotype severity, but most of this variation remains unaccounted for. We conducted whole exome sequencing analyses, comparing SCA patients with mild and severe clinical phenotypes, with the aim of identifying novel genetic modifiers of the disease. SCA patient exomes were sequenced from a cohort at King’s College Hospital, and combined with publicly available SCA exomes recruited in the United States. Nine candidate variants were identified in genes with plausible mechanisms to influence the pathophysiology of the disease. The genes identified in this study affected nitric oxide signalling, haematopoietic regulation, globin gene expression and recovery from ischaemic injury. In order to evaluate these variants, a CRISPR genomic editing pipeline was established and tested on two previously identified candidate modifiers of SCA, in the genes ASH1L and KLF1. These variants were successfully introduced into erythroleukaemic cells and provide a pathway for testing the novel modifier genes identified in the exome sequencing analysis. Preliminary studies indicate that both ASH1L and KLF1 variants alter globin gene expression. In addition to genetic factors, we also hypothesised that epigenetic factors affect the SCA phenotype, and play a role in the therapeutic mechanism of hydroxyurea treatment. We optimised a method for isolating CD45+CD71+GPA- nucleated erythroid progenitors from small volumes of SCA peripheral blood. This was undertaken to evaluate the role of the epigenome in SCA phenotype severity and drug action, but for which patient sample collection proved too challenging within our clinical cohort

The Little Become Big?: Ambit and London's Little Magazines, 1959-1999

In The Little Magazines: A Study of Six Editors (1976), Ian Hamilton described the little magazine as a medium 'which exists, indeed thrives, outside the usual business structure of magazine production and distribution; it is independent, amateur and idealistic' (pp. 7-8). Although this definition applies to many titles of the Thirties, Forties and Fifties, it fails to register radical improvements in magazine design, distribution and book-keeping prompted by the mimeo revolution in the Sixties, Arts Council (ACGB) rationalisation in the Seventies and Eighties, and electronic publishing in the Nineties. This thesis offers a literary-historical account of Ambit's evolution from a scruffy, mimeo-produced pamphlet to a glossy, ACGB-sponsored quarterly. It provides a decade-by-decade analysis of Ambit's principal formal and thematic concerns, relating them to the work of key contributors in poetry, prose fiction and the visual arts, and detailing the emergence of a distinctive Ambit identity. At the same time, Ambit is also treated as a chronologue of the formal development of the contemporary little magazine, registering the complex economic, cultural and technological stimuli that affected the medium between 1959 and 1999. Both approaches are designed to test Hamilton's reading of little magazine history against a. detailed study of a major literary-arts quarterly of the post-1960 period, and identify ways in which contemporary publications like Ambit (1959- ), Agenda (1959- ), New Departures (1959- ) and Stand (1952- ) have departed from Hamilton's highly romanticised conception of 'littleness'. The thesis concludes by suggesting that Ambit and its contemporaries have sacrificed independence, amateurism and idealism for the entrepreneurial pragmatism that has sustained their commitment to the 'non-commercial' publication of 'artistic work from unknown or relatively unknown writers' that Frederick J. Hoffman et al in The Little Magazine: A History and a Bibliography (1946) (p. 2) saw as the little magazine's raison d'être